avi's notes clinical biochemistry
TRANSCRIPT
Avi Sayag Clinical Biochemistry
Table of contents Topic 1: Reference range, requesting a test, sources of error, interpretation of results, specificity and
sensitivity, predictive value…………………………………………………………………….4
Topic 2: Laboratory signs of cellular damage. Basis of clinical enzymology…………………7
Topic 3: Inborn errors of amino acid metabolism and lab diagnosis of CF……………………9
Topic 4: inborn errors of carbohydrate and lipid metabolism…………………………………
Topic 5: Pathobiochemistry of inflammation………………………………………………….
Topic 6: Pathobiochemistry of plasma proteins……………………………………………….
Topic 7: Biochemical effects of tumors……………………………………………………….
Topic 8: Tumor markers in the diagnosis of malignant diseases………………………………
Topic 9: Iron metabolism, hemochromatosis, iron deficiency anemia………………………..
Topic 10: Lab diagnosis of hemoglobinopathies………………………………………………
Topic 11: Lab diagnosis of hemolytic anemia………………………………………………..
Topic 12: Lab diagnosis of megaloblastic anemia……………………………………………..
Topic 13: Major lab characteristics of ALL and CLL…………………………………………
Topic 14: Major lab characteristics of AML and CML…………………………………………
Topic 15: Lab diagnostics of quantitative platelet disorders…………………………………..
Topic 16: Inheritance of ABO blood group system and its clinical significance………………
Topic 17: Inheritance and clinical significance of Rh blood group system……………………
Topic 18: Coagulopathies, lab control of anticoagulant treatment…………………………….
Topic 19: Platelet function disorders…………………………………………………………..
Topic 20: Inherited thrombophilias……………………………………………………………
Topic 21: Acquired thrombophilias……………………………………………………………
Topic 22: Consumption coagulopathies……………………………………………………….
Topic 23: Lab tests of glomerular and tubular functions………………………………………
Topic 24: Clinical biochemistry of acute and chronic renal failures; tubulopathies………….
Topic 25: Disturbances of acid-base balance………………………………………………….
Topic 26: Predominant water depletion; isoosmolar volume depletion………………………
Topic 27: Water and sodium excess; predominant sodium depletion…………………………
Topic 28: Hypokalemia……………………………………………………………………….
Topic 29: Hyperkalemia……………………………………………………………………….
Topic 30: Pathogenesis of diabetes mellitus………………………………………………….
Topic 31: Lab diagnosis and management of diabetes mellitus……………………………….
Topic 32: Acute metabolic complications of diabetes mellitus……………………………….
Topic 33: Hypoglycemia……………………………………………………………………….
Topic 34: Disorders and lab diagnosis of lipid metabolism…………………………………..
Topic 35: Risk factors of atherosclerosis……………………………………………………….
Topic 36: Disturbances of uric acid metabolism………………………………………………
Topic 37: Lab diagnostics of acute MI…………………………………………………………
Topic 38: Hypovitaminosis and hypervitaminosis…………………………………………….
Topic 39: Lab diagnosis of hepatocellular damage; evaluation of liver function…………….
Topic 40: Lab diagnosis of cholestasis and liver cirrhosis…………………………………….
Topic 41: Pathobiochemistry and laboratory diagnostics of the gastrointestinal tract………….
Topic 42: Laboratory diagnosis of acute pancreatitis…………………………………………..
Topic 43: Clinical biochemistry of the hypothalamus and the pituitary……………………….
Topic 44: Pathobiochemistry and laboratory diagnosis of hypothyroidism and hyperthyroidism..
Topic 45: Hypocalcemia…………………………………………………………………………
Topic 46: Hypercalcemia………………………………………………………………………..
Topic 47: Clinical biochemistry of disturbances of the adrenal cortex………………………….
Topic 48: Clinical biochemistry of disturbances of the adrenal medulla……………………….
Topic 49: Clinical biochemistry of the reproductive system…………………………………….
Topic 50: Lab procedure and diagnosis of bone and skeletal disorders………………………….
Topic 51: Lab procedures in the diagnosis of muscle disorders…………………………………. Topic 52: Clinical biochemistry at the extremes of age………………………………………….
Topic 53: Clinical biochemistry and lab diagnosis of porphyrias……………………………….
Topic 54: Lab diagnostics of CNS diseases; lab investigation of the CSF……………………….
Topic 55:
Avi Sayag Clinical Biochemistry
Practical topics Topic 1: Molecular genetic methods for the investigation of inherited diseases………….14
Topic 2: Determination of Hb and Htc…………………………………………………….
Topic 3: Procedure of blood drawing, vacutainer tubes……………………………………
Topic 4:
Topic 5:
Topic 6: Principles of cell counting and differentials by hematology analyzers………….
Topic 7: Characterization of leukemic cells by morphology………………………………
Topic 8: Characterization of leukemic cells by cytochemistry and immunophenotyping….
Topic 9: Determination of ABO and Rh blood group……………………………………..
Topic 10: Determination of PT…………………………………………………………….
Topic 11: APTT assay…………………………………………………………………….
Topic 12: Thrombin time, D-dimer determination……………………………………….
Topic 13: Bleeding time, Detection of fibrin monomers…………………………………...
Topic 14: Lab methods for the determination of urea and creatinine……………………..
Topic 15: Examination of urine (general and sediment analysis)………………………….
Topic 16: Measurement of serum sodium and potassium…………………………………..
Topic 17: Determination of glucose in serum; point of care tests………………………….
Topic 18:
Topic 19: Cholesterol, HDL, LDL assays…………………………………………………..
Topic 20: Triglyceride assay, visual test, lipoprotein electrophoresis……………………..
Topic 21: Tests in the lab diagnosis of MI………………………………………………….
Topic 22: Assay of serum bilirubin. Detection of bilirubin and UBG in the urine…………
Topic 23:
Topic 24: Tests in the investigation of bone metabolism…………………………………..
Topic 25: Principles of chromatography and its application in diagnostics……………….15
Avi Sayag Clinical Biochemistry
Topic 1 Reference range, requesting a test, sources of error, interpretation of results,
specificity and sensitivity, predictive value In clinical lab investigation there are 3 phases:
1. The pre-analytical phase: requesting the test, preparing the patient, collecting the
sample, transporting it and storing it.
2. Analytical phase (manual or automatic)
3. Post-analytical phase: calculation of the results, validation of the results, consultation,
reporting, making the archive and interpreting the results.
Requesting a test:
The test request should include the following:
1. Patient's name, sex, date of birth and insurance number;
2. Ward/clinic/address;
3. The name of the requesting doctor and ways to contact him/her.
4. The diagnosis;
5. The name of the test requested;
6. The type of specimen;
7. Date and time of sampling;
8. Relevant treatment;
9. Indication of potentially hazardous samples;
Sources of error
In the pre-analytical phase, any one of the steps can lead to an error:
1. Requesting the test in an inappropriate manner (switching names of patients, e.g.).
Errors made during collection of the specimens: when blood specimen is collected,
some variables can influence the results, such as posture, venous stasis, hemolysis,
the labeling of the container. Also, the chemical composition of urine can change
during collection: destruction of glucose by bacteria; urea is converted to ammonia,
the pH decreases and phosphate precipitates; urobilinogen and porphobilinogen are
oxidized. Hemolysis interferes with the determination of CK, because RBCs release
adenylate cyclase (not CK!). AC converts 2 ADPs to ATP + AMP, and the ATP
carries the second reaction, leading to overestimation of CK levels.
2. During storage, blood specimens can change: glycolysis occurs in RBCs (glycolysis
in whole blood causes a 5% decrease in blood glucose level per hour), K+ and LDH
are released from RBCs (hemolysis during venipuncture can lead to
pseudohyperkalemia – see topic 29), CO2 is lost from the specimen, organic
phosphate esters are hydrolyzed, and labile enzymes lose their activity.
In the analytical phase, human errors or instrumental errors can occur. In this category, the
error can be systemic: accuracy vs. inaccuracy; precision vs. imprecision; or random errors.
The ideal analytical method is accurate, precise, sensitive and specific.
Precise: the result is the same if the procedure is repeated. It is assessed by repeated analysis
(n=15-20):
Within batch variation: the variation between the results of repeated tests in the same
"batch" (on the same day, for the same person, with the same specimen collected).
Day-to-day variation
Precision is expressed as coefficient of variance: CV(%) = SDx100/mean. Therefore, if the
SD is very small and approaches the mean, the CV(%) is close to 100% (= the test is very
precise).
Accurate: it gives the same result, that is, the deviation from the assigned value ("true" value).
Accuracy is given by: 100(mean-Xt)/ Xt
Avi Sayag Clinical Biochemistry
The mean value is the same in method A and B, but the scatter about the mean is less in method A than
in method B. Therefore, method A is said to be more precise.
Both method C and D are equally precise, but in method D the mean value differs from the true value.
Method C is more accurate.
The last source of variation is the biological one. The variation can be within-individual or
between-individual. The diet of a person, one's posture, the drug the person takes, etc. all
influence the results and variation can occur from test to test for the same person. Age, race
and sex can make the results different for different individuals.
After we have requested the test and considered all possible errors on the way, we have
finally received the results and have to interpret them.
Reference interval: the interval between two reference limits, including the limits. It is
designed as the central interval of values bounded by the lower and upper reference limit at
certain designated percentiles, usually at percentile 2.5 and 97.5 (that is, it is supposed to
include 95% of the reference population).
When choosing the reference population, we have to select for the most appropriate ones, and
exclude those who consume alcohol for example (unless we wish to establish reference range
for alcoholics), obese people, drug abusers, fasting people or non-fasting, etc. Also, we have
to select the group we wish to establish reference range for. Therefore, we partition the
population according to sex, race, age, blood group, stage of menstrual cycle, pregnancy,
exercise, fasting/non-fasting and so forth.
As the reference limit refers to the mean value of the sampled population, there are other
limits as well:
1. Medical decision limit (optimal cut-off values): in glucose levels, for example, DM is
defined as the level of glycemia at which diabetes-specific complications occur rather
than on deviations from a population-based mean.
2. Risk limit: for example, the risk limit for cholesterol is 5.8 mmol/L (a reference range
does not exist, as cholesterol level exceeds the desired value in the majority of the
population).
3. Panic value - results from a specimen that must be reported immediately to a
clinician, i.e., of such severity as to mandate urgent therapy. For example, glucose
levels < 2.6 mmol/L or > 26.9 mmol/L, Na+ < 120 mmol/L or > 158 mmol/L.
When considering a lab test, it is important to know its sensitivity and specificity.
Sensitivity is the incidence of true positive results or the ability of the method to measure low
concentrations of the analyte. It is given by the formula:
Sensitivity = 100×+ FNTP
TP
Specificity is the incidence of true negative results, and the test is not subject to interference
by other substances. It is given by the formula:
Specificity = 100×+ FPTN
TN
When judging the positive or negative results, the predictive value should be considered.
Avi Sayag Clinical Biochemistry
The predictive value of positive results = 100×+ FPTP
TP. This is the proportion of patients
with positive results who are correctly diagnosed. It reflects the probability that a positive
result reflects the underlying condition being tested for.
The predictive value of negative results = 100×+ FNTN
TN. This is the proportion of patients
with negative test results who are correctly diagnosed. For example, the predictive negative
value of a low result of CRP in CSF ruling out bacterial meningitis is 97%. That is, if a person
gets a low CRP result in CSF examination, he has a 97% chance that he really does not have a
bacterial meningitis.
Myoglobin lacks cardiac specificity as a marker. That is, if the myoglobin is elevated 4-8
hours following the onset of pain, but the ECG shows no sign of cardiac condition, then more
cardiac-specific markers should be sought for. However, if myoglobin is not elevated 4-8
hours following the onset of pain, myocardial necrosis can be excluded. That is, myoglobin is
sensitive. It is the only early marker measured (4-6 hours), despite its specificity.
Avi Sayag Clinical Biochemistry
Topic 2 Laboratory signs of cellular damage. Basis of clinical enzymology
Cellular damage can be caused by physical agents, genetic defects, hypoxia, chemical agents,
nutritional imbalance, immunological reactions, infections and aging.
As the cell injury progresses, such as in MI, the ion pumps are the first to fail, leading to ion
leakage to the ECF. Then, metabolites leak out (e.g. lactate, adenosine) and finally, as the
membrane is damaged, macromolecules leak out (enzymes and proteins). Ions and
metabolites drain to the intravascular space and less to the interstitial space, as opposed to
macromolecules that drain to the interstitial space and slowly drained through the lymphatic
system.
There are 2 main groups of enzymes in the plasma:
1. Enzymes released from cells as a result of leakage or cell death. These enzymes have
no known function in the blood. Serum enzymes in health are derived from the
metabolic breakdown and turnover of cells and tissues.
2. Enzymes with clearly defined actions in the blood (e.g. coagulation factors, ACE).
When measuring an enzyme, what influences the value obtained?
1. The rate of release from cells: 4 factors affect the rate
a. Hypoxia/anoxia/drugs � ↑membrane permeability � leakage
b. Cell necrosis � release of mitochondrial enzymes
c. Increased synthesis (e.g. in bile duct obstruction, synthesis of enzymes (ALP,
GGT) is induced)
d. Duct obstruction (e.g. liver and pancreas)
2. The volume of distribution of the enzyme in the ECF
3. The rate of removal from the plasma (catabolism in RES or excretion in bile and
urine). The clearance from the plasma also depends on the half life of the enzyme.
For example, CK has a 1/2 life of 1.4 days and GPT - 6.3 days.
4. The presence of factors in the plasma that may affect the method of assay (i.e.
inhibitors or activators of enzyme activity).
Enzymes can have isoenzymes and isoforms:
Isoenzymes: enzymes that differ in amino acid sequence but catalyze the same chemical
reaction. These enzymes usually display different kinetic parameters (e.g. different KM
values), or different regulatory properties. The existence of isoenzymes permits the fine-
tuning of metabolism to meet the particular needs of a given tissue or developmental stage
(e.g. LDH). In many cases, they are coded for by homologous genes that have diverged over
time. Alleloenzymes represent enzymes from different alleles of the same gene, and
isoenzymes represent enzymes from different genes that process or catalyse the same
reaction. An example of an isozyme is glucokinase, a variant of hexokinase which is not
inhibited by G6P. Its different regulatory features and lower affinity for glucose (compared to
other hexokinases), allows it to serve different functions in cells of specific organs, such as
control of insulin release by the beta cells of the pancreas, or initiation of glycogen synthesis
by liver cells. Both of these processes must only occur when glucose is abundant, or problems
occur. Isoenzymes can be organelle specific (enzymes of the mitochondria e.g.) or tissue
specific. Isoforms: different forms of the same enzyme, which are not the result of genetic
causes. Isoforms can be formed by post-translational modification on the protein component
or on the non-protein component as well as by aggregation of enzyme molecules (CK-MM,
CK-MB, CK-BB). We can use several techniques to differentiate isoenzymes and isoforms:
1. Zone electrophoresis
2. Isoelectric focusing
3. Differences in catalytic properties
4. Immunochemical methods
5. Selective inactivation
6. Ion exchange chromatography
These factors are important in making the choice of enzyme activity measurement:
Sensitivity, selectivity, time-course of elevation and technical errors.
Avi Sayag Clinical Biochemistry
The international unit of enzyme activity: the amount of enzyme which, under given assay
conditions, will catalyze the conversion of 1 µmol of substrate per minute.
Enzyme assays have not been standardized. Therefore, the reference range may change from
lab to lab. The given assay conditions include:
1. The nature and concentration of the substrate
2. The direction of the reaction
3. The temperature
4. The pH, concentration and nature of the buffer
5. The presence of inhibitors and activators
Katal = mol/sec
1 nkatal = 16.7 IU (international unit)
Avi Sayag Clinical Biochemistry
Topic 3 Inborn errors of amino acid metabolism and lab diagnosis of CF
Amino acids (AA) are supplied to the body by diet (essential AA) and by de novo synthesis.
As proteins are synthesized in the liver and in other tissues, the AA pool decreases.
Deamination (degradation) of AA in the liver and kidney, as well as transamination, also
decrease the AA pool of the body.
Under normal conditions, the AA level in the plasma changes during the day (diurnal
variation). In the urine, AA are present: glycine>alanine>serine>glutamine>histidine. During
pregnancy it changes to histidine>phenylalanine>lysine>tyrosine.
Cells have more AA than the plasma, and the plasma level of AA is greater than that of the
CSF.
In the liver, AA and α-ketoglutarate contribute to the synthesis of liver and plasma proteins,
purines, pyrimidines, porphyrines, hormones, etc. Also, they contribute to gluconeogenesis
and the formation of urea.
In the kidney, AA are filtered and completely reabsorbed by active transport systems (one for
neutral AA, one for basic AA (lysine, arginine and histidine), one for proline, hydroxyproline
and glycine, and one for dicarboxylic AA (aspartate and glutamate) ). Homocysteine and
cystathionine are not efficiently reabsorbed.
Aminoaciduria: high blood levels of AA that result in significant renal excretion. This can be
primary (genetic) or secondary (liver/kidney disorders). There are 3 types:
1. Overflow aminoaciduria due to plasma AA level that exceeds tubular capacity. In this
case the plasma AA level is high.
2. Renal aminoaciduria due to a defect in the renal transport. In this case the plasma
level of AA is normal.
3. No-threshold aminoaciduria due to excessive level of AA in the plasma, but with
renal excretion, plasma AA level remains normal.
Effects of enzyme defects: (a) Product D is
synthesized from A by a series of reactions
catalyzed by enzymes a, b and c. Enzyme c'
catalyzes the formation of a small amount of
product E in a minor pathway. (b) In the
absence of the enzyme c, no D is synthesized.
(c) If the conversion of C to D is blocked, the
concentration of the intermediate C, and the
possibly other precursors, may increase. (d)
Increased formation of E may occur if the
concentration of C increases and conversion
of C to D is blocked.
There are 6 disorders that should be mentioned (there are more than 6 inherited AA
disorders, but these are the ones addressed in the course):
1. Hyperphenylalaninemia (and phenylketonuria)
2. Tyrosinemia
3. Alkaptonuria
4. Cystinuria
5. Homocystinuria
6. Urea cycle disorders
Avi Sayag Clinical Biochemistry
1. Hyperphenylalaninemias (HPA)
HPA results from impaired conversion of Phe to tyrosine.
The most common and clinically important is phenylketonuria (PKU). PKU is an autosomal
recessive disorder. There are several HPAs that are not PKU and are called non-PKU HPAs.
HPA is defined as a plasma phenylalanine concentration >120µM. PKU is characterized by
plasma phenylalanine >1000µM and non-PKU hyperphenylalaninemias have plasma
phenylalanine amounts that are <1000µM. PKU is caused by mutation in the phenylalanine
hydroxylase gene (PAH). The HPAs are disorders of phenylalanine hydroxylation. Because
the reaction catalyzed by PAH involves tetrahydrobiopterin (BH4) as a co-factor, the HPAs
can result from defects in any of the several genes required for synthesis and recycling of
BH4. Removal of excess phenylalanine normally proceeds via the tyrosine biosynthesis
reaction and then via tyrosine catabolism. The first reaction in this process is the PAH
catalyzed hydroxylation of phenylalanine. There are 5 types of HPA, of which 3 are PKU
(types I and II are worth mentioning: type I: a defect in Phe hydroxylase, and type II: a defect
in dihydropteridine reductase). The accumulation of Phe inhibits the transport of other AA
required for protein or neurotransmitter synthesis, reduces synthesis and increases degradation
of myelin, and leads to inadequate formation of norepinephrine and serotonin. Phe is a
competitive inhibitor of tyrosinase, a key enzyme in the pathway of melanin synthesis, and
accounts for the hyperpigmentation of hair and skin. Untreated children with classic PKU are
normal at birth, but fail to attain early development milestones, develop microcephaly, and
demonstrate progressive impairment of cerebral function. Hyperactivity, seizures and mental
retardation are major clinical problems later in life. To prevent mental retardation, diagnosis
and initiation of dietary treatment of classic PKU must occur before the child is 3 weeks of
age.
Lab tests:
Screening: Guthrie test (on the 6th-10
th day of life)
Specific diagnosis: determination of plasma Phe and Tyr by HPLC (see following
practical topic 25)
Monitoring: determination of plasma Phe by HPLC
Prenatal diagnosis: DNA analysis (see following practical topic 1)
Treatment consists of diet low in Phe and supplemented with Tyr. (Gene therapy is being
developed).
Guthrie test: A drop of blood is usually obtained by pricking the heel of a newborn infant on
the 6th or 7
th day of life. A small disk of the filter paper is punched out and placed on an agar
gel plate containing Bacillus subtilis and B-2-thienylalanine. Each gel holds 60-80 disks. The
agar gel is able to support bacterial growth but the B-2-thienylalanine inhibits bacterial
growth. However, in the presence of extra phenylalanine leached from the impregnated filter
paper disk, the inhibition is overcome and the bacteria grow. Within a day the bacterial
growth surrounding the paper disk is visible to the eye. The amount of growth, measured as
the diameter of the colony, is roughly proportional to the amount of phenylalanine in the
serum.
Avi Sayag Clinical Biochemistry
2. Tyrosinemia There are three types of tyrosinemia, each with distinctive symptoms and caused by the
deficiency of a different enzyme.
Type I tyrosinemia is the most severe form of this disorder and is caused by a shortage of the
enzyme fumarylacetoacetate hydrolase. Fumarylacetoacetate hydrolase is the last in a series
of five enzymes needed to break down tyrosine. Symptoms of type I tyrosinemia usually
appear in the first few months of life and include failure to gain weight and grow at the
expected rate (failure to thrive), diarrhea, vomiting, jaundice, cabbagelike odor, and increased
tendency to bleed (particularly nosebleeds). Type I tyrosinemia can lead to liver and kidney
failure, problems affecting the nervous system, and an increased risk of liver cancer.
Worldwide, type I tyrosinemia affects about 1 person in 100,000.
Type II tyrosinemia is caused by a deficiency of the enzyme tyrosine aminotransferase
Tyrosine aminotransferase is the first in a series of five enzymes that converts tyrosine to
smaller molecules, which are excreted by the kidneys or used in reactions that produce
energy. This form of the disorder can affect the eyes, skin, and mental development.
Symptoms often begin in early childhood and include excessive tearing, photophobia, eye
pain and redness, and painful skin lesions on the palms and soles. About half of individuals
with type II tyrosinemia are also mentally retarded. Type II tyrosinemia occurs in fewer than
1 in 250,000 individuals.
Type III tyrosinemia is a rare disorder caused by a deficiency of the enzyme 4-
hydroxyphenylpyruvate dioxygenase. This enzyme is abundant in the liver, and smaller
amounts are found in the kidneys. It is one of a series of enzymes needed to break down
tyrosine. Specifically, 4-hydroxyphenylpyruvate dioxygenase converts a tyrosine byproduct
called 4-hydroxyphenylpyruvate to homogentisic acid. Characteristic features of type III
tyrosinemia include mild mental retardation, seizures, and periodic loss of balance and
coordination (intermittent ataxia). Type III tyrosinemia is very rare; only a few cases have
been reported.
Alkaptonuria is an autosomal recessive condition that is due to a defect in the enzyme
homogenistic acid oxidase, which participates in the degradation of tyrosine. As a result, a
toxic tyrosine byproduct called homogentisic acid (or alkapton) accumulates in the blood and
is excreted in urine in large amounts. Excessive homogentisic acid causes damage to cartilage
(HGA binds to collagen). The presentation also includes pigmentation in the ears and
degenerative arthritis in middle age.
3. Cystinuria Cystinuria is an AR disorder caused by defective transporters in the apical brush border of
proximal renal tubule and small intestinal cells. It is characterized by impaired reabsorption
and excessive urinary excretion of the dibasic AA lysine, ornithine and cystine. Because
cystine is poorly soluble, its excess excretion predisposes to the formation of kidney stones,
which are responsible for the signs and symptoms of the disorder.
Lab tests:
Screening: cystine stones in the urine
Specific diagnosis: AA detection in the urine by HPLC
Treatment includes intake of large amount of water, alkalinizing the urine and administration
of penicillamine (captopril) which chelates cystine.
Avi Sayag Clinical Biochemistry
4. Homocystinurias Homocystinurias are 7 biochemically and clinically distinct disorders characterized by
increased concentration of homocystine in blood and urine. The most common one is the
classic homocystinuria, which results from reduced activity of cystathionine β-synthase (the
enzyme that condenses homocysteine with serine to form cystathionine).
Symptoms include vascular complications during the first decade of life and are the major
cause of morbidity and mortality (due to homocysteine). Other symptoms include defects in
collagen metabolism due to cystine, and some patients develop marfanoid habitus and
radiologic evidence of osteoporosis.
Other rare causes include a deficiency in MTHFR and MTHF transferase.
Lab tests:
Screening: Guthrie test
Specific diagnosis: determination of homocystine and methionine by HPLC. False
negative results can occur if the diagnosis is made before 3 days of life, and false
positive result can occur in case of premature enzymes, excessive protein intake,
tyrosinemia and hepatitis.
5. Urea cycle defects Excessive ammonia generated from protein nitrogen is removed by the urea cycle, a process
mediated by several enzymes and transporters. Complete absence of any of these enzymes
usually causes severe hyperammonemia in newborns, while milder variants can be seen in
adults. The accumulation of ammonia and glutamine leads to brain edema and direct neuronal
toxicity. These disorders are AR, except for ornithine transcarbamylase deficiency, which is
X-linked.
Any of the 5 enzymes can be
deficient:
1. CPS: Carbamyl-Phosphate
synthetase
2. OTC: Ornithine
transcarbamylase
3. ASS: Argininosuccinate
synthetase
4. ASL: Argininosuccinase
5. ARG: Arginase
Symptoms include lethargy, vomiting, liver failure and coma.
Lab tests:
Screening: detection of plasma ammonia
Specific diagnosis: detection of plasma and urine AA/enzyme detection.
Avi Sayag Clinical Biochemistry
Lab diagnosis of CF 1. Sweat test: useful in the severe form of CF. Sweat is collected by pilocarpine
iontophoresis, and the concentration of chloride is determined:
a. Borderline values: 40-59 mM
b. Elevated values: >60 mM
2. The presence of IgG against Pseudomonas aeruginosa
3. Immunoreactive trypsinogen (used in newborn screening programs)
4. Tests of exocrine pancreatic functions:
a. Indirect: presence of enzymes in the stool and the fat levels; the level of
carotene in the serum;
b. Direct: analysis of the pancreatic secretion before and after stimulation with
secretin and CCK (cholecystokinin)
5. DNA based CFTR mutation analysis: the tested mutations cover almost 60-95% of
diagnosed CF cases. There are 4 classes of mutations:
a. Class I: caused mainly by premature termination due to splicing defects,
frameshifts due to small deletions or insertions; non-sense mutations. The
CFTR can be reduced or completely absent.
b. Class II: defective protein synthesis; misfolded protein; no transport to the
membrane.
c. Class III: defective regulation; mutant proteins reach the membrane, but the
function of the channel is altered.
d. Class IV: defective conduction. The channel is correctly processed and
delivered to the membrane, but it generates reduced Cl- current. The rate of
ion flow is reduced or the amount of time the channel is open is reduced.
6. ARMS, PCR-OLA, oligonucleotide hybridization assay
a. PCR-ARMS – Amplification Refractory Mutation System (also called: allele
specific oligonucleotide hybridization): a method of PCR which distinguishes
between alleles that differ in even a single nucleotide substitution. In this
procedure the allele specific oligonucleotides (ASO) are immobilized on a
nitrocellulose membrane and hybridized with a labeled PCR product
spanning the variant nucleotide site. Discrimination between the 2 alleles is
based on the fact that in a specific hybridization temperature the perfectly
matched hybrid is more stable than the mismatched. After hybridization and
washing, the detection is mainly colorimetric: if the PCR product is labeled
with biotin, e.g. the detection is based on the strepatavidin-alkaline
phosphatase conjugate enzyme reaction with a substrate. By using 2 ASOs it
is possible to determine the wild type, heterozygous or homozygous.
b. PCR-OLA: PCR followed by oligonucleotide ligation assay (OLA) is a
molecular method that can be used for the detection of nucleotide sequence
variants. PCR-OLA is based on the enzymatic ligation of two
oligonucleotides that anneal next to each other onto the PCR-amplified target
DNA. Even a single-nucleotide mismatch between the oligonucleotides and
the template precludes the ligation.
c. 2 CF mutations + an abnormal sweat test are diagnostic of CF!
7. Nasal potential difference: this test measures the potential difference between the
apical and the basal surface of the nasal mucosa in the presence or absence of an
epithelial sodium channel antagonist (amiloride, e.g.). A decrease in the potential
difference is much higher in CF patients than in healthy ones.
8. Bronchoalveolar lavage (lavage=washing a hollow organ): in this procedure there is a
high percentage of neutrophils in the lavage fluid among CF patients (normally,
neutrophils comprise 3% of the lavage fluid, whereas in CF patients it reaches 50%).
P. aeruginosa may also be present.
9. Prenatal diagnosis is performed when family anamnesis suggests CF:
a. Immunoreactive trypsin in amniotic fluid
b. DNA tests (chromosome 7q31.2)
Avi Sayag Clinical Biochemistry
Molecular genetic methods for the investigation of inherited diseases
(Practical topic 1) There 4 steps in genetic diagnosis of inherited diseases:
1. Isolation of genomic DNA: by traditional method or spin column purification
2. Determination of the concentration and purity of the genomic DNA
3. Storage of DNA
4. Mutation detection: by Southern blot or PCR. Following these, electrophoresis,
restriction digestion, hybridization probes, hybridization with allele specific
oligonucleotides and multiplex ligation-dependent probe amplification (MPLA) can
be used.
1. Isolation of genomic DNA
The sample is taken from the buffy coat from peripheral blood anticoagulated by citrate or
EDTA. In the traditional method:
a. Cell harvesting by SDS1
b. Proteolysis with a special enzyme
c. Multiple extractions by phenol, chloroform, isoamyl alcohol to dissolve
hydrophobic contaminants and remove proteins.
d. DNA is precipitated by 96-100% ethanol
e. DNA is washed by 70% ethanol
f. DNA is dissolved in TRIS buffer
Spin column method: this method is based on the phenomenon that DNA/RNA binds
selectively to silica in high salt concentration. Cell lysates are loaded onto a spin column
containing silica membrane, and then several washing steps follow. This method is fast but
expensive.
2. Determination of DNA purity
DNA yield is determined by measuring the concentration of DNA by absorbance at 260nm.
As the peak absorbance of DNA is at 260nm, that of proteins is at 280nm. Thus, purity is
determined by calculating the ratio of absorbance at 260nm to that at 280nm. Pure DNA has a
ratio of 1.7-1.9. Ratio less than 1.7 indicates protein contamination.
3. Storage
DNA can be stored at 4ºC for months, at -20ºC for years, and at -70ºC for at least 10 years.
4. Mutation detection
Southern blot:
a. Restriction digestion: using enzymes that recognize specific base
compositions that cleave the DNA at these sites. If the recognition sequence
changes (for example due to a point mutation), the enzyme cannot cleave the
DNA.
b. Electrophoresis: separation according to size through an agarose gel or
acrylamide gel. The detection is based on the intercalation of the fluorescent
dye (ethidium bromide).
c. Denaturation
d. Transference to nylon membrane by capillary, vacuum or electric transfer.
e. Immobilization by UV light
f. Hybridization with a labeled probe
g. Washing off the unbound probes
h. Detection (depending on the labeling)
1 SDS – Sodiun Dodecyl Sulfate. This compound disrupts non-covalent bonds in proteins, denatures
them, and causes the molecule to lose its native shape. Also, anions of SDS bind to the main peptide
chain at a ratio of 1 SDS for every 2 AA residues. This imparts a negative charge on the protein that is
proportional to the mass of that protein. This new negative charge is significantly greater than the
original charge of that protein. The electrostatic repulsion created by binding of SDS causes proteins to
unfold into a rod-like shape, thereby eliminating differences in shape as factor for separation in the gel.
Avi Sayag Clinical Biochemistry
PCR: amplification of a well-defined DNA fragment. The reaction consists of cycles:
a. Denaturation at 95ºC to separate the strands of the DNA template
b. Fast cooling to 50-65ºC to hybridize the primers and the polymerases (Taq)
c. Incubation at 72ºC to synthesize the new strands
4 main types of PCR:
1. Standard PCR: to detect small variations in genetic material (e.g. Factor V Leiden
mutation).
2. Multiplex PCR: involves parallel amplification of different pieces of DNA (e.g. Duchene
muscular dystrophy).
3. Long PCR: combines 2 enzymes: Taq polymerase and a proof-reader, which make it
possible to amplify larger DNA fragments (e.g. Friedrich ataxia).
4. Quantitative Real-time PCR: used to quantify the copy number of a specific DNA in the
sample (e.g. translocation 9:22).
Principles of chromatography and its application in diagnostics
(Practical topic 25) Chromatography is used when the substance to be measured needs to be separated from other
disturbing components. A small sample volume is injected in a continuous flow of gas or
liquid (the mobile phase). The mixture of the sample and the mobile phase flows through the
stationary phase, which usually consists of solid particles. On the stationary phase, the
components of the mixture can be separated using different principles, because the different
components spend different period of time in the stationary phase due to their different
chemical properties. There are 3 main methods:
1. Adsorption chromatography: the particles of the stationary phase bind the
components of the sample mixture. Different components remain bound for various
amounts of time according to their chemical properties (those with low affinity to the
stationary phase spend shorter time).
2. Ion exchange chromatography: the stationary phase consists of ion exchange
particles, which bear surface-bound ionic groups with their counter-ions. Components
of the sample replace the counter-ions from the surface of the particles. Different
components bind to the fixed ions with different affinity.
3. Size exclusion chromatography: molecules are separated based on their physical size.
Column chromatography may utilize 2 methods: gas chromatography and high performance
liquid chromatography (HPLC).
Gas chromatography: this method utilizes a high purity gas as eluent with constant pressure
or flow rate. This technique is suitable for the detection of analytes that can be brought to gas
phase and stay stable at the high analysis temperature of 100-350ºC. Gas chromatography can
be used both for quantitative and qualitative analysis.
HPLC: in the standard HPLC, the mobile phase is a stable liquid, and the stationary phase can
be either a liquid or a solid substrate. The eluent circulates in a closed system and the constant
flow is provided by a pump. The effluent coming from the column is transferred into a small
cuvette of a detector. The detectors can be photometric, fluorimetric, electrochemical and
refraction detectors. HPLC is applicable to measure materials which are soluble and stable in
the mobile phase.
The upgraded methods of HPLC are GC/MS and LC/MS (GS: gas chromatography; LC:
liquid chromatography; MS: mass spectrometry). The different analytes reach the detector
(the MS), separated, and analyzed.
Clinical uses: PKU, cystinuria, homocystinuria, detection of HbA1c (on cation exchange
column), hemoglobinopathies, vitamin D disorders, and detection of catecholamines and their
metabolites (for example, catecholamines are elevated in neuroblastoma and
melanoblastoma).
Avi Sayag Clinical Biochemistry
Topic 5 Pathobiochemistry of inflammation
Inflammation can be acute or chronic: the acute one lasts from few minutes to few days, while
the chronic one lasts from days to weeks. Acute inflammation is characterized by the presence
of exudate and neutrophils, and the chronic one is characterized by the presence of
lymphocytes, macrophages and tissue destruction. The chemical mediators released during the
inflammatory process are responsible for the vascular response and for the cellular response.
The vascular changes: in acute inflammation they manifest in a local increase in blood flow
that results in redness and heat, and in increased vascular permeability that results in
transudation. As the intravascular osmotic pressure decreases, the interstitial osmotic pressure
increases and exudation replaces transudation. The accumulation of fluids in the extravascular
tissue contributes to the edema. As the blood becomes more viscous, stasis ensues.
The cellular changes: manifest in emigration of leukocytes from the microcirculation that
accumulate in the inflamed site and are activated there. Leukocytes undergo 5 stages:
1. Margination and rolling: this is facilitated by the selectins expressed on the
leukocytes as they are activated. L-selectins bind to E-selectins and P-selectins.
2. Adhesion and transmigration: adhesion is carried out by expression of integrins that
bind to ICAM-1 expressed on the endothelium.
3. Chemotaxis and activation
4. Phagocytosis and degranulation
5. Leukocyte-induced tissue injury: injury is caused by lysosomal enzymes, by ROS,
and arachidonic acid metabolites. Pain and loss of function result.
Chemical mediators:
1. Chemical mediators of cellular origin:
a. Vasoactive amines stored in secretion granules:
i. Histamine: present in mast cells and basophils. It is released in
response to physical injury, immune reactions, C3a and C5a, proteins
released from leukocytes and IL-1 and IL-8 (cytokines). It causes
arteriolar dilation, contraction of endothelium in venules and it
increases vascular permeability.
ii. Serotonin: present in platelets and is released by platelet-activating
agents. Its effects are similar to those of histamine.
b. Enzymes stored in secretion granules:
i. Acid proteases: present in phagolysosomes only.
ii. Neutral proteases: these include elastase, collagenase and cathepsins.
They cause the destruction of the basement membrane and ECM. The
control of these proteases is carried out by anti-proteases (e.g. α1-
antitrypsin).
c. Newly synthesized mediators:
i. Prostaglandins: synthesized in leukocytes, platelets and endothelium.
PgI2 causes vasodilation and inhibits platelet activation; PgD2, PgE2
and PgF2α cause mainly vasodilation and potentiate edema; TXA2
causes vasoconstriction and activates platelets.
ii. Leukotrients: synthesized in leukocytes. LTB4 is chemotactic; LTC4,
LTD4 and LTE4 cause vasoconstriction, bronchospasm and increased
permeability.
iii. Platelet-activating factor: synthesized in leukocytes and endothelium.
iv. Oxygen radicals: synthesized in neutrophils and macrophages by
NADPH oxidase. These include superoxide, hydrogen peroxide,
hydroxyl free radical and toxic NO derivatives. They cause
endothelial injury and thrombosis, activation of proteases and
inactivation of anti-proteases, and injury of other cells. Normally,
they are counteracted by catalase, superoxide dismutase and
glutathione.
Avi Sayag Clinical Biochemistry
v. NO: synthesized in endothelium and macrophages. It is synthesized
from arginine, in the presence of O2, NADPH and NOS. It causes
vasodilation, inhibits platelet activation and killing of bacteria in
activated macrophages.
vi. Cytokines: synthesized in activated lymphocytes, macrophages and
endothelium. Cytokines are polypeptides that have local effects on
endothelium, leukocytes and fibroblasts, as well as systemic effects.
1. IL-1, IL-6 and TNFα are synthesized in activated
macrophages. They are produced in the acute phase reaction
and are responsible for the fever, lethargy, neutrophilia,
synthesis of acute-phase proteins, corticosteroids and
cachexia. They also cause endothelial activation that results
in leukocyte adhesion, synthesis of PgI, NO and
thrombogenesis.
2. IFN-γ is synthesized in T-cells and activates neutrophils and
macrophages, and induces NO synthesis.
3. Chemokines: these are synthesized in activated macrophages,
endothelium and fibroblasts. They cause chemotaxis and
neutrophil activation (e.g. IL-8).
2. Mediators derived from the plasma:
a. Activation of the kinin system: FXII is activated and cleaves prekallikrein.
The formed kallikrein cleaves off bradykinin from kininogen. Bradykinin
increases vascular permeability, causes vasodilation, bronchospasm and
mediates pain. As FXII is activated, tissue plasminogen activator (t-PA) is
also released.
b. Activation of coagulation: induction of tissue factor in macrophages and
endothelium.
c. Activation of complement system
Avi Sayag Clinical Biochemistry
Topic 6 Pathobiochemistry of plasma proteins
When measuring the proteins in the plasma it is possible to measure the total protein
concentration, the albumin and globulin fractions (semi-quantitative assessment) or specific
proteins.
What is the clinical significance in measuring the total serum protein?
Total serum protein may decrease in the following settings:
1. Decreased protein synthesis (malnutrition, malabsorption, starvation, hepatic failure)
2. Hemodilution
3. Humoral immunodeficiency
4. Increased loss of proteins
5. Catabolic states
Total serum protein may increase in the following settings:
1. Hemoconcentration (dehydration, stasis, posture)
2. Increased synthesis of plasma proteins (hypergammaglubolinemia, paraproteinemia)
What are the most important serum proteins?
Prealbumin
Albumin
α1-antitrypsin α1 globulins
α1-acid glycoprotein
α2-macroglobins
Haptoglobins
α2 globulins
Ceruloplasmin
Transferrin β globulins
Complement components
γ globulins IgG, IgA, IgM, IgD, IgE
1. Prealbumin Prealbumin is a protein status indicator; it has a much shorter half-life and smaller serum pool
than albumin. The half-life of prealbumin is approximately 2 days, making prealbumin a more
timely and sensitive indicator of protein status. Prealbumin is a tryptophan-rich protein, and
like albumin, it is synthesized in the hepatocytes of the liver. Prealbumin's main function is to
serve as a binding and transport protein. The term prealbumin is actually a misnomer-the
prefix pre implies that it is a precursor for albumin, which it is not. The more accurate name
for prealbumin is transthyretin to indicate that it is a serum transport protein for thyroxin and
retinol-binding protein.
Evaluating prealbumin
Like albumin, prealbumin is a negative acute-phase reactant. This limits its use as a screening
tool for malnutrition because low levels could result from either inadequate nutrition or
inflammatory stress. Rather than a diagnostic tool, prealbumin should be used as an indicator
of nutritional improvement and as a measure of how well nutritional interventions are
working. The very short half-life and small serum pool allows small changes in nutritional
status to be identified in a short time frame. Elevated prealbumin levels may be seen in
patients taking corticosteroids and in patients with Hodgkin disease.
2. Albumin Low levels of albumin can result from:
a. Overhydration
b. Decreased synthesis (malabsorption, malnutrition, starvation)
c. Increased loss (nephrotic syndrome, Crohn's disease)
The consequences of low levels of albumin may be:
a. Edema
b. Secondary aldosteronism
c. Hypocalcemia (but ionized Ca+2
is not decreased)
d. Increased risk of kernicterus in infants
e. Altered pharmacokinetics of albumin-bound drugs
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High levels of albumin may result from:
a. Dehydration
b. Venous stasis during blood collection
c. Overinfusion of albumin
Remember: increased albumin synthesis does not cause hyperalbuminemia!
So when do we decide to measure albumin levels in the plasma?
1. When we have to elucidate the cause of edema;
2. To follow up nutritional status (also measuring prealbumin);
3. To evaluate liver function (except for acute hepatitis where albumin is not
informative);
4. To elucidate the cause of low calcium levels in the plasma;
5. When we wish to administer a drug that binds to albumin;
6. When we wish to diagnose analbuminemia.
3. α1-antitrypsin
AAT is an α1-globulin, a protease inhibitor and an acute phase protein. When it is deficient,
homozygotes are prone to develop emphysema and liver disease. The deficient level is
detected by isoelectric focusing, and the genotype (homo- or heterozygote) is determined by
PCR techniques.
4. Haptoglobin
This α2-globulin binds free Hb and is an acute phase protein. Its levels decrease in:
a. Intravascular hemolysis (absent) and extravascular hemolysis (low)
b. Chronic liver disease
c. Metastases of carcinoma
d. Severe sepsis
Its levels increase in:
a. Acute phase reactions
b. Nephrotic syndrome
5. α2-macroglobulin This is a protease inhibitor, whose synthesis is increased in hypoalbuminemia (the decreased
albumin is replaced by the high-molecular-weight macroglobulin)
6. Ceruloplasmin
Ceruloplasmin transports copper. Low levels are associated with Wilson's disease, and high
levels are evident in acute phase reactions, pregnancy and use of oral contraceptives.
Acute phase reactions
This is a systemic, non-specific reaction to acute inflammation, infection, burns, tissue
necrosis and tumor proliferation. It includes fever, leukocytosis, complex hormonal and
metabolic changes (altered levels of acute phase proteins).
Acute phase proteins can be positive or negative.
1. Positive acute phase reactants:
Early, sensitive reactants:
a. α1-antitrypsin
b. α1-acid glycoprotein
c. Haptoglobin
d. Fibrinogen
e. C-reactive protein (CRP)
f. Procalcitonin
Late, weak reactants:
a. Ceruloplasmin
b. C3, C4
2. Negative acute phase reactants:
a. Prealbumin
b. Albumin
c. Transferrin
Avi Sayag Clinical Biochemistry
Cytokines, such as IL-1, IL-6 and TNF, increase the mRNA of positive acute phase reactants,
and decrease the mRNA of the negative ones. Synergethic hormones, such as glucocorticoids,
enhance the reaction.
What is the role of the acute phase proteins?
1. They inhibit the destructive proteases released from leukocytes during
inflammation.
2. Scavanger proteins (CRP, haptoglobin) help to present cellular debris and
breakdown products to the RES in order to process them and retain precious
components.
3. Fibrinogen is required for wound healing.
What are the classic parameters used to detect acute phase reactions?
Fever, leukocytosis, ESR and serum protein electrophoresis. Of the acute phase reactants,
CRP is the most sensitive and specific.
C-reactive protein (CRP) Synthesized in the liver, CRP reacts with the C-polysaccharide of Streptococcus
pneumonia.
In the presence of calcium it bnds to phospholipids, polyanions and galactans.
Through these molecules it binds to many bacteria, fungi, protozoa and cellular
debris.
In protein electrophoresis, it migrates somewhere between the γ band to mid-β region.
Its function is to initiate opsonization, phagocytosis and cell lysis. Once it is
complexed, it activates the classical complement pathway.
CRP is elevated in 4 main scenarios:
1. Inflammation: CRP shows an early rise (4-6 hours), which is more intense than ESR.
When the patient recovers, its levels decrease before those of ESR. Mostly, its levels
decrease when the inflammatory process is suppressed by steroids or salicylates.
Thus, it is an excellent tool to monitor the activity level of rheumatoid arthritis,
rheumatic fever, vasculitides, etc. Of particular interest, CRP is significantly higher in
Crohn's disease than in ulcerative colitis, and its levels correspond to relapse,
remission and response to therapy.
2. Tissue injury and necrosis: in AMI, CRP starts rising within 24-48 hours and reaches
its peak on the 3rd
day. It returns to normal level within 1-2 weeks. If there is tissue
damage, it manifests in permanent increase of CRP. Its levels are elevated in
infarctions of other tissues other than the heart. Other examples of CRP rise are
rejection of kidney and bone marrow transplants, burn injuries and following surgery.
If its levels fail to decrease after surgery, it is highly indicative of complications.
3. Infections: its levels are the highest in bacterial infections, but it is useful in the
diagnosis of both bacterial and viral meningitis. It is useful in monitoring the activity
of the disease and the efficacy of therapy. Last but not least, it is useful in diagnosing
post-operative and intercurrent infections, mainly in active severe SLE and leukemia.
4. Malignancy: its levels are increased particularly in breast cancer, lung cancer and GI
cancer. It can be thus considered a "general" tumor marker (see Topic 8).
Procalcitonin Procalcitonin is a stable precursor of calcitonin coded by CALC-I gene. It is composed of 116
amino acids, and has a half-life of 25-30 hours. It consists of an N-terminal region, calcitonin
and katacalcin domains. Procalcitonin is not synthesized in the thyroid, but in the lung, liver
and intestines.
What is the diagnostic value of procalcitonin?
Procalcitonin levels are increased in sepsis and severe bacterial infections. It is practically
absent (<0.1µg/L) in the serum of healthy individuals. Its levels correlate well with the
activity and extent of systemic bacterial infection. In viral infections, chronic and acute non-
bacterial inflammations, however, its level remains unchanged or only slightly increases. Its
increase precedes the clinical manifestations of severe generalized sepsis and has a prognostic
value to it. Thus, it is useful in monitoring the treatment of such diseases.
Avi Sayag Clinical Biochemistry
Topic 7 Biochemical effects of tumors
The biochemical effects of tumors (malignancies) are indirect: they may secrete biologically
active substances even below the threshold of 109 cells (a tumor formed after 30 divisions,
weighs 1 gram and spans 1 cm in diameter. This is when the tumor is clinically detectable).
A paraneoplastic syndrome (PNS) is a disease or symptom that is the consequence of the
presence of cancer in the body, but is not due to the local presence of cancer cells. These
phenomena are mediated by humoral factors (by hormones or cytokines) excreted by tumor
cells or by an immune response against the tumor. Recognition of a PNS alerts a new
diagnosis of cancer. The frequency of PNS is 10-15%. PNS can be endocrine, neurological,
hematological, dermatological, etc.
1. Endocrine syndromes:
Cushing syndrome: caused by ectopic secretion of ACTH by lung cancers.
SIADH: caused by ectopic secretion of ADH by lung cancers.
Gynecomastia: caused by ectopic secretion of hCG by lung and liver cancers.
Hypercalcemia: caused by ectopic secretion of PTH-rP by lung cancers.
Hypocalcemia: caused by ectopic secretion of calcitonin by breast cancers.
Hypoglycemia: caused by ectopic secretion of IGF-II by sarcomas.
Carcinoid syndrome: caused by ectopic secretion of serotonin by carcinoid tumors.
Carcinoid tumors are discrete, yellow, well-circumscribed tumors that can occur anywhere
along the gastrointestinal tract and in the lung. They most commonly affect the appendix,
ileum, and rectum (90%). Carcinoids are tumors of neuroendocrine nature, that originate in
the cells of the neuroendocrine system (APUD) and are characterized by production of
serotonin (5-hydroxytryptamine; 5-HT).
Multiplex Endocrine Neoplasia (MEN): these are familial cancers (AD) characterized by the
simultaneous appearance of tumors (benign or malignant) of 2 or more endocrine organs.
MEN-I: parathyroid, pancreatic islets, anterior pituitary.
MEN-IIa: thyroid (medullary cell carcinoma), adrenal medulla (pheochromocytoma),
parathyroid.
MENIIb: same as MEN-IIa but with various somatic abnormalities (marfanoid habitus, e.g.).
2. Hematological syndromes:
Anemia: the anemia can be of two types – hypoproliferative or autoimmune hemolytic
anemia. The hypoproliferative anemia is a mild anemia (Hb: 80-120 g/L) due to impaired
erythropoiesis (e.g. insufficient EPO production or impaired response of the BM to EPO). The
autoimmune hemolytic anemia is due to Ab against RBCs. The Abs can be warm reacting (as
in HL, NHL, CLL, MM, breast and lung cancer) or cold reacting (as in chronic granulocytic
leukemia, carcinoid tumors and adrenocorticocarcinoma).
Erythrocytosis: caused by ectopic production of EPO or by hypoxemia (that can be the result
of a direct effect of the tumor, as it narrows the O2 supply highway to the kidney).
Erythrocytosis is mainly seen in hypernephromas (30%), cerebellar hemangioblastoma (20%),
hepatoma (1%) and Wilm's tumor (1%).
Leukocytosis: caused by ectopic production of G-CSF. It is associated with gastric
adenocarcinoma, lung cancer, pancreatic cancer, HL, and NHL. Symptoms appear late in the
course of the malignant process. The WBC count exceeds 20x109/L without a left shift.
Thrombocytosis: caused by thrombopoetin overproduction in 1/3 of cancer patients (HL and
NHL). PLT count is mildly high (1000 G/L).
Thrombocytopenia: caused by ITP-like or hypoplasia, and is associated with HL, NHL,
CLL, lung cancer, breast cancer and testicular cancer. PLT count is less than 30 G/L.
Hemostasis disorders: thromboembolism is the second cause of death among cancer patients
with promyelocytic leukemia, myeloproliferative disease, primary brain tumor, lung cancer
and ovarian cancer. Patients with acute leukemias suffer from hemorrhages due to
consumption coagulopathies. DIC is seen among patients with prostate cancer,
adenocarcinoma, and AML-M3.
Avi Sayag Clinical Biochemistry
3. Neurological syndromes
The paraneoplastic neurological syndromes result from autoimmune processes. Certain
tumors produce large amounts of proteins expressed in the PNS or CNS. These molecules
stimulate the humoral or the cellular immune system.
Lambert-Eaton syndrome: Abs against Ca+2
channels in the presynaptic neuron. Thus,
Ach is not released. It is most frequently associated with lung cancer.
Cerebellar degeneration: Anti-Yo Abs.
Encephalomyelitis: Anti-Hu Abs.
Degeneration of peripheral nerves: Anti-Hu Abs.
Retinopathy: CAR Abs.
4. Dermatological syndromes
These PNS result from autoimmune processes or from mediators produced locally or
systemically such as in carcinoid tumors.
Avi Sayag Clinical Biochemistry
Topic 8 Tumor markers (TM) in the diagnosis of malignant disease
What is TM?
Tumor markers are molecules produced by the tumor or by the host (reacting to the presence
of the tumor). These markers can be enzymes (and isoenzymes), hormones, oncofetal
antigens, carbohydrate epitopes, cytokeratines, receptors and products of oncogenes. TM can
be measured qualitatively and quantitatively by chemical methods, immunochemical methods
or molecular biological methods in the serum or in other body fluids (urine, saliva, CSF,
nipple discharge).
What is the ideal TM?
The ideal TM:
Is produced specifically by the malignant tissue or by the premalignant tissue;
Is produced in all patients with a specific tumor type;
Is produced in an organ-specific manner;
Is measurable in an easily accessible body fluid;
Its concentration in body fluid correlates with tumor volume or with the biological
behavior of the tumor;
Has a short half-life for the post-therapy follow-up;
Informs of the progression and regression by its rise-and-falls;
Is cheap, simple, standardized with an available reproducible assays.
What are the "buts"..?
But the ideal TM test does not exist yet (if you read this after 2009, double-check if
it's still so….)
But TM lack specificity: elevated concentrations can be seen in benign diseases too.
But TM lack sensitivity for early malignancy.
But TM are rarely elevated in all patients of a particular type of cancer.
But no marker so far has an absolute organ specificity.
What are the types of TM?
What are TM used for?
Screening: serum TM are not ideal for population screening because they have low
sensitivity and specificity plus a low positive predictive value of these tests.
Aiding in diagnosis: is the tumor benign or malignant? What is the histological type
of the tumor (e.g. is it a germ-cell tumor, a seminoma or a non-seminoma tumor?) Is
it a metastatic cancer with unknown primary site?
Assessing the prognosis: beside the classical prognostic factors (tumor size, grade,
stage), TM can serve as independent prognostic factors.
Predicting the response to therapy: in breast cancer, for example, the presence or
absence of hormone receptors may predict the response to therapy.
Monitoring patients with diagnosed disease: TM aid in early detection of recurrent
malignancy as well as in monitoring the treatment given to the patient. Those markers
that were positive at the time of diagnosis are applied in the monitoring process.
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What are the tumor markers used for screening?
There are 6 TM that are worth mentioning:
1. VMA (vanillylmandelic acid) and HVA (homovanillic acid) for neuroblastoma (NB):
NB is the most common solid tumor among children younger than 5 years of age. The
tumor originates from the primitive nervous tissue, and it synthesizes adrenaline,
noradrenaline and dopamine. VMA and HVA are breakdown products of these
catecholamines and can be measured in the urine. High levels of these markers can
be found in 85-90% of NB patients at 6 months of age. Screening, however, does not
reduce mortality.
2. PSA for prostate cancer: prostate cancer is the 4th most common malignancy in males.
PSA and a digital rectal examination are used for screening males older than 50 years
of age, and for screening high-risk groups at 40-45 years of age.
3. CA125 for ovarian cancer (OC): OC is the most frequent cancer in females in the
western world. CA125 combined with recto-vaginal pelvic examination and trans-
vaginal ultrasound (TVUS)/transabdominal US (TAUS) and Doppler test are used for
screening. Screening, however, is recommended for high-risk population.
4. AFP for hepatocellular cancer: HC cancer is the 5th most common cancer in males
and the 8th most common in females. It is associated with HBV and HCV infections,
hemachromatosis and liver cirrhosis. AFP combined with TAUS are used for
screening. Screening is recommended for high-risk groups (those with chronic active
hepatitis, cirrhosis, etc.). When performed, AFP is screened for every 3-4 months, and
TAUS is performed every 4-6 months.
5. hCG for choriocarcinoma: this tumor is composed of villous trophoblasts, and may
follow normal pregnancy, non-molar abortion, ectopic pregnancy or hydatidiform
mole. hCG is elevated in almost all patients with choriocarcinoma. It is very sensitive
and is considered a nearly-ideal TM. Even though a good therapy is available,
screening is recommended for high-risk groups only (e.g. hydatidiform mole).
6. Calcitonin for medullary thyroid cancer: this is a rare malignancy that can be
hereditary or acquired, solitary or in combination with other tumors (MEN2A,
MEN2B). The parafollicular cells are malignant. As they produce calcitonin in basal
values or in stimulated values (following the administration of pentagastrin or
calcium gluconate), screening is recommended for high-risk groups once a year.
What are the TM for germ-cell tumors?
Seminoma: AFP
Non-seminoma: hCG
Mixed germ-cell tumor: LDH, P-ALP
What are the gynecological tumor markers?
Ovarian cancer: CA125 (to a lesser degree: CA15-3, CA72-4, TPA)
Cervical cancer: SCC
Endometrial cancer: CA125
What are the lung cancer tumor markers?
Adenocarcinoma: cyfra21-1, CEA (to a lesser extent: CA125)
Squamous cell carcinoma: cyfra21-1, SCC
Small cell carcinoma: NSE, cyfra 21-1
Large cell carcinoma: cyfra21-1, CEA (to a lesser extent: CA125).
What are the breast cancer TM?
CA15-3, CEA (to a lesser extent: TPA, Cyfra21-1)
What are the prostate cancer TM?
PSA, fPSA (to a lesser extent: P-AcP (prostatic acid phosphatase))
What are the GI cancer TM?
Colorectal cancer: CEA
Gastric cancer: CEA<CA19-9<CA72-4
Esophageal cancer: SCC, TPA, cyfra21-1 (to a lesser extent: CA19-9)
Pancreatic cancer: CA19-9
Hepatocellular cancer: AFP
Avi Sayag Clinical Biochemistry
What do CA, TPA, SCC, Cyfra, hCG, AFP, CEA, PSA abbreviate?
CA – carbohydrate antigen;
TPA – tissue polypeptide antigen
SCC – squamous cell carcinoma antigen
Cyfra – cytokeratine 19 fragment
hCG – human chorionic gonadotropin
AFP – alpha-feto-protein
CEA – carcino-embryonic antigen
PSA – prostatic specific antigen
What is the frequency of TM determination?
Before first therapy
2-10 days after therapy (according to the half-life of the TM)
In intervals of 3 months during the 1st and 2
nd year
In intervals of 6 months in the 3rd
, 4th and 5
th year after the first therapy
Before any change of therapy
If a relapse or metastasis are suspected
If restaging is to be carried out
2-4 weeks after the first occurrence of a significant increase of the TM
How do we evaluate the results?
During monitoring of therapy, a decrease of at least 50% in the concentration of the TM
indicate partial remission. TM cannot be used to determine complete remission.
If an increase of the TM concentration is detected, repeat the measurement in 2-4 weeks
(twice; all-in-all: 3 measurements). An increase of at least 25% in the TM concentration is
significant enough to determine progression of the tumor.
What are the factors that alter the serum concentration of TM?
The production of the TM: how much TM is produced.
The release of the TM: the rate of release, and whether it is released or not.
The mass of the tumor: the larger the tumor is, the more TM might be produced.
Blood supply of the tumor: the presence of the TM in the serum depends on that.
Diurnal variation
Position of the body during blood drawing
Iatrogenic influences
Catabolism of the tumor marker
Life-style (habits): smoking, alcohol consumption.
Avi Sayag Clinical Biochemistry
Topic 9 Iron metabolism, hemochromatosis, iron deficiency anemia
(note: this topic includes practical topics 2, 3 and 6) The daily iron intake is about 15 mg, but less than 10% is absorbed (0.6-1.5 mg). The total
body content of iron is ~4 g: 3g in hemoglobin, ~0.5g in myoglobin and enzymes and ~4mg
in the plasma. The body stores up to 1.5g of iron. The average male loses ~1mg per day and a
female loses ~2.5mg per day. The regulation of iron absorption is complex. There are three
major influences:
The state of the body's iron stores (absorption is increased when they are depleted and
decreased when they are replete)
Erythropoiesis (absorption is increased when erythropoiesis is increased, irrespective
of the state of the iron stores)
Recent iron intake (a dietary iron bolus decreases iron absorption for several days).
The main site of iron absorption is the proximal small bowel. Iron is more readily absorbed in
the Fe2+
form but dietary iron is mainly in the Fe3+
form. Gastric secretions are important in
iron absorption in that they liberate iron from food (although heme can be absorbed intact)
and promote the conversion of Fe3+
to Fe2+
. Ascorbic acid and other reducing substances
facilitate iron absorption while phytic acid (in cereals), phosphates and oxalates form
insoluble complexes with iron and decrease its absorption.
Iron is continuously recycled in an almost closed system – there is no significant excretion.
Body stores are determined by the control of intestinal absorption. Free iron is toxic, and thus,
iron is protein-bound (i.e. it is transported bound to transferrin and stored in ferritin and
hemosiderin).
Once absorbed into the intestinal mucosal cells, iron is either transported directly into the
bloodstream, or else combines with apoferritin, a complex iron-binding protein, to form
ferritin. This iron is lost into the lumen of the gut when mucosal cells are shed. In iron
deficiency, the apoferritin content of mucosal cells decreases and a greater proportion of
absorbed iron reaches the bloodstream.
Transport: In the blood, iron is transported mainly bound to transferrin, each molecule of
which binds two Fe3+
ions. Transferrin concentration is increased in pregnancy, among
women who take oral contraceptive, estrogen, and in iron deficiency. Its concentration is
decreased in chronic inflammation, malignancies, nephrotic syndrome and iron overload.
The transferrin saturation is normally 15-45%. In iron deficiency, saturation is < 15% and in
iron overload it is > 60%. Iron can be taken from the plasma by cells other than erythrocytes,
Avi Sayag Clinical Biochemistry
as these cells express transferrin receptors. Transferrin receptor (TfR) is present on the surface
of all cell types (80% on erythroid precursors). It is a transmembrane protein that consists of 2
identical subunits. Its extracellular part is cleaved off by a serine protease and the receptor is
liberated (mainly from late erythroid precursors, i.e. normoblasts and reticulocytes). Once in
the circulation, it is complexed with transferrin. Iron uptake is regulated by the number of
transferrin receptors.
Storage: ferritin and hemosiderin store iron.
Ferritin is found in all cell types. It has a Fe+3
hydroxide-phosphate core covered by 24
subunits of protein shell (apoferritin). On the surface of this "ball" there are channels through
which Fe+3
enters and leaves. There are 2 types of subunits: a heavy chain, which has a
ferroxidase activity, and a light chain that is responsible for the building up of the iron core.
Each subunit (of the 24 subunits) is called an apoferritin. Each apoferritin binds 3000-4500
iron ions. Small amount circulates in the serum and is in equilibrium with that in stores.
Serum ferritin reflects the size of iron stores. Ferritin is an acute phase reactant. It is increased
in iron overload, liver disease, cancer, and in acute and chronic inflammation. It is decreased
in iron deficiency.
Iron regulation of gene expression:
Cells in rest have very low transferrin receptors. When the iron level is low, cells need more
transferrin receptors in order to get enough iron from the circulation. However. They do not
require much storing proteins (ferritin). The opposite is true when the iron concentration in
the blood is high.
Both transferrin receptors and ferritin are regulated at the mRNA level, according to the
cytosolic iron level.
Both mRNAs have a iron-response element sequence, to which binding proteins (IRE-BP)
can bind. When the binding proteins bind to ferritin mRNA, translation is blocked, but when
they bind to transferrin receptor IRE, translation takes place.
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Hepcidin: this is a small peptide hormone synthesized in the liver. It has a regulatory role in
iron homeostasis and an antimicrobial effect. In case of inflammation (IL-6), its levels
increase. It then binds to ferroportin and leads to its degradation. Thus, iron is trapped in the
enterocyte. Some 48 hours following ferroportin degradation, the enterocyte is desquamated
and the iron content spills. Iron is trapped in macrophages as well leading to anemia of
chronic disease.
Iron deficiency anemia:
This anemia is microcytic (MCV<80fL) and hypochromic (MCH < 27 pg).
Iron deficiency can be caused due to increased demand for iron or due to a decreased supply.
Increased demand for iron can be due to:
o Chronic blood loss (GI, urogenital, respiratory system)
o Growth
o Pregnancy and lactation
Decreased supply can be due to:
o Diet with low amount of heme iron
o Impaired iron absorption (intestinal disease, inhibitors of absorption such as
phosphates, phytates that inhibit reduction of Fe+3
, HCl deficiency and
accelerated passage).
Development of iron deficiency anemia:
Lab diagnosis of iron deficiency anemia:
1. hematological tests: Hb, hematocrit, MCV, MCH, MCHC, RDW, blood smear and
bone marrow smear.
2. Biochemical tests: serum iron, serum transferrin, transferrin saturation, serum ferritin,
soluble transferrin receptor.
Procedure of blood drawing, vacutainer tubes (practical topic 3) 1. Vacutainer tubes:
Anticoagulant Common tests Color of stopper
None Chemistry Red
EDTA (ethylene diamine
tetra-acetic acid)
Hematology, molecular
genetic tests
Lavender
Sodium citrate Hemostasis, molecular
genetic tests
Blue
Sodium/lithium-heparin Flow cytometry,
chromosome analysis
Green
Sodium fluoride Glucose determination
from stored samples
Gray
ACD (acid citrate dextrose) Blood culture Yellow
2. Needle size: the gauge is a measurement of the needle diameter – the larger the
number is, the smaller the diameter is (gauge 21 or 23 are usually used).
3. Needle type: multiple draw needle, butterfly needle, blood lancet.
4. Tube holder: barrel (a device for a safe and secure blood drawing)
5. Alcohol swabs: isopropyl alcohol (does not induce hemolysis)
6. Tourniquets
7. Gloves
8. Gauze
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9. Adhesive bandages/tapes
10. Needle disposal container
Procedure:
1. Wear gloves
2. Positioning of the patient
3. Apply tourniquet in the upper arm
4. Locate the vein (middle cubital for example)
5. Assemble the equipment
6. Clean the site
7. Perform venipuncture
8. Release tourniquet
9. Remove the needle
10. Apply the gauze
11. Dispose the needle
12. Label the specimen
Microcapillary blood collection: used in case of newborns and infants. The site chosen is
usually the finger or the heel in case of an infant (for PKU testing for example). The skin can
be preheated if necessary. After cleaning the site, puncture the skin off-center and
perpendicular to the lines of the fingerprint. Then, collect the blood by first wiping off the
first drop and proceed as above-mentioned.
Determination of hemoglobin and hematocrit (practical topic 2) The Hb-cyanide method:
Advantages:
1. Beside Hb, methemoglobin and carboxyhemoglobin (HbCO) can be measured.
2. Stable cyanmethemoglobin standards are available for calibration.
Disadvantages:
1. It does not measure sulphhemoglobin, which is rarely present.
2. The total conversion of HbCO is slow – it takes 30 minutes. If during determination
the extinction of the solution is measured exactly at the 3rd
minute in the presence of
20% HbCO (which may be found in heavy smokers) the Hb concentration is
overestimated by 6% (HbCO absorbs more light at 540nm than does hemoglobin
cyanide).
3. Considering that the reagents contain cyanide, special fluid waste disposal is required.
Procedure:
1. Collection of whole blood anticoagulated with EDTA
2. Lysis of RBCs by SLS (Sodium-Lauryl-Sulphate)
3. Hb ----------> hemiglobin -------------> HiCN.
The reagent contains K3Fe(CN)6 that carries out the first reaction, and KCN (CN-) that
carries out the 2nd
reaction.
4. A non-ionic detergent (Nonidet P40) is added to assure total and rapid hemolysis and
for the prevention of precipitation of proteins and lipoproteins.
5. The extinction of the color product is measured at 540 nm. The transformation of Hb,
HbO2 and Hi occurs in 5 minutes.
If there is a high WBC count, PLT count or high concentration of lipoproteins, turbidity is
visualized and the specimen should be centrifuged before measurement.
Reference interval:
Male: 140-180 g/L
Female: 120-160 g/L
Hematocrit:
Htc can be measured using centrifugation, measuring impedance and by calculation.
Centrifugation:
1. Collection of blood anticoagulated with EDTA or heparin. The blood is mixed to
homogenize RBCs
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2. Fill the blood into the capillary and close with wax. Make sure the closing is perfect
and the surface of the wax is horizontal.
3. Place the capillary in the centrifuge, with the closed end facing outwards.
4. Centrifuge for 5 minutes with RPM corresponding to 12000g.
5. Transfer the capillary to the reading device. The bottom of the sample should fall
exactly on the zero level. The capillary holder should be moved horizontally till the
top of the plasma reaches the line corresponding to the 1.00 value.
6. The line marking the top of the RBCs cylinder gives the Htc value.
7. Multiple the value by 0.97: sedimented RBCs enclose about 3% plasma (trapped
plasma). This value can be higher when the form of the RBC is altered (like in sickle
cell anemia, when the trapped plasma can reach 20%).
Reference interval:
Male: 0.38-0.52
Female: 0.37-0.46
Errors may occur:
1. If the capillary is filled with incorrectly suspended blood.
2. If the capillary is not well closed – some RBC may leak.
3. If the closing wax at the bottom of the capillary is not horizontal (the setting of the
zero line is not correct).
4. If the WBC count is high, and not taken into consideration, it may lead to falsely high
Htc value. In this case, the Htc should be read at the top of RBC layer.
5. Unequal diameter of the capillary, inadequate RPM, short centrifugation time…
Calculation:
Automated hematology analyzers obtain Htc value as a calculated parameter derived from
RBC count and MCV: 1000
MCVRBCHtc
×= . (RBC in T/L (10
12) and MCV in fL (10
-15) ).
Avi Sayag Clinical Biochemistry
Topic 10 Laboratory diagnostics of hemoglobinopathies
Globin chains are synthesized in the liver of the fetus and in the bone marrow of adults.
Adults Hb (HbA) can be of 2 type:
HbA1 – composed of 2α2β chains (96-98%)
HbA2 – composed of 2α2δ (2%)
Fetal Hb (HbF) is composed of 2α2γ (0.5-0.8%)
Gene defects in the Hb molecule are the most common genetic disorders in the world. There
are about 400 Hb variants registered. Abnormal Hb can be detected in electrophoresis. The
most common variants are: HbS, HbC, HbS-C, HbE.
Sickle cell anemia (HbS)
The cause of the disease is a point mutation in the β chain of the globin: the β-globin gene is
found on the short arm of chromosome 11. The association of two α-globin subunits with two
mutant β-globin subunits forms hemoglobin S (HbS). Valine replaces glutamate residue at
position 6 of the β chain located at the surface of the Hb, which is exposed to water. Glu is a
polar amino acid while Val is non-polar. Thus, the replacement causes the formation of a
sticky patch on the surface of the β chains. On the surface of the deoxyHb there is another
sticky patch that causes polymerization of Hb into a fibrous structure.
The loss of red blood cell elasticity is central to the pathophysiology of sickle-cell disease. In
sickle-cell disease, low oxygen tension promotes red blood cell sickling and repeated episodes
of sickling damage the cell membrane and decrease the cell's elasticity. These cells fail to
return to normal shape when normal oxygen tension is restored. Consequently, these rigid
blood cells are unable to deform as they pass through narrow capillaries, leading to vessel
occlusion and ischemia. Thus, the hemolytic anemia dominates the clinical features with
characteristic crises:
1. Infarcts in bones, lungs, spleen (vaso-occlusive crisis)
2. Sequestration in visceral organs
3. Aplastic crises (infections; decreased Hb and reticulocytes)
4. Hemolytic crises (decreased Hb but increased reticulocytes)
25% of Africans are heterozygote for the disease and are therefore resistant to malaria, since
the sickle red blood cells are not conducive to the parasites. In areas where malaria is
common there is a survival value in carrying the sickle-cell genes.
Apart from middle Africa, sickle cell anemia is also common in south Europe and in some
parts of the Saudi Arabia.
Diagnosis 1. In HbSS, Hb levels are in the range of 6–8 g/dL with a high reticulocyte count.
2. A blood film may show features of hyposplenism (target cells and Howell-Jolly bodies).
3. Sickling of the red blood cells, on a blood film, can be induced by the addition of sodium
metabisulfite. The presence of sickle Hb can also be demonstrated with the "sickle solubility
test". A mixture of Hb S in a reducing solution (such as sodium dithionite) gives a turbid
appearance, whereas normal Hb gives a clear solution.
4. Abnormal Hb forms can be detected on electrophoresis
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5. The diagnosis can be confirmed with high-performance liquid chromatography
(HPLC).
6. An acute sickle-cell crisis is often precipitated by infection. Therefore, a urinalysis to
detect an occult urinary tract infection, and chest X-ray to look for occult pneumonia
should be routinely performed.
It should be noted that the sickle cell trait is a benign condition with no anemia. The RBCs
appear to be normal, and crisis can be caused by extreme stress (anoxia, infection).
Note the presence of the A band as opposed to the previous one where no A band is present
(this is because in homozygotes only HbS is present, while in heterozygotes 40% is HbS and
the remainder is HbA).
Hb C disease Hb C is an abnormal Hb with substitution of a lysine residue for a glutamic acid residue at the
6th position of the β-globin chain.
This mutated form reduces the normal plasticity of host erythrocytes causing a
hemoglobinopathy. In those who are heterozygous for the mutation, about 28–44% of total
hemoglobin (Hb) is HbC, and no anemia develops.
In homozygotes, nearly all Hb is in the HbC form, resulting in mild hemolytic anemia.
Target cells (codocytes), microspherocytes and HbC crystals are found in a blood smear from
a homozygous patient. The reticulocyte count is high, as well as serum bilirubin.
Hb E disease Lysine substitutes glutamate on position 26 in the β chain. Most common in south east Asia.
Homozygotes present with mild hypochromic, microcytic anemia with target cells.
Thalassemias Thalassemia is an inherited autosomal recessive blood disease. In thalassemia, the genetic
defect results in reduced rate of synthesis of one of the globin chains that make up
hemoglobin. Reduced synthesis of one of the globin chains causes the formation of abnormal
Hb molecules, and this in turn causes the anemia which is the characteristic presenting
symptom of the thalassemias. The cause of the disease might be a missing gene, improper
processing of mRNA, premature termination of protein synthesis, frameshift mutation, etc.
The thalassemias are classified according to which chain of the hemoglobin molecule is
affected. In α thalassemias, production of the α globin chain is affected, while in β
thalassemia production of the β globin chain is affected.
β globin chains are encoded by a single gene on chromosome 11; α globin chains are encoded
by two closely linked genes on chromosome 16. Thus in a normal person with two copies of
each chromosome, there are two loci encoding the β chain, and four loci encoding the α chain.
α-thalassemia: There are four genetic loci for α globin, two of which are maternal in origin
and two of which are paternal in origin. The severity of the α thalassemias is correlated with
the number of affected α globin loci: the greater the number of affected loci, the more severe
the manifestations of the disease will be.
If 1 copy is defective- silent α-thalassemia carrier; no significant signs or symptoms (except
maybe low MCV and low MCH)
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If 2 copies are defective – α thalassemia triat. Two α loci permit nearly normal erythropoiesis,
but there is a mild microcytic, hypochromic anemia.
If 3 copies are defective – mild-to-moderate hemolytic anemia (HbH disease), resulting in
poor O2 delivery (too high affinity to O2).
If 4 copies are defective - the fetus cannot live once outside the uterus and may not survive
gestation: most such infants are dead at birth with hydrops fetalis, and those who are born
alive die shortly after birth. They are edematous and have little circulating hemoglobin, and
the hemoglobin that is present is all tetrameric γ chains.
β-thalassemia: as mentioned, there are 2 copies of the β-chain gene:
If 1 copy is defective – minor β-thalassemia – mostly asymptomatic (perhaps mild
microcytic, hypochromic anemia may develop). HbF is increased and HbA2 > 3.5%; target
cells are present as well as ovalocytes, poikilocytosis and basophil stippling.
If 2 copies are defective – major β-thalassemia – appears after birth. Severe microcytic
anemia develops (Cooley's anemia), and the patient depends on constant blood transfusion.
This offered treatment is life-saving, but results in iron overload.
The patient may die at 20-25 years of age. The ultimate cure is bone marrow transplantation.
The Hb levels are 30-40 g/L, MCV – 51-61 fL, reticulocytes – 1-8%, and both HbF and HbA2
are elevated.
The bone marrow shows erythroid hyperplasia (E:M is 20:1)
seFe is elevated, Tfsat is > 80%.
There is also thalassemia intermedia in which the morphology is the same as for the major
form, but the HbF is 2-100%, the HbA2 does not exceed 7%.
Diagnosis
Sideroblastic anemia Sideroblastic anemia is caused by abnormal production of red blood cells usually as part of
myelodysplastic syndrome, which can evolve into hematological malignancies (especially
acute myelogenous leukemia). The body has iron available but cannot incorporate it into
hemoglobin. Sideroblasts are seen, which are nucleated erythrocytes with granules of iron in
their cytoplasm
The problem lies in a failure to completely form heme molecules, whose biosynthesis takes
place partly in the mitochondrion. This leads to deposits of iron in the mitochondria that form
a ring around the nucleus of the developing red blood cell.
It can be inherited (X-linked) or acquired. If acquired, it can be primary, as part of a
myelodysplastic syndrome, or secondary due to:
Toxins: lead or zinc poisoning
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Drug-induced: ethanol, isoniazid, chloramphenicol, cycloserine
Nutritional: pyridoxine or copper deficiency
Genetic: ALA synthase deficiency
Diagnosis Specific test: Prussian Blue stain of RBC in marrow. Shows ringed sideroblasts.
Increased ferritin levels
Decreased total iron-binding capacity
Hematocrit of about 20-30%
Serum Iron: High
High transferrin saturation
MCV is usually normal or slightly increased; although it may occasionally be low, leading to
confusion with iron deficiency.
With lead poisoning, there is coarse basophilic stippling of red blood cells on peripheral blood
smear.
Summary and presentation of the topic:
General features of Hb – types, %, structure
Disorders: HbS, HbC, HbC-S, HbE, thalassemia, sideroblastic anemia
Diagnosis of HbS: Hb, ret, blood film, Na-metabisulfite, sickle solubility test,
electrophoresis, HPLC (sickle cell trait)
Diagnosis of thalassemia: history, clinical signs, blood (Hb, MCV, MCH, RDW, ret),
electrophoresis, HPLC, β-chain sequencing
Diagnosis of sideroblastic anemia: Prussian blue stain, ferritin, iron binding capacity,
HTC, Fe, Tfsat, MCV, lead poisoning: basophilic stippling on peripheral smear.
Avi Sayag Clinical Biochemistry
Topic 11 Laboratory diagnostics of hemolytic anemias
An increased rate of red cell destruction;
A compensatory increase in erythropoesis that results in reticulocytosis;
The retention by the body of the products of red cell destruction (including iron). Because
the iron is conserved and recycled readily, red cell regeneration can keep pace with the
hemolysis. Consequently, these anemias are almost invariably associated with a marked
erythroid hyperplasia within the marrow and an increased reticulocyte count in peripheral
blood. In severe hemolytic anemias, extramedullary hematopoiesis often develops in the
spleen, liver, and lymph nodes.
Destruction of red cells can occur within the vascular compartment (intravascular hemolysis)
or within the cells of the mononuclear phagocyte (reticuloendothelial) system (extravascular
hemolysis).
Intravascular hemolysis can result from mechanical trauma (e.g., a defective heart valve)
or biochemical or physical agents that damage the red cell membrane (e.g., fixation of
complement, exposure to clostridial toxins, or heat). Regardless of the cause, hemolysis
leads to hemoglobinemia, hemoglobinuria, and hemosiderinuria. The conversion of the
heme pigment to bilirubin can result in unconjugated hyperbilirubinemia and jaundice.
Haptoglobin, a circulating protein that binds and clears free hemoglobin, is often absent
from the plasma.
Extravascular hemolysis (more common) takes place largely within the phagocytic cells
of the spleen and liver. The RES removes damaged red cells from the circulation. The red
cells need to be highly deformable to travel in the splenic sinusoid. Therefore, any change
in that feature leads to red cells being stuck in the spleen (and phagocytosed there).
Extravascular hemolysis is not associated with hemoglobinemia and hemoglobinuria, but
it often produces jaundice and, if long-standing, can lead to the formation of bilirubin-rich
gallstones. Haptoglobin amounts are always decreased, because some hemoglobin
invariably escapes into the plasma.
In most forms of hemolytic anemia there is a reactive hyperplasia of the RES (splenomegaly).
In chronic hemolytic anemias, changes in iron metabolism lead to increases in iron absorption
from the gut. Because the pathways for the excretion of excess iron are limited, this often
causes iron to accumulate, giving rise to systemic hemosiderosis.
The intracorpuscular abnormalities can be either hereditary or acquired.
The hereditary causes might be:
Membrane defects:
o In the setting of spherocytosis (spectrin deficiency), jaundice may follow,
splenomegaly, gallstones and most probably anemia. Spherocytosis is AD.
Microspherocytes and elevation of reticulocytes (5-20%) are evident in blood film. The
mechanism underlying spherocytosis starts with spectrin deficiency. This leads to
decreased protein density of the RBC's cytoskeleton and as a result parts of the
erythrocyte's bilayer membrane are released as microvesicles. Thus, the surface area of
the RBC is decreased (spherocytosis). This condition impairs the deformability of the
red blood cell, and they are therefore entrapped in the spleen. Trapped cells cause
splenic conditions leading to further loss of surface area, which then again lead to the
release of RBCs' membrane as microvesicles. Other forms of hereditary spherocytiosis
result from mutations that involve ankyrin, band 4.2 and band 3.
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o Diagnosis: the classic laboratory features of HS include minimal or no anemia,
reticulocytosis, an increased mean corpuscular hemoglobin concentration (MCHC),
spherocytes on the peripheral blood smear, hyperbilirubinemia, and abnormal results on
the osmotic fragility test (the most sensitive test to help detect HS is the incubated
osmotic fragility test performed after incubating RBCs for 18-24 hours under sterile
conditions at 37°C - hemolysis of HS cells may be complete at a solute concentration
that causes little or no lysis of normal cells).
o RBC morphology is distinctive yet not diagnostic.
o Other biochemical changes of hemolysis also are present, including increased LDH,
increased unconjugated bilirubin, and decreased serum haptoglobin.
o An increased MCHC obtained from an electronic cell counter is a characteristic feature
of red cells in HS. MCHC values greater than the upper limit of normal (35-36%) are
common. This increased MCHC is a result of mild cellular dehydration. The MCV in
patients with HS is low. This relatively low MCV may reflect membrane loss and cell
dehydration.
o Further characterizing the specific membrane lesion by looking for abnormalities in
spectrin, ankyrin, pallidin, or band 3 is possible. However, these studies are not routine
and are available only in select research laboratories.
o The initial workup if hemolysis is suggested should include the following: � Hb, MCHC, MCV, Reticulocyte count
� Lactate dehydrogenase
� Fractionated bilirubin
� Haptoglobin
� Peripheral smear: Howell-Jolly bodies may be present, indicating remnant splenic
tissue if the patient has had their spleen removed. Findings also may include
megalocytosis.
� Vitamin B-12 and folate: This should be measured to determine the nutritional
stores during recovery from an aplastic crisis.
� Herpes simplex virus, HPV type 19, and infectious mononucleosis: Testing for
these may help identify an infectious etiology for the aplastic crisis.
o Elliptocytosis – due to a mutated band 4.1
o Membrane lipids: a-β-lipoproteinemia
Enzymatic causes: G6PD (x-linked), glutathione synthetase, pyruvate kinase, hexokinase
Disorders of hemoglobin synthesis (hemoglobinopathies): thalassemias, sickle cell
anemia
The acquired cause is due to Paroxysmal Nocturnal Hemoglobinuria (PNH):
o The problem lies in the lack of anchoring proteins for DAF and MIC, leading to non-
inhibition of the complement membrane destruction complex.
Extracorpuscular
The acquired causes can be:
Immune causes: autoimmune (as in transfusion reactions and SLE), alloimmune (as in
the Rh disease of the newborn) or drug-associated (penicillin or methyl-DOPA).
RBC fragmentation (physical damage on abnormal surfaces (artificial heart valves) or
microangiopathic damage: disease of small blood vessels, in DM for example, which
may lead to DIC, HUS or TTP (thrombic thrombocytopenia purpura).
March hemolytic anemia
Infections with meningococci, malaria or Clostridium perfringens
Secondary to liver disease or renal disease.
Due to chemical or physical agents such as snake venom, insect bite and burns.
Avi Sayag Clinical Biochemistry
Topic 12 Laboratory diagnostics of megaloblastic anemias Megaloblastic anemias are macrocytic (MCV>100 fL) and hyperchromic (MCH>31pg/RBC).
The 2 main causes are vitamin B12 deficiency and folic acid deficiency.
Folic acid deficiency
Diet is not a common cause for folic acid deficiency. Although it is present in almost all
foods, it is destroyed in 10-15 minute cooking.
It is distributed widely in green leafy vegetables, citrus fruits, and animal products. Humans
do not generate folate endogenously because they cannot synthesize PABA (p-aminobenzoic
acid), nor can they conjugate the first glutamate.
Folates are present in natural foods and tissues as polyglutamates because these forms serve to
keep the folates within cells. In plasma and urine, they are found as monoglutamates because
this is the only form that can be transported across membranes. Enzymes in the lumen of the
small intestine convert the polyglutamate form to the monoglutamate form of the folate,
which is absorbed in the proximal jejunum via both active and passive transport.
Within the plasma, folate is present, mostly in the 5-methyltetrahydrofolate (5-methyl THF)
form, and is loosely associated with plasma albumin in circulation. The 5-methyl THF enters
the cell via a diverse range of folate transporters with differing affinities and mechanisms
(i.e., ATP–dependent H+ co-transporter or anion exchanger). Once inside, 5-methyl THF may
be demethylated to THF, the active form participating in folate-dependent enzymatic
reactions. Cobalamin (B12) is required in this conversion, and in its absence, folate is
"trapped" as 5-methyl THF.
From then on, folate is no longer able to participate in its metabolic pathways, and
megaloblastic anemia results. Large doses of supplemental folate can bypass the folate trap,
and megaloblastic anemia will not occur.
The biologically active form of folic acid is tetrahydrofolic acid (THF), which is derived by
the 2-step reduction of folate involving dihydrofolate reductase. THF plays a key role in the
transfer of 1-carbon units (such as methyl, methylene, and formyl groups) to the essential
substrates involved in the synthesis of DNA, RNA, and proteins. More specifically, THF is
involved with the enzymatic reactions necessary to synthesis of purine, thymidine, and amino
acid. Manifestations of folate deficiency thereafter, not surprisingly, would involve
impairment of cell division, accumulation of possibly toxic metabolites such as homocysteine,
and impairment of methylation reactions involved in the regulation of gene expression, thus
increasing neoplastic risks.
A healthy individual has about 500-20,000 mcg of folate in body stores. Humans need to
absorb approximately 50-100 mcg of folate per day in order to replenish the daily degradation
and loss through urine and bile. Otherwise, signs and symptoms of deficiency can manifest
after 4 months.
Signs and symptoms:
Anemia: weakness, vertigo, tinnitus, palpitations, angina, pallor with slightly
icteric skin and eyes.
GI: glossitis (sore tongue), cheilosis angularis, diarrhea, weight loss
There are no neurological signs.
B12 deficiency
Vitamin B12 is absorbed in the ileum complexed to intrinsic factor secreted from the parietal
cells of the stomach. It is transported in the blood bound to transcobalamin II. In its
methylated form it facilitates the conversion of homocysteine to methionine – a reaction
which is carried out by the conversion of methyl-THF to THF. As mentioned, THF is required
to synthesize purines (thymidine).
Pathophysiology: vitamin B12 deficiency is caused by failure to absorb the vitamin due to
autoantibodies directed against the gastric parietal cells, the intrinsic factor, the IF-B12
complex or against the receptors in the ileum.
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Signs and symptoms:
Anemia: weakness, vertigo, tinnitus, palpitations, angina, pallor with slightly
ichteric skin and eyes.
GI: glossitis (sore tongue), cheilosis angularis, diarrhea, weight loss
Neurological signs: numbness and parasthesias in the extremities, ataxia,
poor finger coordination, sphincter disturbances, forgetfulness, severe
dementia, and psychosis.
Lab:
MCV > 100 fL (macrocytic anemia)
In the peripheral smear macro-ovalocytes can be found as well as
hypersegmented granulocytes, and the reticulocyte count is low.
Unconjugated serum bilirubin is elevated as well as LDH1. Autoantibodies
can also be found in the serum or in the gastric juice.
In the bone marrow there are megaloblasts and ineffective erythropoesis.
Endoscopy may reveal atrophy of the gastric mucosa that may lead to
achlorhydria. This poses an increased risk for gastric carcinoma.
Serum B12 can be directly measured (plasma immunoassay)
Schilling test is positive and helps to differentially diagnose GI disease from
pernicious anemia:
i. First, IM injection of B12 is given to saturate the transcobalamin II
stores. At the same time, the patient is given labeled B12 per os (the
most commonly used radiolabels are 57
Co and 58
Co).
ii. Urine is then collected over 24 hours. A normal result is documented
when more than 10% of the given labeled B12 is excreted in the
urine.
iii. If less than 10% is collected, the test is repeated with a modification:
labeled B12 and IF are given per os, and urine is collected over 24
hours. If more than 10% is excreted this time, then pernicious anemia
is diagnosed. However, if the result is still not normal, then a GI
disease is suspected and should be further investigated.
Summary and presentation of the topic:
Classify megaloblastic anemia (hyperchromic, macrocytic) and give values of MCV
and MCH
Mention the 2 main causes: folate and B12 deficiency
Speak about folate:
o Forms and evolution: poly � mono (methyl-FH4) � FH4 (active form)
o Absorbance (prox. jejunum, active + passive)
o Participation in metabolism and the role of B12
o Reference range: 3-20 µg/L
o Signs and symptoms of folic acid deficiency
o Diagnosis: plasma immunoassay (RBC is better, as the concentration in
RBCs reflects the body's folate reserves, range >140µg/L , while plasma
concentration reflects recent dietary intake).
Speak about B12:
o Absorbance in the ileum + IF
o Transport in the blood with transcobalamine II
o Functions of B12
o Normal range: 130-700 ng/L
o Recommended daily intake: 3-5µg (the liver stores ~3mg – enough for years)
o Sources of deficiency (autoimmune, gastrectomy, Crohn's, low intake)
o Signs and symptoms of deficiency
o Diagnosis: plasma immunoassay, Schilling test
Lab: Hb, MCV, MCH, B12, folic acid + normal ranges + lab procedures
Additional tests: endoscopy, BM examination, blood smear
Avi Sayag Clinical Biochemistry
Topic 13
Major laboratory characteristics of acute and chronic lymphoid leukemias ALL
Most common between 2-10 years of age.
85% B-cells, 15% T-cells.
More then 20% blasts in peripheral blood and BM.
Risk factors: Down syndrome, Fanconi's anemia, AT, chemical drugs, in utero
radiation.
Involves the enlargement of lymph nodes, spleen and liver.
Complete remission in 90% of cases and while 66% undergo full recovery.
FAB classification:
o L1: small blasts, homogenous population, narrow cytoplasm.
o L2: larger blasts, heterogeneous population, prominent nucleolus, wider
cytoplasm.
o L3: homogenous population, middle-large blasts, basophilic cytoplasm with
vacuoles (Burkitt lymphoma).
Another classification according to the immunophenotyping:
o Pre-B-cell ALL: CD19, CD79a
o Common ALL: CD19, CD79a, CD10
o Late pro-B-cell ALL: CD19, CD79a, CD10, µ-chain
o B-cell ALL: with surface Ig.
B-cell markers: CD 19, CD 20, CD 22, CD 79a, HLA-DR (young: CD 10)
Prognosis:
o L3 - unfavorable.
o CD 10, T-cell ALL - unfavorable.
o Adult > 30 G/L and children > 50 G/L - unfavorable.
o Age: less then 2 years, older then 10 years, older then 35 (adult) -
unfavorable.
o Less then 45 chromosomes – unfavorable.
o More then 50 chromosomes – favorable.
o Translocations t(8:14), t(9:22), t(1:19), t(4:11) – unfavorable.
o Translocations t(12:21) – favorable.
CLL In the spleen the germinal center is surrounded by two separate zones: the mantle
zone (close to the germinal center and IgD positive) and the marginal zone. Both
zones are populated by B-lymphocytes (the marginal zone lymphocytes have irregular
nuclei and the cytoplasm appears more empty).
In the lymph node, there is a mantle zone, and B-cells surround it. However, they do
not form a special zone, but are rather mixed with the lymphocytes in the mental
zone.
CLL is the leukemic counterpart of SLL (small lymphocytic lymphoma)
Usually affects people over 50.
Not aggressive.
Often asymptomatic, those who are symptomatic have general symptoms: general
symptoms of malignancy, lymphadenopathy, hepatosplenomegaly, leukocytosis
(around 200000/µL), hypogammaglobulinemia (prone to infections), anemia,
autoimmune hemolytic anemia, and thrombocytopenia. The organs most commonly
involved are the bone marrow and the blood (these are the primary sites), the lymph
nodes, spleen, liver, skin and tonsils.
Does not transform into ALL
Clonal expansion of mature lymphocytes (98% B-cell, 2% T-cell).
Gumprecht shadow.
More than 30% lymphocytic infiltration of the bone marrow.
Avi Sayag Clinical Biochemistry
In the serum of these patients there are low levels of Igs, paraproteins are rare and
uric acid level is elevated.
Immunophenotyping: B-cell markers (CD19, CD20, CD22), T-cell markers (CD5),
CD23.
Cytogenetics: 12 trisomy; deletions 11,13,17; no translocation involving CycD (no
t(4:11) ), as it must be differentially diagnosed from mantle zone lymphoma
(centrocytic lymphoma) which is characterized by t(4:11)(cycD+), CD5+, no +12,
and CD43.
CLL/SLL ALL
98% B cells 85% B cells
No translocations associated t(4:11), t(8:14), t(1:19), t(9:22), t(12:21)
+12, deletions 11, 13, 17 Chromosomes < 45, >50
CD19, CD20, CD22, CD23, CD5 CD19, CD20, CD22, CD10
Associated with hypo-γ-glubolinemia and
hyperuricemia
Stem cell�early pro-B�late pro-B�large pre-B�small pre-B�immature B�mature B
In general, ALL is characterized by arrested maturation somewhere between late pro-B and
small pre-B/immature B, while CLL is characterized by arrested maturation and proliferation
of activated B cell or memory B cell.
Characterization of leukemic cells by morphology (practical topic 7) Myelopoiesis:
Myeloblast�promyelocyte�myelocyte�metamyelocyte�band form (for PMNs)�
neutrophils/eosinophils/basophils
1. Myloblasts: > 10µm, basophilic plasma, no granulations, loose chromatin in
the nucleus, multiple nucleoli, a halo around the nucleus.
2. Promyelocyte: the largest cell in the lineage, many azurophilic granules,
decreased nucleus:plasma ratio.
3. Myelocytes: less basophilic plasma, lower number of granules, no nucleolus.
4. Metamyelocytes: secondary/specific granules appear; the nucleus is indented
and bean-shaped.
5. Band form: stick-shaped nucleus; grayish-blue granulation
6. Neutrophils: the nucleus has several lobes; bluish granulation.
7. Eosinophils: bi-lobed nucleus, pink plasma with reddish-brown large
granules. 12-17µm.
8. Basophils: rough, purple-black granulation that covers even the nucleus. 10-
14µm.
9. Monocytes: grayish-blue plasma with azurophilic granules. The nucleus is
bean-shaped. 12-20µm.
10. Lymphoblasts: large nucleus:plasma ratio; purplsh plasma; nucleoli are
present; it takes an expert to distinguish them from myeloblasts.
11. Small lymphocytes: small cells (10-12µm); basophilic cytoplasm; the nucleus
is condensed and purple.
12. Large lymphocytes (activated): loose chromatin and less basophilic
cytoplasm.
13. Large granular lymphocytes: large cells, rough azurophilic granules.
14. Plasma cells: blue cytoplasm; dark-purple nucleus in the periphery.
15. Megakaryocyte: large cell; cloudy cytoplasm with azurophilic granulation;
sometimes detached platelets can be seen in the surrounding; the nucleus is
multilobed. 50-70µm.
As a general rule, when cells mature, their size decreases as well as the nucleus:plasma ratio.
Nucleoli disappear from the nucleus, and the nuclear material becomes more condensed.
Avi Sayag Clinical Biochemistry
Topic 14 Major laboratory characteristics of acute and chronic myeloid leukemias AML
AML primarily affects older adults, with the median age being 50 years (nonetheless,
it can affect all age groups).
The clinical signs and symptoms are usually related to marrow failure caused by the
replacement of normal marrow elements by leukemic blasts.
Fatigue and pallor, abnormal bleeding, and infections are common
Splenomegaly and lymphadenopathy are in general less prominent than in ALL, but,
rarely, AML presents as a discrete tissue mass (a so-called granulocytic sarcoma).
Ideally, the diagnosis and classification of AML are based on the results of
morphologic, histochemical, immunophenotypic, and karyotypic studies. Of these
tests, karyotyping is most predictive of outcome:
Most AMLs are associated with acquired mutations in transcription factors that
inhibit normal myeloid differentiation, leading to the accumulation of cells at earlier
stages of development.
t(15;17) translocation occurs in acute promyelocytic leukemia. This translocation
results in the fusion of the retinoic acid receptor α (RARA) gene on chromosome 17
with the PML gene on chromosome 15. The chimeric gene(s) produce abnormal
PML/RARA fusion proteins that block myeloid differentiation at the promyelocytic
stage. Pharmacologic doses of retinoic acid, a vitamin A analogue, overcome this
block and cause the neoplastic promyelocytes to terminally differentiate into
neutrophils and die. Because neutrophils live, on average, for 6 hours, the result is the
rapid clearance of tumor cells and remission in a high fraction of patients. The effect
is very specific; AMLs without translocations involving RARA do not respond to
retinoic acid.
Classification:
o M0 – AML without maturation:
� 2% of AML cases;
� Diagnostic criteria: in the bone marrow – there are no azurophilic
granulation, no Auer rods in the blast cells; MPO is positive in less
than 3% of cells, and immunophenotyping is needed.
o M1 – AML with minimal maturation:
� 10-20% of AML cases.
� Diagnostic criteria (in the bone marrow): azurophilic granules and/or
Auer rods in less than 10% of cells; 90% of non-erythroid cells are
myeloblasts; MPO (or Sudan black) is positive in more than 3% of
blasts.
o M2 – AML with maturation:
� 30-45% of AML cases.
� Diagnostic criteria (in the bone marrow): azurophilic granules and/or
Auer rods in more than 50% of cells; 20-90% of non-erythroid cells
are myeloblasts; less than 20% of cells are monocyte precursors;
MPO (or Sudan black) is positive
o M3 – AML promyelocytic (hypergranular)
� 10-15% of AML cases
� Diagnostic criteria (in the bone marrow): strong granulation and lots
of Auer rods; more than 50% of cells are abnormal promyelocytes;
DIC is characteristic of this class.
o M4 – AML myelomonocytic
� 15-20% of AML cases
� Diagnostic criteria (in the bone marrow): more than 20% of nucleated
cells in the bone marrow should be myeloblasts and promyelocytes;
Avi Sayag Clinical Biochemistry
more than 20% of bone marrow cells should be promonoblasts and
monoblasts (stain with esterases such as NSE).
� Gum hypertrophy and tissue infiltration are characteristic of this
class.
o M5a – AML monoblastic
� Diagnostic criteria (in the bone marrow): more than 80% of non-
erythroid cells in the bone marrow belong to the monocytic lineage;
more than 80% are monoblasts.
� Gum hypertrophy and tissue infiltration are characteristic of this
class.
o M5b – AML monocytic
� Diagnostic criteria (in the bone marrow): more than 80% of non-
erythroid cells in the bone marrow belong to the monocytic lineage;
less than 80% are monoblasts; monocytes and promonocytes
comprise more than 20%.
� Gum hypertrophy and tissue infiltration are characteristic of this
class.
o M6 – AML erythroleukemia
� Diagnostic criteria (in the bone marrow): more than 50% of
nucleated cells in the bone marrow are early erythroid precursors;
more than 20% of non-erythroid cells in the bone marrow are
myeloblasts; PAS+ erythroids.
o M7 – AML megakaryoblastic
� Diagnostic criteria (in the bone marrow): more than 20% of non-
erythroid cells in the bone marrow are blasts showing
megakaryocytic differentiation ("budding" cytoplasm).
Prognosis in AML:
o Age of onset below 2 years and above 60 years is not favorable;
o Pathomechanisms involving deletions in 5, 7 chromosomes, t(9:22) and
t(11q23) are nor favorable.
� However, t(8:21) in M2 and inversion in chromosome 16 in M4 are
favorable.
� t(15:17) in M3 is intermediately unfavorable.
CML It is most common between 40-60 years of age, and more males are affected.
There are 3 phases to the disease:
o The chronic phase: 1/3 of cases are asymptomatic, while those who are have
general symptoms such as anemia, fatigue, dyspnea, tachycardia,
hepatosplenomegaly, abdominal discomfort, weight loss, night sweats and
bleedings.
o The accelerated phase: in this phase, patients exhibit enhanced splenomegaly,
subfever (or fever), there are 5-20% blasts in the bone marrow and in the
peripheral blood, thrombocytopenia or thrombocytosis that is unresponsive to
treatment, an increase in the WBC count, and other genetic alterations that
were not present at time of diagnosis.
o The blastic phase (the crisis phase): in this phase there are more than 20%
blasts in the bone marrow and in the peripheral blood. 1/3 of cases will
transform to ALL and 2/3 to AML.
CML should be differentially diagnosed from leukemoid reaction: in leukemoid
reactions there is a dramatic increase in granulocyte count due to inflammatory
processes going on. Therefore, GAPA score is positive in these reactions
(Granulocyte Alkaline Phosphatase Activity on a 0-4 scale).
Pathogenesis: t(9:22) � BCR-ABL forming mutated tyrosine kinase; 95% of cases
show this Philadelphia chromosome.
Avi Sayag Clinical Biochemistry
Lab:
o Blood count: anemia; thrombocytosis; leukocytosis
o Bone marrow: hypercellular (myeloids > erythroids); eosinophils and
basophils; megakaryocytes are smaller
o Cytochemistry: decreases GAPA
o Cytogenetics: Ph. Chromosome
Therapy:
o Drugs (cytostatics)
o IFN-α (arrests cell cycle)
o TK inhibitors (imatinib)
o BM transplantation (allo).
Characterization of leukemic cells by cytochemistry and
immunophenotyping (practical topic 8) Cytochemical reactions
For the identification of leukemic cells and subtype the evaluation of the intracellular material
is needed (enzymes, stored substances), since undifferentiated cells display a lot of
similarities.
1. Myeloperoxidase (MPO): used to differentiate AML from ALL. Positive reaction:
grayish-black.
2. Sudan black: detects the phospholipids in leukocyte granules. Positive reaction: black.
3. Non-specific esterase: the most abundant activity is detected in monocytes and
promyelocytes. Positive reaction: light brown.
4. PAS: detects the glycogen stored in the cytoplasm. Positive reaction: red.
5. Lysozyme: the enzyme dissolves the bacterial cell wall. Monoblasts and immature
monocytes are positive.
6. Prussian blue: detects iron depletion. Positive in ring sideroblasts. Positive reaction:
blue.
7. GAPA: granulocyte alkaline phosphatase converts α-naphtyl-phosphate into a
brownish precipitate in alkaline pH. The phosphatase reaction is low or absent in
CGL, but its activity is enhanced in myeloproliferative disorders and in leukemoid
reactions.
8. Acid phosphatase: positive reaction: red. Positive in MM and HCL.
Sudan black: positive in – M1, M2, M3, M4. M5: 0/+, all the rest: 0.
MPO: positive in M3, M4. All the rest: 0/+
NSE: positive in M3, M4, M5. ALL: 0, all the rest: 0/+
PAS: positive in M6, ALL, all the rest: 0/+.
Acid phosphatase: positive in MM and HCL (TRAP+).
Type MPO Sudan PAS NSE Lysozyme AP
AML- M1,
M2
0/+ ++ 0/+ 0/+ 0
M3 ++/+++ ++ 0/+ +++ 0
M4 + ++ 0/+ ++ 0/+
M5 0/+ 0/+ 0/+ +++ ++
M6 0/+ 0 + 0/+ 0
M7 0/+ 0 0/+ 0/+ 0
ALL 0 0 + 0 0
MM +++
HCL ++
(TRAP)
Immunophenotyping
Avi Sayag Clinical Biochemistry
This method is based on the detection of cell surface markers and intracytoplasmic markers. It
is based on the identification of specific CD markers on pathological cells by fluorophor
labeled monoclonal Abs. Since these antigens are already present on immature cells, the
method allows the analysis of undifferentiated cells and the determination of subgroups of
leukemias. The primary diagnostic goal in acute leukemias is the characterization of the blast
cells and the detection of abnormal marker expressions.
The most important CDs characteristic for cell lines are as follows:
B-cells:
CD19, CD20, CD22, HLA-DR
CD10: cALLA (common acute lymphoid leukemia antigen) can be found on B cell
precursors, it is absent on B cells in the peripheral blood of healthy individuals.
T-cells:
CD3, CD5, CD7, CD4, CD8
Myeloid cells:
CD13, CD33, CD15, MPO
Monocytes: CD14, HLA-DR
Megakaryocytes: CD41, CD42, CD61
Stem cell marker:
CD34: not detectable in the peripheral blood and less than 5% in the bone marrow of healthy
individuals.
Avi Sayag Clinical Biochemistry
Topic 15 Laboratory diagnostics of quantitative platelet disorders
Platelet disorders
Qualitative Quantitative
Platelet function disorders
(topic 19)
Thrombocytopenia :
Reduced production
Increased destruction
Loss from the body
Abnormal
distribution/increased
trapping in the spleen
Thrombocytosis
Thrombocytopenia
Thrombocytopenia is characterized by spontaneous bleeding, a prolonged bleeding time, and
a normal PT and APTT.
Normal platelet count – 150-400 G/L
Thrombocytopenia – less than 100 G/L
Thrombocytopenia with tendency to bleed – less than 50 G/L
Thrombocytopenia with spontaneous bleeding – less than 10 G/L (in some sources – less than
20 G/L).
Larger hemorrhages into the central nervous system are a major hazard in patients with
markedly depressed platelet count.
In most cases in which the cause is accelerated destruction, the bone marrow reveals a
compensatory increase in the number of megakaryocytes. Hence, bone marrow examination
can help to distinguish the two major categories of thrombocytopenia. It is also worth
emphasizing that thrombocytopenia is one of the most common hematologic manifestations
of AIDS.
1. Decreased production
Congenital Neonatal Acquired
May-Hegglin anomally: a
genetic disorder of platelets
that causes them to be
abnormally large. The cause
is a mutation in the gene of
non-muscle myosin heavy
chain IIA. The pathogenesis
is unknown. It is
characterized by hypoplasia
of megakeryocytes, Dohle
bodies (small inclusions in
PMNs) and giant platelets.
Typical in newborns
infected with rubella, and in
babies of mothers who take
thiazides or tolbutamine.
As part of a general bone
marrow depression with
selective megakaryocyte
depression. Causes include:
1. Chemotherapy/radiation
2. Alcohol (suppresses
production of megak.)
3. Cytotoxic drugs
4. Aplastic anemia
5. Pernicious anemia
6. Infection with CMV,
EBV, VZV, rubella,
measles vaccine
7. Infiltration of BM with
tumor cells.
8. Myeloid dysplasia with
primary myelosclerosis.
Avi Sayag Clinical Biochemistry
2. Increased destruction
This can be due to 2 reasons:
1. Destruction by immune mechanism: a. Chronic idiopathic thrombocytopenia purpura (ITP)
i. The most common form in women between 15-50 years old that
suffer from petechial hemorrhages and are easily bruised.
ii. Can be secondary to SLE, HIV, CLL, etc (but then it is not
idiopathic).
iii. Platelets can be sensitized by Ab directed against GpIIb-IIIa
iv. Premature removal by the RES � life-span is reduced to few hours.
v. The platelet count is 10-50 G/L.
vi. Platelets are large.
vii. Lab detection of Ab in the serum or on platelets.
viii. There is normal to increased number of megakaryocytes.
b. Acute ITP
i. Most common in children
ii. 75 % of them get it after vaccination or infection.
iii. There is a spountandeous remission.
iv. 5-10% become chronic.
c. Drug induced thrombocytopenia
i. Abs against the drug and the carrier protein are produced� the
circulating immune complexes are adsorbed into the platelets�
platelets are removed by the RES or lysed by complement.
ii. Quinine, quinidine, heparin
iii. Count: less then 10 G/L.
iv. Normal to increased number of megakaryocytes.
d. Neonatal alloimmune: the mother becomes sensitized against platelet-specific
antigen of the fetus.
e. Neonatal autoimmune: ITP develops in pregnant women; Ab crosses the
placenta.
f. Post-transfusion thrombocytopenia: Anti-Pl Abs develop following
transfusion.
g. Secondary autoimmune: in CLL, SLE, etc.
2. Destruction by non-immune mechanism:
a. In pregnancy of pre-eclampsia (ischemia � vascular injury � activation of
DIC)
b. Thrombotic thrombocytopenia purpura (TTP)
i. Thrombi in capillaries and arterioles � RBCs and PLTs are
mechanically destroyed by the thrombi � intravascular hemolysis �
reticulocytosis
ii. Women between 20-50 are usually affected.
iii. Severe organ damage.
iv. Count less then 20 G/L.
v. Pathomechanism:
1. Large vWF – deficiency in a MMP called ADAMTS13
which degrades very-high-molecular-weight multimer of
vWF allows multimers of vWF to accumulate in plasma.
2. Endothelial damage.
3. Defective prostacyclin production.
c. In hemolytic uremic syndrome (HUS)
i. Hemolytic anemia (decreased Hb, increased ret., schistocytes)
ii. Renal failure (elevated urea, creatinine, RBCs, proteins)
iii. Thrombocytopenia
iv. Disease of childhood (6 months – 4 years).
Avi Sayag Clinical Biochemistry
v. Often follows acute viral infection or E. coli (verotoxin damages the
endothelium � bleeding, activation and consumption of PLTs)
vi. Resembles TTP with no neurological symptoms.
vii. High mortality rate.
d. DIC
3. Loss from the body
4. Disorders related to distribution/dilution
i. Increased pooling in splenomegaly.
ii. Hypothermia
iii. Extracorporal circulation
iv. Massive blood transfusion
Thrombocytosis 1. Reactive thrombocytosis: blood loss, surgery, post-splenectomy, iron deficiency
due to blood loss, inflammation, stress, exercise.
2. Myeloproliferative disorders: polycytemia vera, myelofibrosis with myeloid
metaplasia.
3. Essential thrombocytemia: megakaryocyte proliferation with high PLT count
(500-2000G/L); recurrent hemorrhage and thrombosis;
abnormally large PLT and megakaryocyte fragments in blood
film; may transform to polycytemia vera, myelofibrosis or
acute leukemia, but may remain stationary for many years.
Avi Sayag Clinical Biochemistry
Topic 16 Inheritance of ABO blood group system and its clinical significance The ABO antigens are added stepwise to proteins or to lipids on the erythrocytes and they
appear on day 40 of gestation. The substrate molecule is L-fucose (the H antigen).
The A antigen is N-acetyl-galactosamine (GalNAc);
The B antigen is galactose (Gal);
A and B genes code for transferase enzymes, such that transferase A is alpha 1-3-N-
acetylgalactosaminyltransferase and transferase B is alpha 1-3-galactosyltransferase.
The antigens are found not only on RBCs, but also on most body cells (leukocytes, platelets,
etc.) The gene coding for blood type lies on chromosome 9q34. However, other separate
genes on chromosome 11 and 19 actually interact with the blood type gene, determining our
ability to secrete our ABO blood type antigens into our body fluids and secretions. This is
called the secretor gene, and by testing for this gene we can determine whether we are
secretors or non-secretors. In the genetics of the secretor system two options exist. A person
can be either a secretor (Se) or a non-secretor (se). This is completely independent of whether
one's blood type is A, B, AB, or O. Thus a person could be an A secretor or an A non-
secretor, a B secretor or a B non-secretor, etc.
Secretors: in a simplified sense, a secretor is defined as a person who secretes one's blood
type antigens into body fluids and secretions like the saliva, the mucus in the digestive tract
and respiratory cavities, etc.
Non-Secretors: non-secretors on the other hand put little to none of their blood type into these
same fluids.
As a general rule, in the US about 15-20% of the population are non-secretors with the
remaining 80-85% being secretors. Aside from the physical implications centering around
whether you have blood type antigens in your body fluids or not, the secretor genetics have
additional significance through the effects of gene linkage: in other words, the outcome of
your secretor genetics ‘links’ to other seemingly unrelated genes and influences their
function. Your secretor status drastically alters the carbohydrates present in your body fluids
and secretions in addition to several important aspects of your metabolism and resistance.
These factors include the activity of an enzyme called intestinal alkaline phosphatase, the
overall composition of bacteria in your intestinal ecosystem, your propensities toward blood
clotting, your level of carbohydrate tolerance, and your resistance to certain parasites and
yeast (based on these features, new diet regimes have been proposed tailored to the blood type
of those who are desperately willing to try anything to lose weight).
The natural ABO antibodies are IgM, which are produced after the first few months of life
(after the 4th month). These antibodies work in "cold" temperatures (room temperature) and
may fade in old age. Other irregular Ab are produced after immunization (incompatible
Avi Sayag Clinical Biochemistry
transfusion or pregnancy). Most complications after such incompatible transfusions are
caused by these Ab (mostly IgG – warm Ab that cross the placenta).
Antigens and antibodies:
Blood group – Ag on RBC – Ab in serum – Genotype
A – A – anti B – AA or Ao
B – B – anti A – BB or BO
AB – A and B – none – AB
O – none (H) – anti A and anti B - OO
Subtype A
The A blood type contains about 20 subgroups, of which A1 and A2 are the most common.
80% of which are A1. These cells carry about 1 million antigens on a single RBC. The A2
cells, however, carry only 200,000 antigens on a single RBC. A1 and A2 differ in such a way
that type A2 people can produce anti-A1 antibodies. As mentioned, there are weaker
subgroups in the A subtype (about 18) due to mutations in the A gene that may lead to
dysfunctional transferase (3-α-N-acetylgalactosaminyl-transferase).
The clinical significance of the ABO group manifest in mismatched transfusions, although
rare. However, should they occur, they may be life-threatening, as they may lead to
intravascular hemolysis. It is more severe in group O patients, as they have very reactive anti-
A and anti-B antibodies.
Universal donors and recipients:
O group carries no A or B antigens.
The packed and processed units have little or no antibody content.
The AB group has no antibodies whatsoever, and therefore cannot lyse
any transfused cells. Other antibodies, however, may be present!
Prevalence:
Bororo (brazil) and Peru (Indian) – 100% type O
North American Indians – 82% type A
Type O is the most prevalent in all parts of the world, except N. American
Indians.
The Bombay phenotype
Individuals with the rare Bombay phenotype (hh) do not express antigen H on their red blood
cells. As H antigen serves as precursor for producing A and B antigens, the absence of H
antigen means the individuals do not have A or B antigens as well (similar to O blood group).
However, unlike O group H antigen is absent, hence the individuals produce isoantibodies to
antigen H as well as to both A and B antigens. In case they receive blood from O blood group,
the anti-H antibodies will bind to H antigen on RBC of donor blood and destroy the RBCs by
complement-mediated lysis. Therefore, Bombay phenotype can receive blood only from other
hh donors (although they can donate as though they were type O). It should be mentioned that
the transferase enzyme IS produced in this phenotype.
Summary and presentation of the topic: Present the blood types and the biochemistry of the various types + prevalence
Explain the difference between them in terms of chemistry, Ag, Ab and transferases
Chromosome 9q34 and interactions with chromosomes 11 and 19 – Se and se (secretor and
non-secretor). The significance of being an secretor or a non-secretor.
Subtype A – distribution of A1 and A2
Clinical significance: blood transfusion – universal donors and recipients
Bombay phenotype
A B
AB
O
Avi Sayag Clinical Biochemistry
Topic 17 Inheritance and clinical significance of Rh blood group system The Rh blood group system is the second most important system and is the most complex. It
is important because it is associated with hemolytic transfusion reactions and with
development of severe hemolytic disease of the newborn (HDN). Rh antigens are proteolipids
and lack carbohydrate. Rh antigens are present on RBCs only, and there are 20,000 antigen
sites on each erythrocyte. They are relatively small (32KDa). The inheritance of Rh antigens
is determined by a complex of 2 closely linked genes on chromosome 1. One gene codes for
the protein carrying D expression; the other codes for the proteins carrying C or c and E or e
expression. Rh-positive individuals have both a D and a CE gene while Rh-negative
individuals have only a CE gene. Depending on which genes are present on a chromosome, 8
common antigen combinations or haplotypes are possible: Dce, DCe, DcE, DCE, dce, dCe,
dcE, dCE. Common phenotypes and genotypes are given below.
D antigen is the most important Rh antigen. Presence of a single D antigen confers upon an
individual the designation Rh-positive; its absence means that the person is Rh-negative.
Eighty five percent of Caucasians, 92% of African Americans and 99% of Asian Americans
are Rh positive. The letter d is commonly used to indicate the lack of D in Rh-negative
individuals, but neither d antigen nor anti-d has been detected. The Fisher-Race nomenclature
has been more widely adopted over the more complex Wiener nomenclature for Rh antigens.
However, an abbreviated version of the Wiener system is useful to describe Rh genotypes.
Wiener is covenient because it uses a single letter, R or r, with superscripts to name a 3 locus
haplotype. It is possible to translate from one nomenclature to the other by remembering a
few rules:
In the Wiener system, D is indicated by an uppercase R and the absence of D is indicated by
lower case r.
In the Wiener system, superscripts or numbers are used to indicate which Cc or Ee genes are
present. Numbers are used with R and primes are used with r.
In the Fisher Race system, loci are lined up in the order Dd, Cc, Ee (e.g. DCE).
In the Wiener system, the Dd position is numbered 0, Cc position is 1 and Ee position is 2.
The Wiener superscript of 0, 1, 2 indicates which of the Fisher Race loci is in its uppercase
form (D, C, or E). For example, 1 or prime indicates that C is capitalized, while a 2 or double
prime indicates that E is capitalized.
Rh Nomenclature and Haplotype Frequencies
Fisher Race Wiener
Dce Ro
DCe R1
DcE R2
DCE Rz
dce r
dCe r'
dcE r"
dCE ry
The most common haplotye in Caucasians and Asian Americans is DCe (R1), while the most
common phenotype in African Americans is Dce (Ro). Red blood cells that fail to react with
all Rh antibodies are called Rh null. An individual's genotype can only be determined with
certainty by performing DNA analysis and family studies.
Unlike the ABO naturally occurring antibodies, Rh antibodies are produced in response to an
incompatible transfusion or pregnancy. The D antigen is the most immunogenic of the Rh
antigens, causing immunization at least 80% of the time when a D-negative person receives a
single unit of D-positive blood. Anti-c is the second most important Rh antibody. Although
anti-E is more common than anti-c, anti-E is frequently a naturally occurring antibody. Anti-c
and anti-e only occur after an antigenic stimulus.
Avi Sayag Clinical Biochemistry
Weak D (Du): some individuals have a weak expression of the D antigen for either which of 3
reasons:
Individuals who lack part of the D antigen (partial D) have a weak expression on their
RBCs. If they are immunized, they produce antibodies to the portion they lack.
The D gene encodes all epitopes of the D antigen, but the antigen number on RBCs is
less than normal.
In some cases a C transposition to a D gene occurs (Dce/Ce or DCe/Ce) which
weakens the expression of D.
Significance:
80% of Rh(D)- persons exposed to Rh(D)+ blood will develop anti-D antibodies.
Anti-D antibodies can also be stimulated by pregnancy with an Rh(D)+ baby.
Sensitization can be prevented by the use of anti-D Ig antenatally and postnatally.
Rh(D)- females of childbearing potential should never be given Rh(D)+ blood.
Antibodies to Rh antigens are primarily IgG antibodies which can cross the placenta. IgG is
too small to make bridges between RBCs, so agglutination doesn't occur in saline. These
antibodies work best in "warm" temperatures.
The D antigen is the most important cause of Hemolytic Disease of the Newborn (HDN).
Other antigens can also cause it (C, c, E, e) as well as other blood groups (rare).
Finally, it should be noted that the inheritance of ABO and Rh are not linked and are inherited
independently.
Summary and presentation of the topic: Rh Ags are proteolipids found only on RBCs, with 20000 Ags on each RBC
Inheritance – 2 genes on chromosome 1 (D and CE) � 8 combinations
Fisher-Race vs. Wiener
Most common DCe (R1) – Caucasians and Asian Americans
Most common Dce (R0) – African Americans
Anti-Rh Abs are produced in response to transfusion, pregnancy, etc.
D Ag is the most immunogenic
c Ag is the second most immunogenic, and NOT naturally occurring
Anti-e is not naturally occurring
Anti-E is frequently naturally occurring and more common than anti-c.
3 causes of weak D (Du)
Significance of Rh: transfusion, pregnancy, transfusion to women in childbearing age
Abs are IgG
Rh and ABO are not genetically linked
Avi Sayag Clinical Biochemistry
Determination of ABO and Rh blood group (practical topic 9) ABO determination
Abs in the ABO group are naturally occurring ones: cross-reacting carbohydrate structures on
environmental agents stimulate the thymus-independent production of IgM anti-A and/or anti-
B in individuals who are not tolerant to these antigens. The IgM Abs then directly agglutinate
the appropriate antigen-positive RBCs, preferentially at room temperature.
The Landsteiner rule: sera of healthy adults contain ABO Ab that reacts with the ABO Ags
missing from the person's RBCs, but it must not contain any Ab that reacts with the Ags on
the person's RBCs.
Blood group Ag on RBC Ab in serum Genotype
A A Anti-B AA or AO
B B Anti-A BB or BO
AB A and B None AB
O None Anti-A and anti-B OO
Bedside blood group determination can be one-sided or two-sided.
1. One-sided This examination is based on agglutination by addition of anti-A, anti-B and anti-AB antisera
at room temperature and in saline medium. We put one drop of each anti serum in its place on
the right around 2 cm apart, and then we put one drop of the patient's serum. Then, we put one
drop of the patient's blood and saline opposite to each antiserum and serum of the patient. We
mix them well using the corner of slide, caring not to contaminate one with the other. We
allow the slide to stand for 30 seconds at room temperature and then tilt the slide backward
and forward by 30º. In the one-sided method, we determine the presence/absence of antigens
on RBCs and only that! Hence, one-sided method.
2. Two-sided In this method, we determine both the Ags on the RBCs and the Abs in the serum of the
patient (hence "two-sided").
We divide a tile into 2 parts by an imaginary line. We put one drop of anti-A, anti-B and anti-
AB sera with proper distance from each other on the left side, and mark their places for safety
(see figure on page 55 of the practice book). We then make a 10% suspension of the blood
sample in physiological saline and put one drop opposite each of them. On the right side we
put 4 drops of the requested serum with sufficient space from each other. We put one drop of
known A1, A2, B and O test RBCs opposite the blood sera. We mix them carefully and let
them stand for 10 minutes at room temperature. We again tilt it and read the results visually.
Rh determination The test is performed on a glass slide or white glass bottle filled with hot water and with a
surface temperature of 37-42ºC. We place one drop of anti-D serum, one drop of the
appropriate control reagent and to both drops we add one drop of a well-mixed 50%
suspension of the investigated RBCs in their own serum or plasma. We then mix the RBC
suspension and the reagent, and put it in a wet chamber at 37ºC for 20 minutes. We gently tilt
the slide and observe for agglutination. After 2-3 minutes we record and interpret the results.
We should use Rh+ and Rh- controls with the same method at the same time.
Papain enzyme treatment of RBCs cleaves the sialoglycoproteins from the RBCs, and the net
surface charge decreases. This enhances the agglutination reaction. It has a special importance
in cases when allo-Abs or auto-Abs are bound to the surface of the RBCs as a result of
previous incompatible transfusion.
Rh+: the suspension is agglutinated and the autocontrol is homogenous.
Rh-: if the suspension ad autocontrol are the same and homogenous. In case of an uncertain
result (positive autocontrol, weak reaction, etc.), the sample should be sent to special
departments for further examination.
Avi Sayag Clinical Biochemistry
Topic 18
Coagulopathies, laboratory control of anticoagulant treatment The coagulopathies can be inherited, acquired, iatrogenic or therapeutic.
The coagulopathies can be caused by 3 main mechanisms:
1. Decreased levels or absence of clotting factors: In this category, the decreased levels can be caused by inherited disorders, acquired disorders
(e.g. in liver failure which leads to decreased synthesis of clotting factors as well as in
consumption coagulopathies) and by therapeutic mechanisms (agents leading to
thrombolysis).
2. Synthesis of abnormal clotting factors In this category, the synthesis of abnormal clotting factors can be caused by congenital
defects, by acquired disorders (such as in dysfibrinogemia in liver diseases and in vitamin K
deficiency), by therapeutic agents (e.g. Syncumar, aka Coumarin) and by iatrogenic factors
(such as administration of cephalosporin).
3. Inhibitors of coagulation In this category, the coagulopathies can be caused by neutralizing or non-neutralizing
antibodies that are directed against clotting factors. This condition can be accompanied by
inherited factor deficiency but not necessarily. However, not only antibodies can cause
coagulopathies, but global inhibitors can as well (e.g. heparin).
Bearing in mind the coagulation pathway, 4 main screening tests of blood coagulation should
be mentioned: APTT, PT, TT and bleeding time.
Avi Sayag Clinical Biochemistry
Intrinsic pathway Extrinsic pathway
APTT PT
28-40 seconds + 9 seconds � prolonged
After collecting blood samples in vacu-tubes
with oxalate or citrate to arrest coagulation
by binding calcium, the specimen is then
delivered to the laboratory. In order to
activate the intrinsic pathway, phospholipid,
an activator (such as silica, celite, kaolin,
ellagic acid), and calcium (to reverse the
anticoagulant effect of the oxalate) are mixed
into the plasma sample . The test is termed
"partial" due to the absence of tissue factor in
the reaction mixture. The time is measured
until a thrombus forms. If the clotting does
not occur within 200 seconds, the result is
said to be APTT > 200 sec.
Causes of prolonged APTT:
1. Deficiency or decreased "intrinsic
factors": hemophilia A and B
2. Presence of heparin in the sample
3. Presence of inhibitors directed
against clotting factors or
phospholipids (lupus anticoagulant)
4. Inappropriate ratio of Na-
citrate:blood (citrate in excess)
5. Consumption coagulopathies
When do we use APTT?
1. To monitor unfractionated heparin
therapy (UFH)2
2. Control of fibrinolytic therapy:
before thrombolytic therapy,
screening for hemorrhagic diathesis
should be performed.
3. Diagnosis of DIC
4. Diagnosis of thrombophilia
5. In liver diseases
6. If a patient with severe bleeding is
treated only with RBC concentrate,
and does not receive the plasma
clotting factors.
8-12 seconds + 4 seconds � prolonged
The prothrombin time is most commonly
measured using blood plasma. Blood is
drawn into a test tube containing liquid
citrate. The blood is mixed, then centrifuged
to separate blood cells from plasma. An
excess of calcium is added (thereby reversing
the effects of citrate), which enables the
blood to clot again. Tissue factor (also known
as factor III or thromboplastin) is added, and
the time the sample takes to clot is measured
optically or using the KC-1. TF is both the
receptor and activator of FVII. If the clotting
does not occur within 100 seconds, the result
is said to be PT > 100 sec. Factors II, V, VII,
IX and X are vit.-K dependent, therefore the
PT test is good to monitor coumarin therapy.
If so, the result should be given as
INR =
ISI
contorl
pt
PT
PT
. ISI is the International
Sensitivity Index. The smaller the ISI is, the
more sensitive the reagent is. The INR should
be kept between 2-3. INR of patients with
prosthetic heart valve should be between 2.5-
3.5. PT determination and INR should be
performed every 2 weeks during the first 6
weeks of therapy, and then, if the INR is
stable, once in a month. If INR is > 5, there is
a risk of spontaneous bleeding.
Causes of prolonged PT:
1. Coumarin therapy
2. Hereditary/acquired absence of
"extrinsic" factors/abnormal
synthesis
3. Inappropriate ratio of Na-
citrate:blood (citrate in excess)
4. Fibrinolytic therapy
When do we use PT?
1. To monitor coumarin therapy
2. Before thrombolytic therapy:
screening for hemorrhagic diathesis
should be performed.
3. Diagnosis of DIC
4. Liver disease
2
APTT taken after: 0.5-1 hr 2-3 hr 4-6 hr
UFH continuous X
I.V bolus heparin X
Subcutaneous heparin X
Avi Sayag Clinical Biochemistry
Thrombin time (TT): TT evaluates the last phase of the clotting cascade and
represents the time (in seconds) that elapses between the addition of thrombin
(usually bovine thrombin) and the onset of clotting. The values of both the
control's and the patient's plasma are reported. The reference interval is 14-22
seconds. TT is considered prolonged if the patient's TT exceeds the control
value by 8 seconds. If clotting does not occur within 100 seconds, the result is
given as TT > 100 seconds. Causes of prolonged TT:
i. Heparin treatment
ii. Pathologic levels of fibrinogen/fibrin split products (acute DIC,
primary hyperfibrinolysis, dissolution of a thrombus) that inhibit the
thrombin activity and fibrin polymerization
iii. Severe hypofibrinogenemia or afibrinogenemia
iv. Dysfibrinogenemia
v. Certain hepatic diseases due to hypo- or dysfibrinogenemia.
vi. If a patient with severe bleeding is treated only with RBC
concentrate, and does not receive the plasma clotting factors.
Bleeding time: apply 40 mmHg tourniquet pressure to the upper arm and
maintain it during the entire process. Wipe the inner surface of the forearm
with ethanol and, by avoiding larger visible veins, cut the skin using a special
disposable device. The device is attached to the forearm without pressure,
and pushing a trigger two 5-mm blades are released that make two 1-mm-
deep cuts. Dry the blood with a sterile blotting paper every 30 seconds
without touching the wound. The time when the last drop of blood is visible
on the blotting paper is the bleeding time. The reference interval is 2.5-9.5
minutes. If bleeding does not stop within 20 minutes, the result is given as
bleeding time > 20 minutes. Unfortunately, in most labs and hospitals the
bleeding time is determined by pricking the fingertip. This is a completely
unreliable method of no clinical significance, for the bleeding time in this
case depends on the thickness of the skin of the fingertip and on the depth of
the pricking, rather than on platelet function. Bleeding time is the most
important screening test of platelet function, as bleeding of a small wound
stops when platelets adhere to the injured vessel wall forming a primary
platelet plug. Bleeding time is normal in coagulopathies with the exception of
afibrinogenemia (i.e. it is normal in hemophilias!) Diseases that cause
prolonged bleeding time include:
i. Thrombocytopenia
ii. DIC
iii. Aspirin and other cyclooxygenase inhibitors can prolong bleeding
time significantly.
iv. While warfarin and heparin have their major effects on coagulation
factors, an increased bleeding time is sometimes seen with use of
these medications as well.
v. People with von Willebrand disease usually experience increased
bleeding time, as von Willebrand factor is a platelet agglutination
protein, but this is not considered an effective diagnostic test for this
condition.
vi. It is also prolonged in hypofibrinogenemia.
Several coagulopathies should be mentioned:
1. Hemophilia A
2. Hemophilia B
3. Afibrinogenemia and dysfibrinogenemia
4. Other factor deficiencies
5. Acquired coagulopathies
Avi Sayag Clinical Biochemistry
Hemophilia A This bleeding disorder is caused by a mutation in FVIII gene, which is located on
chromosome X (Xq28) and is the most common hemophilia.
FVIII is a big glycoprotein synthesized in the liver, and perhaps in endothelial cells.
It is a cofactor of FX activation (along with FIX). Thrombin is required to activate FVIII.
The deficiency in FVIII can be inherited (a mutated FVIII gene) or acquired (antibodies
directed against FVIII).
Symptoms: hemophilia leads to a severely increased risk of bleeding from common injuries.
The first symptoms that should suspect of hemophilia are bleeding at labor and during
delivery, circumcision, vaccination and onset of walking. The sites of bleeding include the GI
and the brain (less common) and the joints and muscles (most common). Hemarthrosis occurs
primarily in the knee and elbow joints. As RBCs are lysed, iron is deposited in the synovium.
This leads to chronic synovitis, synovial fibrosis, joint stiffness, limited motion and pain.
Diagnosis: APTT is elevated, PT is normal, TT is normal and bleeding time is normal. Factor
assay can also be performed.
Hemophilia A should be differentially diagnosed from:
1. von Willenbrand disease;
2. Combined FVIII and FV deficiency; and
3. Consumption coagulopathies.
4. Hemophilia B
5. Vitamin K deficiency
6. Vitamin K antagonist drugs.
Treatment: administration of FVIII.
Hemophilia B Hemophilia B is caused by a mutation in the FIX gene located on the X chromosome (Xq27).
FIX is synthesized in the liver and its function depends on vitamin K. Its cleavage is carried
via FXI and FVII. In the presence of FVIII and Ca+2
it cleaves FX.
Symptoms: Factor IX deficiency leads to an increased propensity for hemorrhage. This is in
response to mild trauma or even spontaneously, such as in joints (haemarthrosis) or muscles.
Diagnosis: APTT is elevated, with normal PT, normal TT and normal bleeding time. Factor
assay can also be carries out.
Hemophilia B should be differentially diagnosed from:
1. Liver disease
2. Vitamin K deficiency
3. Vitamin K antagonist drugs. 4. von Willebrand disease
5. Afibrinogenemia/dysfibrinogenemia
6. Fibrinolytic diseases
7. Hemophilia A
Both hemophila A and B can be classified as severe (if the activity/level of the defective
factor is less than 1% - leading to spontaneous bleeding); moderately severe (1-5% - bleeding
due to minor trauma or surgery); or mild (5-30% - bleeding due to major trauma and surgery).
Afibrinogenemia and Dysfibrinogenemia In this autosomal recessive disorder there is failure of the fibrinogen chains to assemble, or
fibrinogen fails to be secreted (in afibrinogenemia there is no fibrin at all, whereas in
dysfibrinogenemia the levels are low).
Both are characterized by severe bleeding diathesis and platelet function disorder, as well as
by spontaneous abortion, as there is a need for more than 1g/L of fibrinogen to maintain
pregnancy.
Diagnosis: APTT, PT, Fibrinogen levels, TT, and Bleeding time are all abnormal.
Avi Sayag Clinical Biochemistry
Other factor deficiencies:
FXI deficiency: APTT is elevated (only)
Deficiency in FX, FVII, FV, FV-FVIII (combined), prothrombin, combined FII-FVII-
FIX-FX. In these deficiencies the APTT and the PT are elevated (only).
FXIII deficiency
α2 plasmin inhibitor deficiency.
Acquired coagulopathies 1. Neutralizing and non-neutralizing antibodies which inhibit the function of clotting
factors and accelerate their clearance. These antibodies can occur spontaneously or
after replacement therapy of a certain factor that is absent in the patient's blood.
2. Non-antibody inhibitors (such as heparin)
3. Liver diseases which lead to a decreased factor synthesis
4. Vitamin K deficiency due to malabsorption or elimination of the intestinal bacterial
flora.
5. Vitamin K antagonists: coumarin (a precursor of warfarin (Coumadin)),
cephalosporin. The APTT and PT will be elevated.
For APTT, PT and TT:
1. Blood is collected into a vacutainer tube containing 0.105M Na-citrate as an anticoagulant.
Citrate prevents coagulation by forming complex with calcium that is required for some steps
in the clotting process. Clotting test should be performed within 2 hours following blood
collection.
2. To obtain platelet poor plasma, samples should be centrifuged at 3000/min (1500g) for 15
minutes at room temperature. After centrifugation, the upper 3/4 of platelet poor plasma
should be transferred into plastic tubes.
3. Magnetic sensor coagulometer (we use the KC-1 in our lab): a cup with a steel ball is
rotated in a heated compartment of the coagulometer and the ball is held on its place by a
magnet. The plasma and reagents are pipetted into this cup. The timer of the instrument is
started by the addition of the starting reagent with an automatic pipette connected to the
instrument. The clot formed during coagulation pulls the steel ball out of place, and this
displacement detected by a magnetic sensor. The timer then stops, and the clotting time is
recorded.
For fibrin monomer test and D-dimer test, see Topic 22, as part of DIC panel.
Avi Sayag Clinical Biochemistry
Topic 19 Platelet function disorders Normal blood flow is laminar, such that platelets flow centrally in the vessel lumen, separated
from the endothelium by a slower moving clear zone of plasma.
vWF gene is located on chromosome 22. The product (vWF) is synthesized and stored in
endothelial cells (Weibel Palade bodies), in megakaryocytes (synthesis) and platelets
(storages in α-granules) and is also found in the ECM (subendothelium) with collagen fibers.
The half-life of vWF is 12-20 hours and ADAMTS13 inhibits vWF by cleaving it.
The functions of vWF are:
Supports platelets adhesion to collagen and other subendothelial structures,
especially at high-shear rates. The platelets bind via the GpIb receptors to the vWF
located in the subendothelium and exposed at endothelial injuries.
Supports platelets aggregation at high-shear rates.
Associates with FVIII in the circulation and protects it from degradation (it also
promotes the secretion of FVIII).
Von Willebrand Disease An autosomally (dominant mostly) inherited disorder of platelet adhesion, with reduced
amount/function of vWF. The disease is marked by spontaneous bleeding from mucous
membranes, excessive bleeding from wounds, menorrhagia, and a prolonged bleeding time in
the presence of a normal platelet count. Individuals with von Willebrand disease have a
compound defect involving platelet function and the coagulation pathway. The amounts of
factor VIII are only moderately depressed, and it is the defect in platelet function that
dominates the clinical picture.
Lab:
1. Anamnesis with focus on bleeding time (should be prolonged in vWD)
2. PFA-100 (Platelet Function Analyzer): The PFA-100 test is a new in vitro test of
platelet function. The test measures the time taken for blood, drawn through a fine
capillary, to block a membrane coated with collagen and epinephrine (CEPI) or
collagen and ADP (CADP). This is referred to as the Closure Time (CT) and is
measured in seconds. The test is therefore a combined measure of platelet adhesion
and aggregation. If CEPI is <180 seconds - normal platelet function.
A normal CEPI value excludes the presence of a significant platelet function defect.
CEPI >180 s; CADP <116 s (normal value) - "Aspirin Effect". If this result is normal,
the most likely explanation is that the patient has ingested aspirin or similar
medication. CEPI >180 s; CADP >116 seconds - abnormal platelet function.
However, thrombocytopenia (<100K) and anemia (Hct <0.28) should first be
excluded.
3. Platelet count (normal values are 150-400 G/L)
4. APTT
5. RIPA (Ristocetin Induced Platelet Aggregation): Ristocetin induces the binding of
vWF to platelets, and induces their aggregation.
6. vWF:Ag – this test measures the antigenic characteristics of vWF (measured by
immunoassay).
7. vWF:RCo (Ristocetin cofactor) – this test measures the binding capacity of plasma
vWF to platelets in the presence of Ristocetin (it is measured by agglutination of
isolated platelets).
8. vWF:CB – this test measures the binding capacity of plasma vWF to collagen.
9. vWF:FVIII – this test measures the binding capacity of vWF to FVIII
10. Examining the structure of the multimeric vWF (by SDS-agarose elctrophoresis)
11. vWF gene analysis (chromosome 22)
Avi Sayag Clinical Biochemistry
Types of vWF disease:
Type I: partial quantitative deficiency in vWF. Autosomal dominant (AD). The
multimer distribution is normal, but there is a reduced amount of vWF:Ag and FVIII.
In type IIA, the high-molecular-weight multimers (the largest) are not synthesized,
leading to a true deficiency. Thus, the activity of vWF:Rco is reduced. AD.
In type IIB, functionally abnormal high-molecular-weight multimers are synthesized
but are rapidly removed from the circulation. These high-molecular-weight
multimers cause spontaneous platelet aggregation (a situation reminiscent of the
very-high-molecular-weight multimer aggregates that are seen in TTP), and indeed
some individuals with type IIB von Willebrand disease have chronic mild
thrombocytopenia that is presumably caused by platelet consumption. AD. The
vWF:Rco is normal but the RIPA may be enhanced.
Type IIM: vWF defect associated with specific defects in platelet/vWF interaction,
but with a normal range of multimers. AD.
Type IIN: vWF defect resulting from defective vWF binding to FVIII. This leads to
low levels of FVIII. AR.
Type III: severe quantitative disorder resulting from a huge reduction in or absence
of vWF in the plasma and in the platelets (less than 5%). FVIII will also be reduced.
AR.
Individuals with blood group O have lower levels of vWF than those with blood group A, B
and AB (more than 65% of patients with vWD have blood group O).
In some cases, the disease can be acquired due to:
1. Reduced rate of synthesis;
2. Increased rate of clearance from the circulation (might result in abnormal multimeric
structure);
3. Antibodies directed against vWF (that might be inhibitory or non-inhibitory)
The acquired form may accompany several diseases:
1. Hypothyrodism (like in type I vWD);
2. Wilms tumors (tumors of the kidneys);
3. Congenital cardiovascular defects (in case of aortic valve stenosis it may lead to GI
bleeding – Heyde's syndrome);
4. Absorption on certain tumors;
5. Autoimmune vWD.
Avi Sayag Clinical Biochemistry
Topic 20 Inherited thrombophilias Thrombophilia is the propensity to develop thrombosis (blood clots) due to an abnormality in
the system of coagulation. Hereditary defects in one or more of the clotting factors can cause
the formation of potentially dangerous thrombosis.
The most common symptoms of thrombophilia are
DVT at a relatively young age (<55 years of age)
Pulmonary embolism
Thrombosis at an unusual location (not in the lower limbs)
Recurrent thrombosis
Familial occurrence (inherited thrombophilias)
Causes of inherited thrombophilias:
1. Antithrombin III deficiency
2. Protein C deficiency
3. Protein S deficiency
4. Prothrombin mutation (20210A allele)
5. Elevated factor VIII
6. Activated protein C resistance (FV Leiden) – the most common cause
7. Dysfibrinogenemia
Antithrombin III deficiency
The physiological target proteases of antithrombin are those of the intrinsic pathway: Xa, IXa,
XIa, XIIa and thrombin, and also VIIa from the extrinsic pathway.
The incidence of inherited antithrombin deficiency has been estimated to be between 1:2000
and 1:5000 in the normal population.
Maintenance of an adequate level of antithrombin activity, which is at least 70% that of a
normal functional level, is essential to ensure effective inhibition of blood coagulation
proteases. Typically as a result of type I or type II antithrombin deficiency, functional
antithrombin levels are reduced to below 50% of normal.
Type I antithrombin deficiency is characterized by a decrease in both antithrombin activity
and antithrombin concentration in the blood of affected individuals.
Most cases of type I deficiency are due to point mutations, deletions or minor insertions
within the antithrombin gene.
Type II antithrombin deficiency is characterized by normal antithrombin levels but reduced
antithrombin activity in the blood of affected individuals.
Diagnosis: antithrombin III activity should be measured first. If low, then antithrombin
antigen is measured to look for mutations consistent with type II disease.
Protein C deficiency
The prevalence of protein C deficiency has been estimated to be about 0.2% to 0.5% of the
general population (that is, between 1:200 and 1:500). Protein C deficiency is associated with
an increased incidence of venous thromboembolism (relative risk 8-10), whereas no
association with arterial thrombotic disease has been found.
The main function of protein C is its anticoagulant property as an inhibitor of coagulation
factors V and VIII. There are two main types of protein C mutations that lead to protein C
deficiency:
Type I: Quantitative defects of protein C (low production or short protein half life)
Type II: Qualitative defects, in which interaction with other molecules is abnormal. Defects in
interaction with thrombomodulin, phospholipids, factors V/VIII and others have been
described.
The majority of people with protein C deficiency lack only one of the functioning genes, and
are therefore heterozygous.
Protein S deficiency Protein S, a vitamin K-dependent physiological anticoagulant, acts as a nonenzymatic
cofactor to the activated protein C in the proteolytic degradation of factor Va and factor VIIIa.
Avi Sayag Clinical Biochemistry
Decreased (antigen) levels or impaired function (activity) of protein S, leads to decreased
degradation of factor Va and factor VIIIa and an increased propensity to venous thrombosis.
In healthy individuals, approximately 30-40% of total protein S is in the free state. Only free
protein S is capable of acting as a cofactor in the protein C system.
There are three types of hereditary protein S deficiency:
Type I - decreased protein S activity: decreased total protein S (=both bound and free protein
S) levels AND decreased free protein S levels (quantitative defect)
Type II - decreased protein S activity: normal free protein S levels AND decreased total
protein S levels (qualitative defect)
Type III - decreased protein S activity: decreased free protein S levels AND normal total
protein S levels (quantitative defect)
Protein S deficiency can also be acquired due to vitamin K deficiency or treatment with
warfarin, systemic sex hormone therapy and pregnancy, liver disease, and certain chronic
infections (for example HIV). Vitamin K deficiency or treatment with warfarin generally also
impairs the coagulation system itself (factors II, VII, IX and X), and therefore predisposes to
bleeding rather than thrombosis. Protein S deficiency is the underlying cause of a small
proportion of cases of DIC, DVT and pulmonary embolism. Hereditary protein S deficiency is
an autosomal dominant condition.
Prothrombin 20210A mutation
This mutation in the gene encoding the clotting factor prothrombin is found in about 1 in 50
persons in the US. The mutation gives rise to slowed mRNA degradation and to an increase in
circulating prothrombin levels. This appears to create a hypercoagulable state.
The mutation is inherited in an autosomal dominant manner. Testing for prothrombin
mutation G20210A is therefore useful in determining a person's predisposition to thrombosis
and can assist in determining the need for anticoagulant therapy.
People who have prothrombin mutation G20210A have a 2-to-3 fold increase in the risk of
DVT. Persons who have this mutation plus the Factor V Leiden mutation have a 10-to-20 fold
increase in thrombotic risk. Prothrombin mutations have also been linked with thrombotic
events other than DVT, including recurrent miscarriages. In addition, there may be
interactions with other risk factors for thrombosis (e.g. pregnancy, oral contraceptives).
Activated Protein C Resistance (FV Leiden)
Factor V Leiden is the name given to a variant of human factor V that causes a
hypercoagulability disorder in 1:20 Caucasians in N. America. In this disorder factor V and
FVIII cannot be inactivated by activated protein C due to structural changes in these factors. It
is named after the city Leiden (The Netherlands), where it was first identified in 1994.
Diagnosis: APC added to the plasma degrades these factors and prolongs clotting time tests
that include these factors. In case of APC resistance, structural changes in factor V (and rarely
in FVIII) prevent the effect of APC and the prolongation of the clotting test becomes more
moderate. FV is a cofactor of FX that converts prothrombin to thrombin. FV is activated by
thrombin and inactivated by protein C. Protein C cleaves FV at Arg306, Arg506 and Arg679.
In FV Leiden mutation guanine exchanges adenine at nucleotide 1691 resulting in
Arg506Gln.
The test sample is Na-citrate anticoagulated plasma. The patient's plasma should be diluted 5-
fold by FV deficient plasma. The samples are tested for APTT in the presence and absence of
APC. The 2 samples are first incubated for 5 minutes (after diluting them). Calcium is added
to both samples, but one gets APC and the other one does not. APTT is measured for both
samples. The normal ratio should be > 2. A ratio < 2.0 suggests APC resistance (sample with
APC divided by the sample without APC).
Molecular genetic test for FV Leiden mutation
DNA is prepared from peripheral WBCs. The exon containing the FV Leiden mutation is
amplified by PCR, and the products are digested by specific restriction endonucleases. The
digestion products are separated by agarose gel electrophoresis.
Interpretation: when Leiden mutation is present, the restriction endonuclease loses one of its
cleavage sites on the PCR product. (Note: APC resistance is measured by APTT-based assay,
while Leiden mutaion is based on a molecular genetic method).
Avi Sayag Clinical Biochemistry
Topic 21 Acquired thrombophilias Acquired thrombophilias can occur in the settings of:
Post-operative conditions
Pregnancy, estrogen therapy, pills (oral contraceptives)
Prosthetic valves
Anti-phospholipid syndrome
Atrial fibrillation
Immobilization
Initial phase of syncumar therapy
Heparin-induced thrombosis
Varicose veins
Malignancy
Thrombus and malignancy In cancer patients, thromboembolism is the second most frequent cause of death.
Thrombosis and embolism may indicate occult malignancy.
Malignant diseases are frequently associated with thromboembolic complications
o Promyelocytic leukemia
o Primary cerebral tumors
o Pancreas carcinoma (mucin-secreting adenocarcinoma)
Hypercoagulability in cancer patients results from the response of the host
(monocytes/macrophages are stimulated by tumor cells, which provoke a
procoagulant activity) as well as from features of malignant cells: they express tissue
factor and platelet activating substances.
Thrombosis promoting factors in cancer patients:
o Surgical intervention
o Aggressive chemotherapy
o Hormone therapy
o Permanent central venous catheter
Anti-phospholipid syndrome (APS)
An autoimmune condition in which a group of autoAb (antiphospholipid) play a
direct role in the pathogenesis of thrombosis, fetal loss and other symptoms.
APS can be primary or secondary (related to SLE, other autoimmune diseases,
neoplasias, etc.)
Clinical criteria for APS:
o Vascular thrombosis: one or more episodes of arterial/venous/small vessel
thrombosis confirmed by imaging or Doppler (or histopathology)
o Pregnancy morbidity: 3 or more unexplained consecutive miscarriages or 1 or
more unexplained fetal deaths at the 10th week of gestation or afterwards. One
or more premature births at week 34 or before, associated with severe pre-
eclampsia or placental insufficiency.
Lab:
o Lupus anticoagulant (LA): present in the plasma at 2 or more occasions, at
least 12 weeks apart, and detected according to the guidelines of the ISTH
(International Society on Thrombosis and Haemostasis).
o Anticardiolipin Ab (ACA): IgG and/or IgM isotypes present in medium or
high titer at 2 or more occasions, at least 12 weeks apart, and measured by a
standardized ELISA for β2 glycoprotein I-dependent ACA.
o Anti-β2-glycoprotein-I-Ab: IgG and/or IgM isotypes present in medium or
high titer at 2 or more occasions, at least 12 weeks apart, and measured by a
standardized ELISA.
LA or other AP-Ab are directed against epitopes on certain proteins, most commonly
against β2-glycoprotein I and prothrombin. The epitopes become exposed upon
binding to anionic phospholipids or negatively-charged surfaces.
Avi Sayag Clinical Biochemistry
LA: these are immunoglobulins (IgG and/or IgM) that interfere with phospholipid-
dependent clotting tests. They are directed against phospholipid-protein complexes
(epitope on the protein) but not against clotting factors. In vitro, they are
anticoagulants, but in vivo they may predispose for thrombosis.
Lab diagnosis of LA:
o Prolongation of APTT
o Demonstration of an inhibitor by mixing studies. The inhibitor has to be
demonstrated to be phospholipid-dependent by:
� Relative correction by increasing the amount of phospholipids
� The effect is increased by diluting the phospholipid.
Mechanism of thrombophilias induced by AP-Ab:
o Inhibition of protein C/protein S pathway
o Interference with the synthesis of PgI2 (aka prostacyclin – a potent inhibitor
of platelet aggregation and a powerful vasodilator)
o Inhibition of the binding of heparan-sulphate with AT-III on the surface of
endothelial cells
o Induction of the expression of tissue factor in endothelial cells
o Involvement in Ab-induced platelet activation.
Avi Sayag Clinical Biochemistry
Topic 22 Consumption coagulopathies. DIC There are 4 consumption coagulopathies:
1. Acute DIC;
2. Large thrombus; 3. Primary hyperfibrinolysis; and 4. Thrombolysis
The bleeding symptom is due to the consumption of clotting factors.
Disseminated Intravascular Coagulation
An acute, subacute, or chronic thrombohemorrhagic disorder. DIC occurs as a secondary
complication in a variety of diseases. It is caused by the systemic activation of the coagulation
pathways, leading to the formation of thrombi throughout the microcirculation. As a
consequence of the widespread thromboses, there is consumption of platelets and coagulation
factors and, secondarily, activation of fibrinolysis. Thus, DIC can give rise to:
either tissue hypoxia and microinfarcts caused by many microthrombi; or
a bleeding disorder related to
o Pathologic activation of fibrinolysis
o The depletion of the elements required for hemostasis (hence the term
consumptive coagulopathy). This is probably a more common cause of
bleeding than all of the congenital coagulation disorders all together.
Two major mechanisms can trigger DIC: the release of TF and massive endothelial damage.
Release of tissue factor
The placenta in
obstetric
complications;
amniotic fluid
embolism; abruptio
placentae (wherein the
placental lining has
separated from the
uterus of the mother.
Trauma, hypertension,
or coagulopathy,
contributes to the
avulsion of the
anchoring placental
villi from the
expanding lower
uterine segment,
which, in turn, leads to
bleeding into the
decidua basalis. This
can push the placenta
away from the uterus
and cause further
bleeding); dead fetus
syndrome; septic
abortion;
The cytoplasmic
granules of acute
promyelocytic
leukemia cells:
treatment leads to
destruction of
granulocytes �
proteolytic enzymes
are released �
vessel damage �
TF is exposed.
Mucin-secreting
adenocarcinoma
cells. Some
tumors express
tissue factor on
the cell
membrane
(mainly lung
adenocarcinoma
and pancreatic
adenocarcinoma)
In gram-negative and
gram-positive sepsis
(important causes of
DIC), endotoxins or
exotoxins cause
increased synthesis,
surface expression, and
release of tissue factor
from monocytes.
Furthermore, activated
monocytes release IL-1
and TNF, both increase
the expression of tissue
factor on endothelial
cells and simultaneously
decrease the expression
of thrombomodulin. The
net result is the enhanced
activation of the extrinsic
clotting system and the
decrease of inhibitory
pathways that tend to
prevent coagulation
(protein C and S).
Avi Sayag Clinical Biochemistry
Widespread endothelial cell damage
Severe endothelial cell injury can initiate DIC by causing the release of tissue factor and by
exposing subendothelial collagen and von Willebrand factor (vWF), which act together to
promote platelet aggregation and the activation of the intrinsic coagulation cascade.
Widespread endothelial injury can be produced by:
1. The deposition of antigen-antibody complexes (e.g., in SLE),
2. By temperature extremes (e.g., following heat stroke or burns), or
3. By infections (e.g., meningococci and rickettsiae). Endothelial injury is an
important consequence of endotoxemia; therefore, DIC is a frequent complication
of gram-negative sepsis.
DIC has two phases:
The thrombotic phase:
The hemorrhagic phase:
Lab
1. INR and PT: a high INR level such as INR=5 indicates that there is a high chance of
bleeding, whereas if the INR=0.5 then there is a high chance of having a clot. Normal
range for a healthy person is 2-3, and for people on warfarin therapy or prosthetic
valves: 2.5–3.5. PT: 8-12 seconds (+ 4 seconds � prolonged)
2. APTT: 28-40 seconds (+8 seconds � prolonged).
3. Platelet count (reveals thrombocytopenia. Range: 150-400 G/L);
4. Soluble fibrin test: fibrin monomers and oligomers at low concentrations form
complexes with fibrinogen and remain soluble. The advantage of measuring soluble
fibrin over fibrinopeptide A to detect thrombin action on fibrinogen is the
considerably longer half-life of soluble fibrin in the circulation.
Fibrin deposition
Obstructed vessels
ischemia
Hemolysis of RBCs
(RBCs are traumatized in
passing though narrowed
vessels)
Fibrinolysis (FDP produced)
Impair fibrin
polymerization
Depletion of
PLTs Release of plasminogen activators
Antithrombin
activity Inhibit PLT
aggregation
FV and FVIII are
cleaved by
plasminogen
Consumption of
factors
Avi Sayag Clinical Biochemistry
5. D-dimers: these are products of fibrin degradation (among other fibrin degradation
products – FDPs). Their detection is made possible using monoclonal antibodies that
do not cross react with fibrinogen.
6. TT: 14-22 seconds (+ 8 seconds � prolonged)
7. Fragmentocytes (aka schistocytes)
8. Tests indicating the consumption of antithrombin III (ATIII).
9. Other markers for the intravascular activation of blood coagulation are the
prothrombin fragments 1 and 2, the fibrinopeptide A and the thrombin-antithrombin
(TAT complex). As prothrombin is cleaved it forms thrombin and the remaining is 2
fragments. Thrombin formation results in irreversible TAT complex. Fibrinopeptide
A is cleaved off by thrombin from the N-terminal end of Aα-chain of the fibrinogen
(the products of this cleavage are FPA, FPB and fibrin monomers).
The first 6 comprise the DIC panel and the last 3 complete the diagnostic procedure.
Primary hyperfibrinolysis
Fibrinolysis is responsible for fibrin breakdown. Hyperfibrinolysis occurs when fibrinolytic
activity is greater than fibrin formation such that clot integrity is threatened. The central event
of fibrinolysis is the generation of plasmin, which cleaves fibrin and fibrinogen. Free plasmin
is rapidly inhibited by its inhibitor – α2 antiplasmin. Fibrinolytic activity is initiated by the
plasminogen activators: t-PA and u-PA, which convert plasminogen to plasmin. t-PA is
released by endothelial cells, has a short half-life of 3-5 minutes and is regulated by specific
inhibitors: PAI (plasminogen activator inhibitor) types 1 and 2. PAI-1 is the main systemic
inhibitor and is produced by several cell types including endothelial cells, smooth muscle
cells, fibroblasts and hepatocytes. PAI-2 is found in the placenta. Platelets are the source of
90% of the circulating PAI-1, which is released at the site of a forming thrombus.
Hyperfibrinolysis occurs in the setting of imbalance between fibrinolytic activators and their
inhibitors.
The consequences of hyperfibrinolysis affect other aspects of hemostasis. Plasmin may reduce
platelet adhesion and aggregation by degradation of GpIb receptors and IIb/IIIa receptors. The
consumption of the clotting factors due to the direct effect of plasmin and the formation of
fibrinogen degradation products, which inhibit fibrin polymerization, results in poor fibrin
generation.
Fibrinolytic activation has been separated into primary and secondary: the primary form
represents fibrinolytic activity independent of other factors, whereas the secondary form is a
consequence of activation of coagulation and thus thrombin generation which stimulates the
endothelium to produce increased amount of t-PA.
Chronic liver disease is a common cause of hyperfibrinolysis, and is characterized by both
primary and secondary hyperfibrinolytic changes. There is reduced clearance of t-PA, and
reduced concentrations of α2-antiplasmin due to diminished protein synthesis. In addition,
primary hyperfibrinolysis can occur in the setting of prostate cancer and neoplasia as tumors
produce plasminogen activators (AML – M3 tumors produce uPA).
Among the lab marker specified for DIC, only fibrin degradation product (FDP) is positive.
Detection of fibrin monomers (topic 13 in the practical topics) Fibrin monomers are coupled to human RBCs (type O, Rh negative) in the reagent. In the
presence of soluble fibrin monomer complexes in the test plasma, hemagglutination will
occur. Positive and negative fibrin monomer controls are supplied by the manufacturer. The
method is fast, simple and specific.
Avi Sayag Clinical Biochemistry
Detection of D-dimers (topic 12 in the practical topics) In coagulation, the activated factor XIII crosslinks fibrin, which results in high molecular
weight crosslinked fibrin polymers. The secondary fibrinolysis degrades fibrin. Fibrinogen is
cleaved to D and E fragments. If fibrin was cross-linked before fibrinolysis, the products of
fibrinolysis are E fragment and D-dimer.
The reagent contains latex conjugated with specific antibodies directed against the D-dimer
domain of the cross-linked fibrin. In the presence of excess amount of D-dimers, the latex
beads will agglutinate and the test is considered positive. The D-dimer test detects only the
presence of split products of fibrin cross-linked by FXIII. Thus, the D-dimer test is positive
only in secondary hyperfibrinolysis.
If the sample is agglutinated, the D-dimer level is said to be > 2mg/L. In normal conditions,
agglutination cannot be detected and the D-dimer is said to be < 0.25mg/L.
In the clinical practice, the only acceptable method for D-dimer measurement is the
quantitative D-dimer determination where the agglutination is evaluated by automated
methods.
The presence of rheumatoid factor (anti-human IgG) can lead to false positive results.
FX
Prothrombin Prothrombin fragments 1 & 2 + thrombin TAT
Fibrinogen FPA, FPB,
fibrin
monomers
Avi Sayag Clinical Biochemistry
Topic 23 Laboratory tests of glomerular and tubular function Assessing renal function, we have to test 3 aspects:
1. The glomerular function (the glomerular filtration and integrity);
2. The renal tubular functions; and
3. The renal endocrine function.
Estimation of the glomerular function
The estimation consists of 4 parameters:
1. Clearance determinations
2. Plasma creatinine
3. Plasma urea
4. Plasma level of low molecular weight proteins: β2-microglobulins, retinol binding
protein, cystatin C.
We shall consider each of these:
1. Clearance Determinations
We can use exogenous or endogenous substances that are completely filtered, not reabsorbed
and not secreted in the tubules. Whichever substance chosen, we calculate its concentration in
the urine collected, in the plasma collected, and we measure the urine output over 24 hours. In
order to avoid problems, the collection has to be carried out twice. The exogenous substances
are inulin and Cr-EDTA, and the endogenous one is creatinine. The formula for clearance is
given by: P
UxVC = .
Creatinine clearance in adults is normally of the order of 120 mL/min, corrected to a standard
body surface area of 1.73 m2. It should be noted that the clearance formula is only valid for a
steady state, that is, when renal function is not changing rapidly. The accurate measurement
of creatinine clearance is difficult, especially in outpatients, since it is necessary to obtain a
complete and accurately timed sample of urine. The usual collection time is 24 h, but patients
may forget the time or forget to include some urine in the collection. Creatinine is actively
secreted by the renal tubules and, as a result, the creatinine clearance is higher than the true
GFR (by 15%). The difference is of little significance when the GFR is normal, but when the
GFR is low (<10 mL/min), tubular secretion makes a major contribution to creatinine
excretion and creatinine clearance significantly overestimates the GFR. The effect of
creatinine breakdown in the gut also becomes significant when the GFR is very low. Certain
drugs, including spironolactone, cimetidine and amiloride, decrease creatinine secretion and
thus can reduce creatinine clearance. Lastly, in the calculation of creatinine clearance, two
measurements of creatinine concentration and one of urine volume are required. Each of these
has an inherent imprecision that can affect the accuracy of the overall result. Even in well-
motivated subjects, studied under ideal conditions, the coefficient of variance of
measurements of creatinine clearance can be as high as 10%, and it can be two or three times
greater than this in ordinary patients.
The Cockcroft-Gault formula gives the creatinine clearance: )/(
)140(
LmolneseCreatini
xKgxKage
µ−
where
K (constant) is 1.227 for men and 1.04 for women.
As mentioned, the creatinine clearance estimates the GFR. However, the GFR can be
estimated using the 4-variable Modification of Diet in Renal Disease study group, or in short,
the 4-v MDRD formula:
GFR=203.0154.1 )()0113.0(186 −− agexScrx . The result should be multiplied by 0.742 if the
patient is a female, and by 1.21 if the patient is black. Scr is serum creatinine given in
µmol/L. As implied by the name of the formula, the GFR is tested in renal disorders, and it is
valid only at high plasma creatinine concentrations.
Another estimation tool to calculate GFR is the Mayo Quadratic formula. This formula was
developed in an attempt to better estimate GFR in patients with preserved kidney function. It
is well recognized that the MDRD formula tends to underestimate GFR in patients with
Avi Sayag Clinical Biochemistry
preserved kidney function. Notwithstanding, the formula estimates the GFR in patients
suffering from renal disorders, and it is valid at low plasma creatinine concentrations (the
quadratic formula).
None of the formulae is valid for children under 18 years of age and for pregnant women! The reference range for GFR is 90-120 ml/min normalized to body surface of 1.73m
2.
GFR between 60-90 ml/min/1.73m2 suggests mild kidney disorder.
GFR between 30-60 ml/min/1.73m2 suggests moderate kidney disorder.
GFR between 15-30 ml/min/1.73m2 suggests severe kidney disorder.
GFR < 15 ml/min/1.73m2 suggests end-stage kidney disorder.
2. Plasma Creatinine Measurement of plasma creatinine concentration is a reliable test of glomerular function.
Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced
at a fairly constant rate by the body (depending on muscle mass). Meat intake may increase
creatinine serum level by 10%. It is not reabsorbed by the renal tubules, but a small amount is
secreted.
The reference range- for men: 62-106 µmol/L; for women: 44-97 µmol/L.
The standard laboratory measurements for creatinine can suffer from interference, for
example from bilirubin and ketones3. The laboratory should be able to advise on whether this
may be a problem in individual cases.
Several factors influence plasma creatinine concentration:
It decreases with age and among females. It also decreases among vegetarians and in
cases of malnutrition. Immediately after surgery and in patients treated with
corticosteroids the concentration also decreases.
It increases among blacks, in cooked meat, among athletes (increased muscular mass)
and if certain medications are taken: creatinine levels may increase when ACE
inhibitors (ACEI) or angiotensin-II receptor blockers (ARBs) are used in the
treatment of chronic heart failure (CHF). Using both ACEI & ARB concomitantly
will increase creatinine levels to a greater degree than either of the two drugs would
individually. An increase of <30% is to be expected with ACEI or ARB use.
Obesity does not affect creatinine concentration.
3. Plasma Urea
Urea is synthesized in the liver, primarily as a by-product of the deamination of amino acids.
Its elimination in the urine represents the major route for nitrogen excretion. It is filtered from
the blood by the glomeruli (90%) but significant tubular reabsorption occurs through passive
diffusion (neither active reabsorption nor secretion occurs). Plasma urea concentration is a
less reliable indicator of renal glomerular function than creatinine.
Urea production depends on non-renal factors!
The reference interval is 2.9-8.2 mmol/L.
Urea production is increased by a high protein intake, in catabolic states, and by the
absorption of amino acids and peptides after gastrointestinal hemorrhage. Dehydration and
urinary stasis also increase its levels. Conversely, production is decreased in patients with a
low protein intake and sometimes in patients with liver disease. Tubular reabsorption
increases at low rates of urine flow (e.g. in fluid depletion) and this can cause increased
plasma urea concentration even when renal function is normal.
3 Bilirubin decreases creatinine and ketones increase creatinine.
Avi Sayag Clinical Biochemistry
Lab methods for the determination of urea and creatinine (Practical
topic 14) Urea There are 2 methods to determine the urea concentration:
1. Direct chemical methods
2. Indirect enzymatic method
1. Direct chemical method
This method is outdated because they cannot be automated. The direct chemical reaction of
urea and special chemical substances results in the formation of colored products that can be
measured by spectrophotometry.
2. Indirect enzymatic method
The ammonia, formed after the reaction of urease on urea, is determined.
Urea + H2O ------------> 2NH4+ + CO2 (carried by urease)
From this point, there are 3 ways to determine urea concentration:
- Enzymatic UV kinetic method:
2NH4+ + 2α-ketoglutarate + 2NADH ---------> 2 glutamate + 2NAD + 2H2O
From ammonia and α-ketoglutarate, in the presence of NADH and under the effect of
glutamate dehydrogenase, NAD and glutamate are formed. The decrease in the amount of
NADH correlates with the amount of ammonia formed.
- Conductometric method:
The amount of ammonia formed from urea can be detected by the change of conductivity in
the solution.
- Ionselective electrode:
The changes in the electrode potential are proportional to the ammonia concentration
(ammonium ions).
Creatinine There are 2 methods:
1. Methods based on Jaffe reaction
2. Enzymatic methods
1. Methods based on Jaffe reaction
Creatinine in alkaline solution gives an orange condensation product with picric acid.
There are 2 types of reactions:
End-point Jaffe reaction: one reaction with deproteinization and the other one
is without deproteinization. In the first method we deproteinize the solution,
because proteins interfere with the reaction. This makes the method
laborious. The other method, in which we give up the deproteinization
process, is considered outdated. Thus, the end-point Jaffe reaction is no
longer practical. (In other words, in the oral exam, just mention the reaction
and move on. In the written SCT, remember that it exists…).
Kinetic Jaffe reaction: the kinetic Jaffe reaction is a modified end-point Jaffe
reaction. It is fast, automated, and measures the rate of color development.
The Jaffe reaction is not specific for creatinine because other serum
components (proteins, glucose, ascorbic acid and α-ketoacids) also react with
picric acid. The reaction rate in case of creatinine and interfering substances
is different, so prior deproteinization is not necessary. A part of non-specific
reactions are completed within 30 seconds, others only cause interference
after 2 minutes. The change in absorbance after 30 seconds to 2 minutes is
mainly caused by creatinine. In case of diabetic ketoacidosis, falsely high
creatinine concentration can be detected due to ketone bodies, whereas in
icteric patients (jaundice) bilirubin and its metabolites may cause falsely low
results.
Avi Sayag Clinical Biochemistry
2. Enzymatic methods
Here, too, there are 2 options:
Partial enzymatic method: creatinine is degraded under the effect of creatinine
aminohydrolase producing ammonia. The ammonia can be detected by ionselective
electrode. The other possibility is to measure the amount of chromogens from Jaffe
reaction before and after the enzyme reaction. The difference will be proportional to
the creatinine concentration.
Completely enzymatic UV method: creatinine is converted to creatine under the
effect of creatine hydrolases, which can be detected by creatine kinase.
(cont. of topic 23:)
4. Cystatin C This low molecular weight peptide (13 kDa) is produced by all nucleated cells. It is a cysteine
protease inhibitor. Due to its low Mw and high isoelectric point (9.2), it is cleared from the
plasma by glomerular filtration only and its plasma concentration reflects the GFR. However,
its plasma concentration is more variable than that of creatinine; it is increased in malignancy
and by treatment with corticosteroids. Although advocated as being a more sensitive and
specific indicator of moderately impaired renal function than creatinine, measurement of
cystatin C does not at present have a clear role in the assessment of patients with suspected
renal impairment
Assessment of glomerular integrity Impairment of glomerular integrity results in the filtration of large molecules that are
normally retained and it manifests as proteinuria. 'Clinical proteinuria' is proteinuria that can
be reliably detected by dipstick testing of urine, and is >300 mg/L. With severe glomerular
damage, RBCs are detectable in the urine (hematuria). While hematuria can occur as a result
of lesions anywhere in the urinary tract, the RBCs often have an abnormal morphology in
glomerular disease. The presence of red cell casts (cells embedded in a proteinaceous
matrix) in urinary sediment is strongly suggestive of glomerular dysfunction.
Between 7-10 g of proteins are filtered per day; however, only less than 150 mg is excreted!
(thus, most is reabsorbed). Half of the excreted proteins is given by Tamm-Horsfall protein: a
human gene. This gene encodes uromodulin, the most abundant protein in normal urine.
Uromodulin may act as a constitutive inhibitor of calcium crystallization in renal fluids.
Excretion of uromodulin in urine may provide defense against urinary tract infections caused
by uropathogenic bacteria. Defects in this gene are associated with the autosomal dominant
renal disorders: medullary cystic kidney disease-2 (MCKD2) and familial juvenile
hyperuricemic nephropathy (FJHN). Less than 30 mg of albumin is excreted.
Proteinuria can result from 3 causes:
1. Increased filtered load;
2. Decreased tubular reabsorption;
3. Postglomerular secretion/leakage;
We shall consider each cause separately:
1. Increased filtered load The increased load can be the result of:
1. Increased glomerular permeability – there is a progressively increasing excretion of
higher Mw proteins as the permeability increases;
2. Increased plasma concentration of a relatively freely-filtered proteins (such as Bence-
Jones proteins and myoglobin);
3. Decreased number of glumeroli – thus, there is an increased filtered load per nephron.
2. Decreased tubular reabsorption The decreased reabsorption can be the result of:
1. Damage to the proximal tubules – the indication to such damage is the presence of
low Mw proteins, such as α1-microglobulins in the urine;
Avi Sayag Clinical Biochemistry
2. Enzymuria – damage of the tubular epithelial cells results in increased cell turnover
and cell lysis. Therefore, enzymes such as those in the brush border, in the cytosol
and lysosomes are released.
3. Postglomerular secretion/leakage
This can be the result of:
1. Increased protein secretion by the tubular system (Tamm-Horsfall glycoprotein)
2. Leakage of various plasma proteins into the urinary space as a result of
tubulointerstitial damage (in cases of inflammation, e.g.).
Proteinuria is screened by using dip-stick, which detects albumin sensitively (>200 mg/L),
and is less sensitive to other proteins such as Bence Jones protein.
False positive reactions can be obtained in alkaline urine and if X-ray contrast medium is
used.
External causes of proteinuria should be excluded (fever, strenuous exercise, orthostatic
proteinuria).
The source of proteinuria can be tubular damage or glomerular damage. If the source is
tubular, then low Mw proteins will be detected in the urine (the high Mw ones will not be
filtered in the glomerulus, as it is intact). In particular, retinol binding protein and α1-
microglobulin will be present. If the glomerulus is the source of the proteinuria, then higher
Mw proteins will also be detected in the urine.
Hematuria can be the manifestation of glomerular disease, tubulointerstitial disease or post-
renal disease. It can manifest in the presence of RBCs in the urine, which are detected as
sediments seen by phase-contrast microscopy, or in hemoglobinuria, which is detected by
chemical methods that detect Hb.
Estimation of the renal tubular function
1. Detection of low-Mw molecules (α1-microglobin and retinol binding protein).
2. Assessment of renal concentrating ability: the osmolality of the urine, or its specific
gravity, is measured following water deprivation.
3. Tests for renal tubular acidosis: the excretion of fractional bicarbonate is measured,
and the ammonium chloride loading test is performed: the acid loading test (pH)
measures the ability of the kidney tubules to acidify urine when there is increased
plasma acidity. The patient is told to take ammonium chloride capsules by mouth for
3 days. Then, urine and blood samples are taken. (The blood sample is needed to
show that the ammonium chloride made the blood slightly acidic.) The laboratory
measures the level of acid found in both samples.
4. Tests for aminoaciduria: amino acids are measured using the HPLC method
5. Measurement of glucose in the plasma and urine (renal glucosuria)
Urinalysis – what can be detected using the dipstick method?
(check this website for clear details: http://www.irvingcrowley.com/cls/urin.htm)
Avi Sayag Clinical Biochemistry
Examination of urine (general and sediment analysis)
(Practical topic 15) 1. Types of samples: random, first-morning urine (the most preferred sample.
Concentrated, acidic, enhances the stability of the cellular elements), collected (24
hours, day, night. Urine sediment analysis must not be performed from collected
sample!)
2. Collection of urine: mid-stream specimen. Collection via a catheter, collection bags
(infants, children), collection via urostomy4. In rare cases, suprapubical aspiration is
performed (when other techniques are not applicable).
3. Storage: urine examination and urine sediment analysis are better completed within
30 minutes. Urine samples can be kept at 4ºC (refrigerator) for maximum 2 hours.
Urine examination by test strips The samples should be first examined macroscopically. The color of urine
sampled from a healthy person is yellowish.
For urine analysis by test strips, fresh (not older than 2 hours), uncentrifuged,
carefully mixed samples have to be used.
Submerge the sample for 1-2 seconds and remove the unnecessary urine off
the strip.
After 1-2 minutes, the color reactions can be visually evaluated. Remember
that vitamin C (ascorbic acid) and certain drugs (captopril, phenazopyridin)
can alter the results (inquire the patient about these drugs).
The reactions on the urine test strips are based on dry chemical principles (the
reagents are impregnated into a membrane attached to a plastic base).
The parameters measured:
1. Specific gravity: detects the ion concentration of the urine (in the presence of cations,
protons are released through a complex formation reaction, which lead to a change in
color of the bromothymicblue indicator). Reference range: 1.005-1.03
2. pH: the indicators are methyl-red, phenophtalein and bromothymicblue. Reference
range: 5-6 (higher values are recorded in vegetarians and after a long period of
sample standing).
3. WBC: the esterase enzyme (present in granulocytes) cleaves indoxylester into
indoxyl, which forms a purple color with a diazonium acid. The healthy urine does
not contain enough WBCs to induce this color reaction (if the sample is not fresh, we
may get a false positive result, because the esterase from leukocytes may pass out of
the cells).
4. Nitrite: the most pathogenic bacteria (e.g. E. coli) in the urine reduce nitrate into
nitrite, which can be visualized by the Griess probe (false negative results in the
presence of vitamin C; false positive in the presence of phenazopyridine – an anti-
inflammatory drug).
5. Proteins: the test strip contains bromophenolblue indicator set to an acidic pH by a
buffer. When there are no proteins, the color of the indicator at pH 3 is yellow, and in
the presence of proteins, the color changes into greenish-blue, depending on the type
of the protein and its concentration. The indicator is much more sensitive to albumin
than to globulin.
6. Glucose: detected by glucoseoxidase/peroxidase/chromogen-sulphate method. In
healthy sample glucose is not detectable.
7. Ketone bodies: acetone and acetoacetate can be detected but not β-hydroxybutyrate
(this is kind of problematic if ketoacidosis is suspected, because β-hydroxybutyrate is
the most abundant ketone body in this condition).
4 A urostomy is a stoma (artificial opening) for the urinary system. A urostomy is made in cases where
long-term drainage of urine through the bladder and urethra is not possible, e.g. after extensive surgery
or in case of obstruction.
Avi Sayag Clinical Biochemistry
8. Urobilinogen: it is important to keep the sample at dark to avoid the effect of direct
sunshine that can yield false negative results. If UBG is present in high concentration,
it can be observed macroscopically by the yellowish-brown color. Chemically, the
presence of UBG gives a red color due to the formation of diazonium salt.
9. Bilirubin: the detection is based on the transformation of diazonium salt into a
colored substance. As bilirubin dissociates by light, the sample has to be kept at dark
prior to the test.
10. Blood: Hb (and myoglobin) catalyze the oxidation of tetramethylbenzidine. If the
sample stands for too long before analysis, false positive results may be obtained due
to hemolysis.
Urine sediment analysis 1. Microscopic evaluation of urine sediment: 10mL of urine is put into a tube,
centrifuged for 5 minutes, the supernatant is suctioned, the sediment is stirred,
pipetted onto a slide, covered by a coverslip and examined under the microscope.
2. Analysis of sediment under the microscope: evaluate 10 low-power fields (count the
casts); evaluate 10-20 high-power fields (count all cells and other elements).
Reference range: 1 isomorphic RBC, 1-2 leukocytes, 1 hyaline cylinder in 1 HPF, 1
superficial urothelium per 4-5 HPF.
3. The analysis can be automated: in one class of analyzers, the cellular components
flow by an optical lens. The other types of analyzers are based on a flow cytometry
principle: the cellular elements are labeled with 2 types of fluorescent dyes by the
machine – one labels nucleic acids and the other labels the negatively-charged cell
membrane, the nuclear membrane and the mitochondria. After hydrodynamic
focusing, we measure the impedance of the cellular components, and a laser beam
determines the scattering and fluorescent parameters of each element. According to
this data, the computer of the analyzer can identify the right type of the cellular
components, and can calculate the number of these elements in 1 µL of urine.
4. Components of the urine sediment: there are organic components, inorganic
components and artifacts/contaminating substances
Organic components Cellular elements
RBCs WBCs Epithelium
4-7µm
Isomorphic cells indicate post-
renal bleeding
Acantocytes suggest glomerular
disease
Ghost cells (lysed cells)
DD: calcium-oxalate
monohydrate crystals, bubbles,
fat drops and yeast (2% acetic
acid does not lyse yeast
particles)
Mainly PMNs, but all can be
present
Usually form clumps
Usually associated with
infections
May be due to contamination
Tubular epithelium – larger than
WBCs (13µm)
Can appear during fever, ATN,
interstitial nephritis, acute
rejection of kidney
transplantation
Urothelium – large (30µm): can
be:
Superficial type: oval with
smaller nucleus
Deeper type: smaller cells
Squamous epithelium: large
(50-60µm) – usually suggests
contamination
Casts Hyaline casts – contain only the base matrix of Tamm-Horsfall protein. Barely visible.
Granular casts – lysosomes of tubular cells or degraded cells (acute renal disease)
Waxy casts – chronic renal disease (sharp edges with indented borders)
Fatty casts – contain lipid particles (nephrosis)
Cellular casts – RBCs (renal parenchymal bleeding), WBCs (acute pyelonephritis, acute interstitial
nephritis), tubular epithelial cells (glomerular disease)
Hb and myoglobin casts – renal parenchymal bleeding
Bacterial and yeast casts – among immunocompromised patients with pyelonephritis
Avi Sayag Clinical Biochemistry
Microorganisms Bacteria: if they are associated with high WBC count �infection; if WBCs are present without bacteria
� TB
Yeasts – mainly candida
Trichomonas vaginalis
Parasites
Inorganic components Crystals
Urine sediment analysis suggests the possibility of renal calculus only if the urine is examined right
after urine passing and only if the repeated analysis of the sediment still shows crystals in bulk
quantity.
Cystine Uric acid Ca+2
oxalate Ca+2
oxalate
monohydrate
Struvite (Mg-
NH4-Phosphate)
Amorphous
The major components are urates (in acidic urine) and phosphates (in alkaline urine).
They appear as granular particles, often in clumps: "sedimentum lateritium"
Artifacts/contamination
Vaginal floor
Sperm
Feces
Hair
Pollens
Glass particles
Bubbles
Textile filaments
Avi Sayag Clinical Biochemistry
Topic 24 Clinical biochemistry of acute & chronic renal failure (ARF CRF); tubulopathies ARF is a rapid loss of renal function due to damage to the kidneys, resulting in retention of
nitrogenous (urea and creatinine) and non-nitrogenous waste products that are normally
excreted by the kidney. It is a serious disease and treated as a medical emergency. Signs of
renal failure are:
Symptoms manifest when the renal functional capacity decreases below 50-60% and
the GFR falls below 50 ml/min. It thus follows that the kidneys have considerable
reserve capacity!
Uremia: manifests as nausea, vomiting and lethargy
Disorders of micturition: frequency of urination increases, nocturia, retention of urine
and dysuria (pain when urinating)
Disorders of urine volume: polyuria, oliguria and anuria
Alteration in urinary composition: hematuria, proteinuria, bacteriuria, leukocyturia
and calculi
Pain (not necessarily present)
Edema: due to hypoalbuminemia and retention of salt and water
ARF can be completely resolved, develop into CRF or lead to death in 50% of cases!
ARF is usually categorized according to pre-renal, renal and post-renal causes:
1. Pre-renal ARF Pre-renal (causes in the blood supply):
Hypovolemia: usually from shock, dehydration, fluid loss or excessive diuretics use,
trauma, burns and surgery. When cardiac output is decreased (as in cardiogenic
shock, in congestive heart failure and pulmonary embolism) it may result in ARF.
Hepatorenal syndrome in which renal perfusion is compromised in liver failure
Vascular problems, such as atheroembolic disease and renal vein thrombosis (which
can occur as a complication of the nephrotic syndrome). The effective plasma volume
is also decreased due to sepsis and shock.
Infection: usually sepsis and systemic inflammation due to infection.
When there is renal hypoperfusion, the GFR decreases. Parallelly, there is intense renal
vasoconstriction that leads to redistribution of renal blood flow. This preserves tubular
function. If hypoperfusion is left untreated, acute tubular necrosis develops.
In case of hypovolemia, RBF
decreases, which leads to increased
levels of renin, angiotensin and
aldosterone. Aldosterone increases
tubular reabsorption of Na+, thus
leading to decreased Na+ in the
urine. Elevated levels of Na+
increase plasma osmolality, which
activates the hypothalamus, leading
to secretion of ADH from the
neurohypophysis. There is water
retention to correct for the
hypovolemia, and small amount of
concentrated urine is excreted.
Regardless of this mechanism to correct for the hypovolemia, the decreased plasma volume
results in decreased GFR. This leads to increased serum urea and creatinine. In addition, less
sodium is delivered to the distal tubule (because less sodium is filtered and NOT because
more is reabsorbed in the proximal tubule due to aldosterone secretion, as aldosterone acts on
the distal tubule and the collecting ducts). Since less sodium reaches the distal tubule, less K+
and H+ are secreted (normally, in the distal tubule, sodium is reabsorbed actively and
potassium and hydrogen ions are secreted passively). This leads to hyperkalemia and acidosis.
Avi Sayag Clinical Biochemistry
2. Renal (intrinsic) ARF Toxins or medication (e.g. some NSAIDs, aminoglycoside antibiotics, cephalosporin,
iodinated contrast, lithium, phosphate nephropathy due to bowel preparation for
colonoscopy with sodium phosphates)
Rhabdomyolysis - the resultant release of myoglobin in the blood affects the kidney;
it can be caused by injury (especially crush injury and extensive blunt trauma),
statins, stimulants and some other drugs.
Hemolysis - the hemoglobin damages the tubules; it may be caused by various
conditions such as sickle-cell disease, and lupus erythematosus.
Multiple myeloma, either due to hypercalcemia or "cast nephropathy" (multiple
myeloma can also cause chronic renal failure by a different mechanism).
Acute glomerulonephritis which may be due to a variety of causes, such as anti
glomerular basement membrane disease/Goodpasture's syndrome, Wegener's
granulomatosis or acute lupus nephritis with SLE.
Renal hypoperfusion: due to hypotension, bleeding and sepsis. Ischemia may result
from low cardiac output, burns and trauma.
Intrarenal obstruction: Bence-Jones proteinuria.
Some features distinguish pre-renal ARF from renal ARF:
1. In prerenal ARF, the GFR is decreased while the tubules are preserved. In the renal
form, tubular necrosis is frequent. Although glomerular damage is uncommon in
intrinsic ARF, the GFR falls as a result of glomerular hypoperfusion.
2. In pre-renal ARF, urine [Na] is less than 20 mmol/L, while in the intrinsic form it
rises above 40 mmol/L.
3. In pre-renal ARF, urine urea is concentrated by a factor of 20, that is, the urine-to-
plasma urea concentration is more than 20:1 (it doesn't contradict the fact that the
plasma concentration of urea is elevated in pre-renal ARF). In the intrinsic form, the
ratio is less than 10:1.
4. The urine-to-plasma osmolality ratio is more than 1.5:1 in the pre-renal form. In the
intrinsic form, the ratio is less than 1.1:1.
5. In the intrinsic form, proteinuria is always present.
There are typically three phases to the course of acute tubular necrosis (renal ARF): the initial
oliguric phase, a diuretic phase and a recovery phase. The oliguric phase typically lasts for 8-
10 days but sometimes is much shorter or persists for several weeks. When it occurs, the
oliguric phase is followed by a diuretic phase, with increasing urine volume. This is the result
of an increase in GFR, and initially there is often little improvement in tubular function. The
composition of the urine is similar to that of protein-free plasma. During this phase, urine
volume may exceed 5 L/day and, because of its high ionic concentration, there is a
considerable risk of both dehydration and depletion of sodium and potassium.
Although the onset of the diuretic phase often heralds clinical improvement, plasma
concentrations of urea and creatinine do not fall immediately since the GFR is still much
lower than normal and insufficient to allow excretion of the surplus. The persisting high urea
concentration in the blood, and hence in the glomerular filtrate, contributes to the diuresis by
an osmotic effect. The acidosis also persists until tubular function is restored. Plasma Ca+2
concentration may rise during this phase, particularly after crush injuries, owing to the release
of Ca+2
from damaged muscles. Temporary persistence of any elevation in the plasma
concentration of parathyroid hormone will stimulate calcitriol synthesis and this may also
contribute to hypercalcaemia.
Gradually, in the recovery phase, as the tubular cells regenerate and tubular function is
restored, the diuresis subsides and the various abnormalities of renal function resolve. Patients
who survive the acute illness usually recover completely. Some residual impairment of renal
function is often demonstrable but it is not usually of clinical significance.
Post-renal ARF In post-renal ARF the hydrostatic pressure increases and opposes glomerular filtration. If this
state lasts for too long, it may lead to secondary renal tubular damage.
Avi Sayag Clinical Biochemistry
Causes include:
Medication interfering with normal bladder emptying (e.g. anticholinergics).
Benign prostatic hypertrophy or prostate cancer.
Kidney stones.
Abdominal malignancy (e.g. ovarian cancer, colorectal cancer).
Obstructed urinary catheter.
Common to all these is the presence of any obstruction to bladder outflow or ureteric
obstruction.
In summary, in ARF there is an increase in K+, H
+, urea, creatinine, phosphate, Mg
+2 and uric
acid, while there is a decrease in Na+, HCO3
-, and Ca
+2.
Chronic Renal Failure (CRF) CRF is a progressive, irreversible loss of renal function over a period of months or years. The
symptoms of worsening kidney function are unspecific, and might include feeling generally
unwell and experiencing a reduced appetite. Often, CRF is diagnosed as a result of screening
of people known to be at risk of kidney problems, such as those with hypertension or diabetes,
glomerulonephritis and pyelonephritis, renal vascular diseases and those with a blood relative
with CRF. CRF may also be identified when it leads to one of its recognized complications,
such as cardiovascular disease, anemia or pericarditis.
CRF is identified by a blood test for creatinine. Higher levels of creatinine indicate a falling
GFR and as a result a decreased capability of the kidneys to excrete waste products.
Creatinine levels may be normal in the early stages of CRF, and the condition is discovered if
urinalysis shows that the kidney is allowing the loss of protein or RBCs into the urine. To
fully investigate the underlying cause of kidney damage, various forms of medical imaging,
blood tests and often renal biopsy are employed to find out whether there is a reversible cause
for the kidney malfunction.
Recent professional guidelines classify the severity of chronic kidney disease in 4 stages:
Decreased renal reserve: when 50-75% of the renal function are preserved;
Renal insufficiency: when 25-50% of the renal function are preserved;
Renal failure: when 10-25% of the renal function are preserved;
Renal end-stage: when less than 10% of renal function are preserved (uremic
syndrome).
There is no specific treatment unequivocally shown to slow the worsening of chronic kidney
disease. If there is an underlying cause to CRF, such as vasculitis, this may be treated directly
with treatments aimed to slow the damage. In more advanced stages, treatments may be
required for anemia and bone disease. Severe CRF requires one of the forms of renal
replacement therapy; this may be a form of dialysis, but kidney transplant is ideal.
Signs and symptoms
Initially, it is without specific symptoms and can only be detected as an increase in serum
creatinine or protein in the urine. As the kidney function decreases:
Blood pressure is increased due to fluid overload and production of vasoactive
hormones, increasing one's risk of developing hypertension and/or suffering from
congestive heart failure.
Urea accumulates, leading to azotemia and ultimately uremia (symptoms ranging
from lethargy to pericarditis and encephalopathy). Urea is excreted by sweating and
crystallizes on skin ("uremic frost").
Hyperkalemia with a range of symptoms including malaise and potentially fatal
cardiac arrhythmias. When K+ levels are above 6.5mmol/L it is an indication for
hemodialysis.
Erythropoietin synthesis is decreased (potentially leading to anemia, which causes
fatigue).
Fluid volume overload - symptoms may range from mild edema to life-threatening
pulmonary edema.
Hyperphosphatemia - due to reduced phosphate excretion, associated with
hypocalcemia (due to vitamin D3 deficiency). The major sign of hypocalcemia is
tetany.
Avi Sayag Clinical Biochemistry
Later, this progresses to tertiary hyperparathyroidism, with hypercalcaemia, renal
osteodystrophy and vascular calcification that further impairs cardiac function.
Metabolic acidosis, due to accumulation of sulfates, phosphates, uric acid etc. This
may cause altered enzyme activity by excess acid acting on enzymes and also
increased excitability of cardiac and neuronal membranes by the promotion of
hyperkalemia due to excess acid.
People with chronic kidney disease suffer from accelerated atherosclerosis and are
more likely to develop cardiovascular disease than the general population. Patients
afflicted with CRF and cardiovascular disease tend to have significantly worse
prognoses than those suffering only from the latter.
Insulin is also increased as a response to the hyperkalemia (it activates the Na-K
pump, in order for K+ to enter the cell).
Testosterone and estrogen are also decreased.
HCO3- and Na
+ are also decreased
In summary:
Increased: K+, urea, creatinine, phosphate, Mg
+2, H
+, insulin, uric acid;
Decreased: Na+, HCO3
-, estrogen, testosterone, Ca
+2, erythropoietin, Hb
Several toxins are potential uremic toxin:
1. Urea: in very high concentrations it may lead to fatigue, vomiting and headache
2. Creatinine: affects glucose tolerance and RBC survival
3. Uric acid: uremic pericarditis
4. Cyanate: causes irreversible carbamylation of proteins, drowsiness and
hyperglycemia
5. Polyols: may cause peripheral neuropathy
6. Phenols: may cause membrane toxicity
7. Medium Mw molecules might be responsible for most of symptoms
8. β2-microglobulins: may cause renal amyloidosis
CRF may necessitate dialysis or later kidney transplant. Thus, the clinical and biochemical
monitoring of patients include:
1. Monitoring of graft functions and graft rejection. The rejection manifests as oliguria
and fever;
2. Increase in creatinine levels (due to rejection or ciclosporin toxicity)
Most patients with CRF become hypocalcaemic and, in time, many develop renal
osteodystrophy.
Avi Sayag Clinical Biochemistry
Renal Tubular Diseases Renal tubular disorders can be congenital or acquired; they can involve single or multiple
aspects of tubular function. The congenital conditions are inherited and all are rare: their
clinical sequelae relate to the consequences of loss of substances that are normally completely
or partially reabsorbed by the tubules.
1. The Fanconi syndrome
This is a generalized disorder of tubular function characterized by glycosuria, amino aciduria,
phosphaturia and acidosis. It can occur secondarily to a variety of conditions. One of these is
cystinosis, or Lignac-Fanconi disease, a rare inherited disease in which there is a defect in the
transport of cystine out of lysosomes. This leads to cystine accumulation and the deposition of
cystine crystals in many body tissues, including the kidneys. Affected infants fail to thrive,
develop rickets and polyuria with dehydration and eventually progress to renal failure. There
is no specific treatment. Cystinosis should not be confused with cystinuria, a disorder of
tubular transport.
Cystinosis is one of idiopathic inherited metabolic diseases along with galactosemia, fructose
intolerance, glycogen storage disease, tyrosinemia, and Wilson's disease.
In Fanconi's syndrome, apart from the idiopathic inherited metabolic diseases, there are also:
- Nephrotoxins (heavy metals and drugs)
- Paraproteinemia
- Amyloidosis
2. Renal tubular acidosis (RTA)
In this condition there is acidosis due to:
1. Increased loss of bicarbonate (impaired reabsorption in the proximal tubule) and/or
2. Insufficient acidification of distal tubular fluid (impaired secretion of hydrogen ions
in the distal tubule)
There are 4 types:
RTA-1 - distal renal tubular acidosis: it can be either inherited (autosomal dominant)
or acquired (sporadic, non-familial). Other causes are drugs (such as amphotericin,
gentamycin and lithium) and autoimmune diseases. There is a defect in H+ excretion
and the urine cannot be acidified. Consequences include growth retardation, rickets,
hypophosphatemia, osteomalacia, hypercalciuria, and often hypokalaemia. In general,
hyperkalaemia is more usual in acidotic states, but in these types of RTA, the
impaired ability of the kidneys to excrete H+ necessitates increased K
+ excretion when
Na+ is reabsorbed in the distal tubules, and this may cause H
+ depletion and
hypokalaemia. Treatment of type 1 RTA involves the administration of bicarbonate in
sufficient quantities to buffer normal H+ production and K
+ supplements. Diagnosis
involves the ammonium chloride loading test, and the urinary pH is more the 5.5.
RTA-2 – proximal renal tubular acidosis: the lesion in proximal RTA is impairment
of bicarbonate reabsorption. It can be primary/idiopathic or secondary to Fanconi's
syndrome. It can also be associated with other non-inherited diseases such as MM,
Sjogren syndrome, renal transplantation, hyper-D-vitaminosis, nephrotic syndrome,
etc. Bicarbonate can be completely reabsorbed if the plasma bicarbonate
concentration is low, and thus patients may excrete normal amounts of acid but at the
expense of systemic acidosis. At nearly normal bicarbonate levels, bicarbonate is lost,
but the urine pH does not fall below 5.5! Treatment consists of administering large
amounts of bicarbonate. It is characterized by normokalemia or hypokalemia,
hypocalcemia, hypophosphatemia, glycosuria, aminoaciduria, Na+ and K
+ wasting.
RTA-3 – combined renal tubular acidosis
RTA-4 – selective aldosterone deficiency: It is associated with hypoaldosteronism,
either secondary to adrenal disease, or to renal disease in which there is decreased
renin secretion (hyporeninaemic hypoaldosteronism, e.g. in diabetic nephropathy) or
resistance to aldosterone (e.g. in obstructive nephropathy). In contrast to the other
types of RTA, there is hyperkalaemia. The urine can be maximally acidified, but the
pH may decrease below 5.5.
Avi Sayag Clinical Biochemistry
The diagnosis of RTA requires a high index of suspicion. Typically, there is hyperchloraemia
and a normal anion gap.
3. Defects of urinary concentration
Impairment of urinary concentration is a feature of nephrogenic diabetes insipidus, a group of
primary tubular disorders. It is also a feature of cranial diabetes insipidus and chronic renal
failure and can occur with hypercalcaemia, hypokalaemia and certain drugs, notably lithium.
In inherited nephrogenic diabetes insipidus, ADH secretion is normal, but there is a mutation
either affecting its receptor (the V2 receptor) or aquaporin 2. Hypercalcaemia and
hypokalaemia interfere with the intracellular cAMP-mediated signalling pathway that leads to
the insertion of aquaporins into the cell membranes of the collecting ducts.
4. Glycosuria
Renal glycosuria can also occur in association with other tubular abnormalities, for example
as part of the Fanconi's syndrome.
5. Amino aciduria: can also be part of Fanconi's syndrome.
6. Hypophosphataemic rickets
This condition, also known as vitamin D-resistant rickets, has a dominant X-linked pattern of
inheritance. A defect in tubular phosphate reabsorption leads to severe rickets. This does not
respond to treatment with vitamin D alone, even if administered in massive doses, but can be
treated effectively with a combination of oral phosphate supplements and vitamin D, usually
given as a 1α-hydroxylated derivative. Hypophosphataemic rickets should not be confused
with inherited vitamin D-dependent rickets type I, an autosomal recessive condition. The
defect is in the 1α-hydroxylation of 25-hydroxycholecalciferol. This condition can be treated
with 1α-hydroxylated derivatives of vitamin D alone.
Avi Sayag Clinical Biochemistry
Topic 25 Disturbances of acid-base balance Generally, acid is a proton donor, while a base is a proton acceptor. The plasma pH is
normally between 7.35 and 7.45. That is, ~40 nmol/L H+ are in the plasma. Below 7.35
acidosis occurs (below 7.25 it's a severe one) and above 7.45 alkalosis occurs (above 7.55 it's
a severe one). Death occurs when pH drops below 6.8 or rises above 7.8. pH is measured
using ion-selective glass electrode.
Optimal pH provides conditions for optimal intracellular functions, such as enzymes, and the
intracellular trapping of metabolite intermediates is maximized at a neutral intracellular pH.
The main intermediate groups are phosphate, ammonium and carboxylic acids.
There are 2 types of acids produced intracellularly:
1. Volatile acids (respiratory acids): CO2 derived from H2CO3. Between 12000 and
13000 mmol of CO2 is produced per day (basal production).
2. Fixed acids (metabolic acids): these are not excreted by the lungs, and are referred by
their anions: lactate, phosphate, sulphate, acetoacetate, β-hydroxybutyrate.
In order to keep the balance, the amount of acid excreted must equal the amount of acid
produced per day. As acids are produced intracellularly, they are buffered within the cell, and
leave to the extracellular space while buffering is kept during transport. Then, acids are
excreted.
In the interstitial fluid, bicarbonate buffers metabolic acids.
In the blood, bicarbonate buffers metabolic acids, and Hg buffers CO2. Proteins and
phosphates are also present in these 2 compartments, but they are insignificant.
In the intracellular fluid, however, proteins and phosphates are important buffers.
In the urine, phosphate and ammonia are important buffers, while in the bone calcium
carbonate is a the major player (in chronic metabolic acidosis).
The ECF buffering comprises 43% of the total buffering capacity, and the buffers are the
bicarbonate and proteins. The ICF buffering comprises the remaining 57%, and the buffers
are proteins, phosphate and bicarbonate. This is due to entry of hydrogen ions via the Na-H
exchangers (36%), K+-H
+ exchangers (15%) and other transporters (6%).
The respiratory part in keeping the acid-base balance is determined by the arterial pCO2
(changes in arterial pCO2 changes arterial pH). Central and peripheral chemoreceptors sense
these changes (the PCR can also detect pO2, but the CCR can only detect pCO2) and signal
the respiratory centers in the medulla (the apneustic center and the inspiratory center). These
centers (mainly and directly through the inspiratory center) control the respiratory muscles,
and lead to increased minute ventilation (hyperventilation) that ultimately reduces arterial
pCO2 to its initial setpoint. This mechanism takes some 2-3 minutes to control the pH, and is
thus very fast. The renal compensatory mechanism, however, is slower (takes 2-3 days to
control pH). The mechanism compensates for the pH change by reabsorbing the filtered
bicarbonate and by excretion of the fixed acids. These occur in the proximal and distal
tubules. In the proximal tubule bicarbonate is reabsorbed (85%) and ammonium is produced.
In the distal tubule bicarbonate is reabsorbed to a much lesser extent (0-5%), ammonium is
added to the urine, and the H+ is buffered mainly by phosphate. Creatinine also contributes to
the formation of titrable acidity. The remaining bicarbonate is reabsorbed in the thick
ascending limb of the loop of Henle.
The anion gap is a good indicator of metabolic acidosis. It is the difference between the sum
of the concentrations of the principal cations (Na+ and K
+) and the principal anions (Cl
- and
HCO3-). It represents the concentration of all the unmeasured anions in the plasma. Proteins
(negative charge) account for about 10% of plasma anions and make up the majority of the
unmeasured anions under normal circumstances. The acid anions (lactate, acetoacetate,
sulphate) produced during metabolic acidosis are not measured as part of the usual lab tests.
As known, H+ reacts with HCO3
- and the CO2 produced in this reaction is excreted via the
lungs. The net effect is a decrease in the concentration of the measured anions (bicarbonate)
and an increase in the concentration of the unmeasured anions (the acid anions). Thus, the
anion gap increases. The reference range for anion gap is 8-16 mmol/L.
Avi Sayag Clinical Biochemistry
The metabolic acidosis can be organic (the lost bicarbonate is replaced by the acid anion,
which is not normally measured) or inorganic (as in HCl infusion for example). The anion gap
can help differentiate between these two: if the anion gap is normal, inorganic metabolic
acidosis is present, as the chloride anions replace the lost bicarbonate; if the anion gap is
increased, organic acidosis is present. The anion gap also helps determine the severity of the
metabolic acidosis and follow the response to treatment.
Blood gas analysis is another measure to determine alkalosis or acidosis (and whether they are
respiratory or metabolic):
pH: normally between 7.35 and 7.45;
pCO2: gives information about the respiratory side. In arterial sample the reference
range is 34-46 mmHg;
pO2: normal reference range > 60 mmHg
Standard bicarbonate: this is the concentration of bicarbonate in the plasma of fully
oxygenated blood. In other words, it tells us what the bicarbonate would be if there
were no respiratory disturbance. Therefore, it gives us information about the
metabolic side. Normal reference range 22-26 mmol/L
Actual bicarbonate: reflects the metabolic side. Normal reference range 25 mmol/L
The difference between the actual and the standard bicarbonate concentrations
indicates a respiratory alkalosis or acidosis:
o When the actual bicarbonate is higher than the standard bicarbonate, it is a
sign of respiratory acidosis.
o When the actual bicarbonate is lower than the standard bicarbonate, it is a
sign of respiratory alkalosis.
Buffer base (BB): reflects the overall base contents of the organism. It gives
information about the metabolic side. Normal reference range 44-52 mmol/L
Base excess (BE): reflects the metabolic side. Normal reference range (-2.5)- (+2.5)
mmol/L. Positive values mean base excess or lack of acids.
In order to calculate how much bicarbonate is needed to be administered to correct for
metabolic acidosis (ml) = BE x 0.3 x body weight
Causes of acidosis and alkalosis
Acidosis Alkalosis
Increased intake of acids
Increased acid production (diabetic
ketoacidosis, alcoholic ketoacidosis,
increased protein metabolism)
Impaired H+ removal (renal disease)
Loss of base (diarrhea, renal diseases, loss of
GI fluid)
Impaired removal of CO2 (lung disease)
Increased intake of basic material (increased
intake of bicarbonate in ulcers or
overcompensation with bicarbonate)
Impaired excretion of bicarbonate (kidney
disese)
Loss of acids (vomiting)
Hyperventilation (hysteria, fever, intracranial
inflammatory disease, chest wall irritation,
irritation of the breathing center,
inappropriate mechanical ventilation)
Avi Sayag Clinical Biochemistry
Topic 26 Predominant water depletion, isoosmolar volume depletion Predominant water depletion can occur when there is loss of fluid with Na
+ concentration
lower than that of the plasma, or when there is deficient water intake. Excessive loss of water
without any sodium loss is unusual, except in diabetes insipidus, but, even if there is loss of
sodium as well, provided that this is small, the clinical consequences will be related primarily
to the water depletion. There are 2 main scenarios of water depletion:
1. Water depletion in the presence of normal homeostatic mechanisms, in which
excessive water loss is due to sweating, vomiting, diarrhea, excessive respiratory loss
and excessive burns, or due to deficient water intake due to inadequate supply and
mechanical obstructions.
2. Water depletion in the absence of (or failure) of the homeostatic mechanisms for
water retention, in which there is inadequate response to thirst, diabetes insipidus,
osmotic diuresis and nephrogenic diabetes insipidus5.
Features of predomiant water depletion
With normal homeostatic mechanisms With deficient homeostatic mechanisms
Hyperosmolality
Hypovolemia (late stage)
Oliguria Polyuria (but not in case of damage to the
thirst center)
Hypernatremia
Hemoconcentration
Mild uremia
Decreased urinary Na+
Concentrated urine Diluted urine (only in diabetes insipidus and
nephrogenic diabetes insipidus)
If the patient is unconscious, predominant water depletion manifests as:
1. Pyrexia (fever)
2. Overbreathing
3. Osmotic diuresis (osmotic diuresis is increased urination caused by the presence of
certain substances in the small tubes of the kidneys. The excretion occurs when
substances of high molecular weight, such as glucose, enter the kidney tubules. The
substances cause an increase in the osmotic pressure within the tubule, causing
retention of water within the lumen, and thus reduce the reabsorption of water,
increasing urine output).
4. Diabetes insipidus
5. Inability to drink
The block in predominant water depletion is between the hypothalamus and the secretion of
ADH.
Hypernatremia accompanies predominant water depletion (details in next topic).
Isoosmolar volume depletion can occur due to:
1. Loss of small-intestinal secretion (in the presence of normal homeostatic
mechanisms). This can occur if there is a fistula in the small intestine, or if there is
obstruction or paralytic ileus in the small intestine.
2. Tubular damage with normal glomerular function (in the absence of normal
homeostatic mechanisms). This can occur during the recovery phase of acute renal
failure or when there is polyuric chronic renal failure.
5 Diabetes insipidus is a condition characterized by excessive thirst and excretion of large amounts of
severely diluted urine, with reduction of fluid intake having no effect on the latter. There are several
different types of DI, each with a different cause. The most common type is central diabetes insipidus,
caused by a deficiency of ADH, also known as antidiuretic hormone (ADH). The second common type
of DI is nephrogenic diabetes insipidus, which is caused by an insensitivity of the kidneys to ADH.
Avi Sayag Clinical Biochemistry
The hypovolemia that results from this condition manifests in hypotension, oliguria, uremia
and hemoconcentration.
The management of water depletion involves treatment of the underlying cause and
replacement of the fluid deficit. Water should preferably be given either orally or via a
nasogastric tube. If this is not possible, either 5% dextrose or, if there is also some sodium
depletion, dextrose-saline should be administered (4% dextrose, 0.18% NaCl) intravenously.
The aim should be to correct approximately two-thirds of the deficit in the first 24 h and the
remainder in the next 24 h, but plasma osmolality should not be allowed to fall too rapidly. However, 2 side effects should be borne in mind during fluid replacement:
1. If the Na+ concentration is low in the fluid replacement, predominant sodium
depletion can pursue;
2. Overcorrection with protein-free fluid will lead to increased hydrostatic pressure (and
reduced oncotic pressure), which will eventually lead to increased loss of fluid.
Avi Sayag Clinical Biochemistry
Topic 27
Water and sodium excess; predominant sodium depletion Predominant Na
+ depletion is one of 3 causes of hyponatremia, the second being water excess
and the third – excess of water and Na+.
Hyponatremia As mentioned, there are three causes for hyponatremia:
1. Depletion of Na+ (hypovolemic hyponatremia)
2. Excess of water (euvolemic hyponatremia)
3. Excess of water and sodium (hypervolemic hyponatremia)
Each one will be considered separately:
1. Predominant sodium depletion
This condition can be caused due to 2 main reasons:
a. Volume replacement with fluid of incorrect composition: in the first phase of
this condition the clinical symptom is hypovolemia alone, while in the second phase
hypoosmolality and polyuria are the dominant symptoms (hyponatraemia is a very
common finding in postoperative patients on IV fluid infusions. It usually reflects
excessive administration of hypotonic fluids (5% dextrose or 'dextrose-saline') at a
time when the ability of the body to excrete water is depressed as part of the normal
metabolic response to trauma, which includes increased release of ADH). The lab
findings in the first phase are normonatremia, hemoconcentration, increased urea
and decreased urinary Na+ concentration, while in the second phase there are
hyponatremia, hemodilution, decreased urea and increased urinary [Na+].
b. Failure of homeostatic mechanisms for Na+: this cause is primarily due to
Addison's diseases or pseudoaddison's disease. Addison's disease is a rare endocrine
disorder in which the adrenal gland produces insufficient amounts of steroid
hormones. Pseudoaddison's disease is a condition in which there is no dysfunction
of the adrenal gland, but failure of the renal tubules to respond to aldosterone (the
distal tubules and the collecting ducts). The clinical signs span from hypovolemia to
late hypoosmolality (same as in the previous cause), and the lab findings are
hemoconcentration, mild uremia, late hyponatremia and inappropriately high
urinary [Na+] (again, same as in the previous condition).
2. Predominant excess of water
There are 4 main factors leading to predominant excess of water:
Glomerular dysfunction if fluid of low [Na+] is replaced in excess to fluid loss;
Inappropriate ADH secretion (SIADH);
Oxytocin or other narcotics given in infusion (in 5% glucose or dextrose-saline);
Psychogenic polydipsia
Water excess gives rise to a dilutional hyponatremia with reduced plasma osmolality. It can
occur acutely purely due to excessive water intake, but this is rare. Normal kidneys are
capable of excreting 1 L of water per hour: water intoxication and hyponatremia will thus be
seen only when very large quantities of fluid are ingested rapidly, as is seen in some patients
with psychoses. It can also occur in people who drink large quantities of weak beer. Far more
frequently, however, the acute development of water excess and hyponatremia is a result of a
combination of excessive hypotonic fluid intake and impairment of diuresis. Since osmolality
is normally precisely controlled, the persistence of dilutional hyponatremia implies a failure
of diuresis, which must be due to either continued (and inappropriate) production of ADH (or
the presence of a drug having a ADH-like action) or an impairment of the renal diluting
mechanism.
SIADH is essentially a diagnosis of exclusion. It is frequently diagnosed on insufficient
evidence with no regard to other possible causes of hyponatremia. Both clinical information
and laboratory data are important. It is essential to measure urine and plasma osmolalities: the
urine may not be more concentrated than the plasma but must be less than maximally dilute
(osmolality >50 mmol/kg). Edema is not a feature of SIADH: the excess of water is shared by
the ICF and the ECF and the effect on ECF volume is insufficient to cause edema.
Avi Sayag Clinical Biochemistry
Measurement of ADH concentration is seldom helpful in differential diagnosis: raised values
are present in the majority of patients with hyponatremia, irrespective of the cause.
There is undoubtedly more than one type of SIADH. Tumors may produce the hormone
(ectopic production), but patients with many other conditions can also fulfill the diagnostic
criteria for SIADH. In some of these, there may be an inappropriate stimulus to ADH release,
such as stimulation of volume receptors during artificial ventilation, and in others the
'osmostat' appears to be reset, so that osmolality is still controlled but at a lower level.
Decreased intracellular organic solute ('osmolyte') content may be one mechanism whereby
the osmostat can be reset. Patients have been described in whom suppression of ADH release
when osmolality falls is incomplete (a 'ADH leak'), while in others the production of ADH is
entirely normal and antidiuresis must be presumed to reflect an abnormal response to the
hormone.
3. Water and Na+excess
Water excess
This is usually related to an impairment of water excretion. However, the limit to the ability
of the healthy kidneys to excrete water is about 20 mL/min and, occasionally, excessive
intake is alone sufficient to cause water intoxication. This can sometimes occur in patients
with psychiatric disorders. It has also been described in people drinking large amounts of beer
with a low solute content, because this results in a low osmotic load for excretion and there is
a minimum osmolality below which the urine cannot be diluted further. Increased thirst can
occur in organic brain disease (particularly trauma, and following surgery). Hyponatremia is
invariably present in water overload. The increased water load is shared by the ICF and ECF.
The clinical features of water overload are related to cerebral over-hydration, the incidence
and severity depending upon the extent of the water excess and its time course. A patient with
a plasma Na+ concentration of 120 mmol/L, in whom water retention has occurred gradually
over several days, may be asymptomatic, while one in whom this is an acute phenomenon
may show signs of severe water intoxication.
Na+excess
Na+ excess can result from increased intake or decreased excretion. The clinical features are
related primarily to expansion of ECF volume (peripheral edema, pulmonary edema, venous
congestion and hypertension). When related to excessive intake (e.g. the inappropriate use of
hypertonic saline), a rapid shift of water from the intracellular compartment may also cause
cerebral dehydration. When Na+ overload is due to excessive intake, hypernatremia is usual.
Na+overload is more usually due to impaired excretion than to excessive intake. The most
frequent cause is secondary aldosteronism. This is seen in patients who, despite clinical
evidence of increased ECF volume (e.g. peripheral edema), appear to have a decreased
effective arterial blood volume, for example due to venous pooling or a disturbance in the
normal distribution of ECF between the intravascular and extravascular compartments. This
phenomenon is particularly associated with cardiac failure, hypoalbuminaemia and hepatic
cirrhosis, damage to renal vessels, and pregnancy. The clinical symptoms of such patients are
determined by the primary condition. Lab findings may reveal mild hyponatremia or
normonatremia, a decrease in urinary Na+ concentration, hypokalemia (if loop diuretics are
administered) and other findings related to the primary abnormality. That many such patients
with Na+ excess are, paradoxically, hyponatraemic, implies the coexistence of a defect in free
water excretion. This is probably in part due to an increase in ADH secretion as a result of the
decreased effective blood volume. Also, the decrease in GFR and consequent increase in
proximal tubular Na+ reabsorption decreases the delivery of Na
+ and Cl
- to the loops of Henle
and distal convoluted tubules. This reduces the kidneys' diluting capacity, thereby
compromising water excretion. Renal disease is a relatively uncommon cause of Na+ excess,
as is increased mineralocorticoid secretion due to primary aldosteronism (PA) (as in Conn's
syndrome). It is noteworthy, however, that edema is not a feature of Conn's syndrome. The
clinical features of PA are hypertension and hypokalemia. Lab findings will reveal
hypokalemia, elevated plasma HCO3-, plasma Na in the upper limit, decreased urinary sodium
concentration, elevated aldosteron levels and decreased renin levels.
Avi Sayag Clinical Biochemistry
Measurement of serum Na+ and K
+ (practical topic 16)
There are 4 methods to measure these ions:
1. Atomic absorption spectrophotometry This is the reference method. The electrons of the atoms in the atomizer can be promoted to
higher orbitals for a short amount of time by absorbing a set quantity of energy (i.e. light of a
given wavelength). This amount of energy (or wavelength) is specific to a particular electron
transition in a particular element, and in general, each wavelength corresponds to only one
element. This gives the technique its elemental selectivity. This method is no longer used in
routine diagnostics.
2. Flame photometry If the salt of a metal is vaporized into a flame at an appropriate temperature, a part of the
metal's atoms will be excited. Light emitted from the thermally excited ions directed into a
photometer results in an electric signal. The calibrators contain Na+ and K
+ in known
concentrations. In practice, lithium salt of a constant concentration is mixed into the diluting
solution. Then, signals of the Na+ and K
+ are compared to the lithium (lithium is an ideal
internal standard as it is not present in the serum of normal individuals and its light emission
if different from that of Na+ and K
+).
Reference values: Na+: 135-145 mmol/L; K
+: 3.6-5 mmol/L.
3. Ionselective electrodes This electrode is a membrane producing electric potential, which is proportional to the
concentration of the ion. The membrane is a thin film made of solid material or plastic into
which components responsible for selectivity are mixed. In cases of an ion selective
membrane electrode, a membrane separates the internal electrolyte (known concentration of
ions) from the external electrolyte (unknown concentration), and it is selectively permeable
for an ion. This selective permeability can generate a measurable change in membrane
potential that depends on the ion activity in the sample. In direct potentiometry the ion
activity is measured in undiluted samples, while in the indirect potentiometry samples are
diluted before analysis. Reference values: Na+: 137-150 mmol/L; K
+: 3.5-5.3 mmol/L.
False results: homolytic samples (K+ concentration in RBCs is 23 greater than that of plasma);
stored sample; contamination with Na/K infusion.
Pseudohyponatremia is a falsely low [Na+] in a hyperlipemic or hyperproteinemic sample
when measured by flame photometry or indirect potentiometry.
4. Spectrophotometry
Avi Sayag Clinical Biochemistry
Topic 28 Hypokalemia Hypokalemia refers to the condition in which the concentration of K
+ in the blood is low.
Normal serum K+ levels are between 3.5 to 5.2 mmol/L; at least 95% of the body's K is found
inside cells, with the remainder in the blood. This concentration gradient is maintained
principally by the Na+/K
+-ATPase pump.
Pathophysiology
K+ is essential for many body functions, including nerve activity and skeletal and cardiac
muscle contraction and rhythm. The electrochemical gradient of K+ between the intracellular
and extracellular space is essential for nerve function; in particular, K+ is needed to repolarize
the cell membrane to a resting state after an action potential has passed. Decreased K+ levels
in the extracellular space will cause hyperpolarization of the resting membrane potential. This
hyperpolarization is caused by the effect of the altered K+ gradient on resting membrane
potential as defined by the Goldman equation. As a result, a greater than normal stimulus is
required for depolarization of the membrane in order to initiate an action potential.
In certain conditions, this will make cells less excitable. However, in the heart, it causes
myocytes to become hyperexcitable. Lower membrane potentials in the atrium may cause
arrhythmias because of more complete recovery from Na+-channel inactivation, making the
triggering of an action potential more likely. In addition, the reduced extracellular K+
(paradoxically) inhibits the activity of the IKr K current and delays ventricular repolarization.
This delayed repolarization may promote reentrant arrhythmias.
Regularly, K+ depletion leads to hypokalemia. However, an exception to this rule is
hypokalemia without K+ depletion. This occurs when there is ECF-to-ICF shift of K
+.
Causes
Hypokalemia can result from one or more of the following medical conditions:
1. Perhaps the most obvious cause is insufficient consumption of K+ (that is, a low-K
+ diet).
However, without excessive K+ loss from the body, this is a rare cause of hypokalemia.
Chronic starvation, anorexia nervosa and alcoholism reduce K+ intake.
2. A more common cause is excessive non-renal loss of K+, often associated with heavy fluid
losses that "flush" K+ out of the body. Typically, this is a consequence of vomiting, diarrhea,
laxatives, intestinal fistula6, mucus-secreting villous adenomas (K
+ loss is secondary to mucus
secretion), excessive perspiration, or losses associated with surgical procedures
3. Certain medications can accelerate the removal of K+ from the body, including:
1. Thiazide diuretics such as hydrochlorothiazide: act on the distal convoluted tubule,
where it blocks the reabsorption of Na+. Thus, in the more distal part of the tubule there
is an accelerated reabsorption of Na+ ions into the bloodstream in exchange for K
+.
2. Loop diuretics such as furosemide: for the same reason (act on the thick ascending
loop of Henle)
3. Carbonic anhydrase inhibitors, such as acetazolamide: in the proximal convoluted
tubule, this enzyme promotes reabsorption of bicarbonate. Inhibiting this enzyme will
result in non-reabsorption of NaHCO3. It will lead to metabolic acidosis, and to an
accelerated reabsorption of Na+ ions into the bloodstream in exchange for K
+ in the
more distal part of the tubule .
4. Osmotic diuretics, such as mannitol: they act on the descending loop of Henle. The
drug annexes water to it along K+.
5. Amphotericin B: the drug is toxic to the kidney (RTA-1). Nephrotoxicity can lead to
increased K+ secretion and thus to hypokalemia.
4. A special case of K+ loss occurs with diabetic ketoacidosis. In addition to urinary losses
from polyuria and volume contraction, there is also obligate loss of K+ from kidney tubules as
6 A fistula is a narrow passage or duct formed by disease or injury, as one leading from an abscess to a
free surface, or from one cavity to another.
Avi Sayag Clinical Biochemistry
a cationic partner to the negatively charged ketone, β-hydroxybutyrate. Note that this is a
special case of hyperkalemia with K+ depletion.
5. Hypomagnesemia can cause hypokalemia. Magnesium is required for adequate processing
of K+. This may become evident when hypokalemia persists despite K
+ supplementation.
Other electrolyte abnormalities may also be present.
6. Alkalosis can cause transient hypokalemia by two mechanisms. First, the alkalosis causes a
shift of K+ from the plasma and interstitial fluids into cells; perhaps mediated by stimulation
of Na+-H
+ exchange and a subsequent activation of Na
+/K
+-ATPase activity. Second, an acute
rise of plasma HCO3- concentration (caused by vomiting, for example) will exceed the
capacity of the renal proximal tubule to reabsorb this anion, and K+ will be excreted as an
obligate cation partner to the bicarbonate. It should be noted that metabolic alkalosis is often
present in states of volume depletion, and thus alkalosis is typically not the main cause of
hypokalemia seen in volume-depleted states.
7. Disease states that lead to abnormally high aldosterone levels can cause hypertension and
excessive urinary losses of K+. These include renal artery stenosis and tumors (generally non-
malignant) of the adrenal glands. Hypertension and hypokalemia can also be seen with a
deficiency of the 11-beta-hydroxysteroid dehydrogenase type 2 enzyme which allows cortisol
to stimulate aldosterone receptors. This deficiency can either be congenital or caused by
consumption of glycyrrhizin, which is contained in extract of licorice, sometimes found in
herbal supplements, candies and chewing tobacco. It follows that increased secretions of
mineralocorticoids may lead to hypokalemia (hyperaldosteronism, Cushing syndrome with
steroid therapy, ACTH therapy or ectopic ACTH secretion, renal hemangiopericytoma, and
licorice/tobacco chewing).
11-β-OH-steroid DH -----------| cortisol � aldosterone R � hypokalemia
(aldosteron R � increased Na+ reabsorption � increased K
+ secretion � hypokalemia)
Rare hereditary defects of renal salt transporters, such as Bartter syndrome or Gitelman
syndrome, can cause hypokalemia, in a manner similar to that of diuretics. As opposed to
disease states of primary excess of aldosterone, blood pressure is either normal or low in
Bartter's or Gitelman's.
Rare hereditary defects of muscular ion channels and transporters that cause hypokalemic
periodic paralysis can precipitate occasional attacks of severe hypokalemia and muscle
weakness. These defects cause a heightened sensitivity to the normal changes in K+ produced
by catechols and/or insulin and/or thyroid hormone, which lead to movement of K+ from the
extracellular fluid into the muscle cells.
Lastly, renal dysfunction leads to hypokalemia: renal tubular acidosis types I and II (RTA-I
and RTA-II), recovery phase of acute oliguric renal failure, chronic pyelonephritis, polycystic
kidney, interstitial nephritis, and amphotericin B.
Summary of Causes of Hypokalemia:
There are 3 main causes: low intake, a shift from the ECF to the ICF, and increased loss in the
urine:
1. Decreased intake: due to starvation or clay ingestion (perhaps K+
is trapped there)
2. ECF � ICF : there are 4 main causes:
1. Metabolic alkalosis
2. Hormonal: insulin (stimulates the Na-H antiporter and secondarily the Na-K
pump), α-antagonists, and β2 agonists (β2 receptors participate in glycogenolysis.
Stimulating them will result in hyperglycemia, and increased insulin level. Moreover,
hyperglycemia will lead to osmotic diuresis and K+
wasting).
3. Anabolic states: vitamin B12/folic acid, GM-CSF – pernicious anemia treated with
vitamin B12/folic acid, or neutropenia treated with GM-CSF, lead to rapid cell
growth and an increased anabolic state. Since K+ is the major intracellular cation,
there is an increased shift of K+
into the cells.
4. Others: hypokalemic periodic paralysis, hypothermia, barium toxicity.
Avi Sayag Clinical Biochemistry
3. Increased loss: this can be further subdivided into:
1. Non-renal loss: GI loss (diarrhea) or integumentary loss (sweating)
2. Renal loss: due to increased K+
secretion (RTA1, RTA2, aldosteron excess,
diabetic ketoacidosis, hypomagnesemia, amphotericin B) or increased distal flow
(diuretics, osmotic diuresis).
Signs and symptoms
Mild hypokalemia is often without symptoms, although it may cause a small elevation of
blood pressure, and can occasionally provoke cardiac arrhythmias. Moderate hypokalemia,
with serum K+ concentrations of 2.5-3 mEq/L, may cause muscular weakness, myalgia, and
muscle cramps (owing to disturbed function of the skeletal muscles), and constipation (from
disturbed function of smooth muscles). With more severe hypokalemia, flaccid paralysis,
hyporeflexia, and tetany may result. There are reports of rhabdomyolysis occurring with
profound hypokalemia with serum K+ levels less than 2 mEq/L. Respiratory depression from
severe impairment of skeletal muscle function is not uncommon. Other symptoms include
impaired glucose tolerance, symptoms of extracellular alkalosis, renal tubular damage,
impairment of memory, disorientation and confusion.
Some (ECG) findings associated with hypokalemia are flattened (or low) T waves, increased
U waves, ST segment depression, and prolongation of the QT interval. The prolonged QT
interval may lead to arrhythmias.
Treatment
The most important treatment in severe hypokalemia is addressing the cause, such as
improving the diet, treating diarrhea or stopping an offending medication. Patients without a
significant source of K+ loss and who show no symptoms of hypokalemia may not require
treatment.
Mild hypokalemia (>3.0 mEq/L) may be treated with oral K+ chloride supplements (Klor-
Con, Sando-K, Slow-K). As this is often part of a poor nutritional intake, K+-containing foods
may be recommended, such as leafy green vegetables, tomatoes, citrus fruits, oranges or
bananas. Both dietary and pharmaceutical supplements are used for people taking diuretic
medications.
Severe hypokalemia (<3.0 mEq/L) may require intravenous supplementation. Typically,
saline is used, with 20-40 mEq KCl per liter over 3-4 hours. Giving intravenous K+ at faster
rates (20-25 mEq/hr) may predispose to ventricular tachycardias and requires intensive
monitoring. A generally safe rate is 10 mEq/hr.
Difficult or resistant cases of hypokalemia may be amenable to a K+-sparing diuretic such as
amiloride. In contrast to the more commonly used diuretics like hydrochlorothiazide and
furosemide, these K+-sparing diuretics actually reduce the kidney's excretion of K
+.
When replacing K+ intravenously, infusion via central line is encouraged to avoid the frequent
occurrence of a burning sensation at the site of a peripheral IV, or the rare occurrence of
damage to the vein.
Avi Sayag Clinical Biochemistry
Topic 29 Hyperkalemia Extreme degrees of hyperkalemia are considered a medical emergency due to the risk of
potentially fatal arrhythmias (levels above 8.5 mmol/L � cardiac arrest).
Signs and symptoms
Symptoms are nonspecific and generally include malaise, palpitations and muscle weakness;
mild hyperventilation may indicate a compensatory response to metabolic acidosis, which is
one of the possible causes of hyperkalemia. Often, however, the problem is detected during
screening blood tests for a medical disorder, or it only comes to medical attention after
complications have developed, such as cardiac arrhythmia or sudden death.
Diagnosis
In order to gather enough information for diagnosis, the measurement of K+ needs to be
repeated, as the elevation can be due to hemolysis in the first sample. The normal serum level
of K+ is 3.5 to 5.2 mmol/L. Generally, blood tests for renal function (creatinine, blood urea
nitrogen), glucose and occasionally creatine kinase and cortisol) will be performed.
Calculating the trans-tubular K+ gradient can sometimes help in distinguishing the cause of
the hyperkalemia.
In many cases, renal ultrasound will be performed, since hyperkalemia is highly suggestive of
renal failure.
Also, ECG may be performed to determine if there is a significant risk of cardiac arrhythmias.
Causes include: 1. Ineffective elimination from the body
Renal insufficiency
Medication that interferes with urinary excretion:
ACE inhibitors and angiotensin receptor blockers
K-sparing diuretics (e.g. amiloride and spironolactone)
NSAIDs
The calcineurin inhibitor immunosuppressants ciclosporin and tacrolimus
The antibiotic trimethoprim
The antiparasitic drug pentamidine
Mineralocorticoid deficiency or resistance, such as:
Addison's disease
Aldosterone deficiency, including reduced levels due to heparin
Some forms of congenital adrenal hyperplasia
Type IV renal tubular acidosis (resistance of renal tubules to aldosterone)
Gordon's syndrome (“familial hypertension with hyperkalemia”), a rare genetic disorder
caused by defective modulators of salt transporters, including the thiazide-sensitive Na+-Cl
-
cotransporter.
2. Excessive release from cells
Rhabdomyolysis, burns or any cause of rapid tissue necrosis, including tumor lysis syndrome.
Massive blood transfusion or massive hemolysis
Shifts/transport out of cells caused by acidosis, low insulin levels (which lead to decreased
activity of the Na+-K
+ pump), beta-blocker therapy, digoxin overdose (blocks the Na
+-K
+
pump), or the paralyzing anesthetic succinylcholine (depolarizing muscle relaxants can
increase the plasma K+).
3. Excessive intake
Intoxication with salt-substitute, K+-containing dietary supplements, or KCl infusion. Note
that for a person with normal kidney function and nothing interfering with normal
elimination, hyperkalemia by K+ intoxication would be seen only with large infusions of KCl
or massive doses of oral KCl supplements. Administration of high doses of penicillin may
also lead to hyperkalemia, since penicillin is given as K+ salt or Na
+ salt.
4. Lethal injection
Hyperkalemia is intentionally brought about in an execution by lethal injection, with KCl
being the third and last of the three drugs administered to cause death.
Avi Sayag Clinical Biochemistry
5. Pseudohyperkalemia
Pseudohyperkalemia is a rise in the amount of K+ that occurs due to excessive leakage of K
+
from cells, during or after blood is drawn. It is a laboratory artifact rather than a biological
abnormality and can be misleading to caregivers. Pseudohyperkalemia is typically caused by
hemolysis during venipuncture; excessive tournequet time or fist clenching during
phlebotomy (which presumably leads to efflux of K+ from the muscle cells into the
bloodstream); or by a delay in the processing of the blood specimen. It can also occur in
specimens from patients with thrombocytosis, leukocytosis, or erythrocytosis
(hematocrit>55%).
The renal elimination of K+ is passive (through the glomeruli), and reabsorption is active in
the proximal tubule and the ascending limb of the loop of Henle. There is active excretion of
K+ in the distal tubule and the collecting duct; both are controlled by aldosterone.
Increased extracellular K+ levels result in depolarization of the membrane potentials of cells.
This depolarization opens some voltage-gated Na channels, but not enough to generate an
action potential. After a short while, the open Na channels inactivate and become refractory,
increasing the threshold to generate an action potential. This leads to the impairment of
neuromuscular, cardiac, and gastrointestinal organ systems. Of most concern is the
impairment of cardiac conduction which can result in ventricular fibrillation or asystole.
During extreme exercise, K+ is released from active muscle and the serum K
+ rises to a point
that would be dangerous at rest.
Patients with the rare hereditary condition of hyperkalemic periodic paralysis appear to have a
heightened sensitivity of muscular symptoms that are associated with transient elevation of K+
levels. Episodes of muscle weakness and spasms can be precipitated by exercise or fasting in
these subjects.
ECG findings: peaked T waves.
Treatment: Acute: When arrhythmias occur, or when K+ levels exceed 6.5 mmol/l, emergency
lowering of K+ levels is mandated: Ca
+2 supplementation (calcium gluconate) does not lower
K+ but decreases myocardial excitability, protecting against life threatening arrhythmias.
Insulin will lead to a shift of K+ ions into cells.
Bicarbonate therapy is effective in cases of metabolic acidosis.
Salbutamol (Ventolin), a β2-selective
Refractory or severe cases may need dialysis to remove the K+ from the circulation.
Avi Sayag Clinical Biochemistry
Topic 30 Pathogenesis of diabetes mellitus Diabetes mellitus (DM) is characterized by a tendency to chronic hyperglycaemia with
disturbances in carbohydrate, fat and protein metabolism that arise from a defect in insulin
secretion or action or both.
Diabetes can occur secondarily to other diseases (see table). Most cases of DM are primary.
There are two distinct types. In type 1 DM, there is destruction of pancreatic cells, leading to
a decrease in, and eventually cessation of, insulin secretion. Approximately 10% of all
patients with diabetes have type 1. They have an absolute requirement for insulin. In type 2
DM, insufficient insulin is secreted to prevent hyperglycaemia, often because of resistance to
its actions. Most patients with type 2 DM can initially be successfully treated by diet, with or
without oral hypoglycaemic drugs, but many eventually require treatment with insulin to
achieve adequate glycaemic control. Type 1 DM usually presents acutely in younger people,
with symptoms developing over a period of days or only a few weeks. However, there is
evidence that the appearance of symptoms is preceded by a 'prediabetic' period of several
months during which growth failure (in children), a fall in insulin response to glucose and
various immunological abnormalities can be detected. Type 2 DM tends to present more
chronically in the middle-aged and elderly (although it is increasingly being diagnosed in
obese young people), with symptoms developing over months or even longer. The prevalence
of type 2 DM is over 10% in people over the age of 75 years.
The previously used terms, 'insulin-dependent' and 'juvenile-onset' diabetes (for type 1) and
'non-insulin-dependent' and 'maturity-onset' diabetes (for type 2) are obsolete! It has become
apparent that some young patients with diabetes are not insulin dependent, while
approximately 10% of patients developing diabetes over the age of 25 have 'latent
autoimmune diabetes of adulthood' (LADA). Patients with LADA may be misclassified as
having type 2 DM. However, in comparison with patients with true type 2 DM, they tend to
present at a younger age, are less likely to be overweight, have plasma markers of
autoimmunity and, although often treated successfully with diet alone or diet and oral agents
initially, develop a requirement for insulin, often within a year of diagnosis.
Type 1 diabetes mellitus
Type 1 DM is an autoimmune disease. There is a familial incidence, though to a lesser extent
than with type 2 DM, and there is a strong association with certain histocompatibility
antigens, for example HLA-DR3, DR4 and various DQ alleles. An individual's HLA antigens
are genetically determined but it is clear that type 1 DM is a genetically heterogeneous
disorder. Environmental factors are also important and there is considerable circumstantial
evidence that viral antigens (e.g. Coxsackie B) may initiate the autoimmune process in some
genetically susceptible individuals. Proteins in cows' milk have also been implicated.
The pancreatic islets of newly diagnosed patients with type 1 DM show characteristic
histological features of autoimmune disease. Islet cell antibodies (ICA) are frequently present
in the plasma (and may be detectable long before the condition presents clinically), together
with antibodies to insulin and glutamic acid decarboxylase (GAD), which, like ICA, are
sensitive markers of risk of progression to clinical diabetes in the apparently healthy members
of patients' families.
It is thought that β-cell destruction is initiated by activated T-lymphocytes directed against
antigens on the cell surface, possibly viral antigens or other antigens that normally are either
not expressed or not recognized as 'non-self'.
Type 2 diabetes mellitus
Type 2 is characterized by impaired insulin secretion, insulin resistance (a defect downstream
the insulin receptor), excessive hepatic glucose production, and abnormal fat metabolism.
The exact pathogenesis of type 2 DM is uncertain. It is undoubtedly a heterogeneous disease.
In established cases, β-cell dysfunction with an inadequate insulin response to hyperglycemia
and insulin resistance usually coexist but it is not clear which is the primary defect:
hyperglycemia itself causes insulin resistance and β-cell dysfunction (glucotoxicity); so, too,
does hyperlipidemia (lipotoxicity), which is frequently present in diabetes. Immune
Avi Sayag Clinical Biochemistry
mechanisms are thought to contribute to the loss of insulin secretion in approximately 10% of
patients.
Type 2 DM shows a strong familial incidence.
Several single gene defects have been identified in specific subsets of patients with type 2
DM, notably in the dominantly inherited forms that typically develop in the young (MODY,
maturity-onset diabetes of the young). The commonest mutations responsible for MODY are
in the glucokinase gene (MODY type 2: six types of MODY have been described, each due to
a different mutation – see table). Glucokinase is the rate-limiting enzyme of glucose
metabolism in pancreatic β-cells and through acting as a 'glucose sensor' is key to the
regulation of pancreatic insulin secretion. Such specific mutations are, however, rare in type 2
DM considered overall, where the tendency to develop diabetes is polygenic and there is no
clear pattern of inheritance.
Environmental factors are also important. Many patients with type 2 DM are obese,
particularly tending to have visceral obesity, which is known to cause insulin resistance.
Reduced physical activity also causes insulin resistance, and various drugs, including
corticosteroids, thiazides in high doses and some β-adrenergic antagonists, are diabetogenic.
The interaction between genetic and environmental factors in the pathogenesis of type 2 DM
is exemplified by the high prevalence of the condition in certain ethnic groups (e.g. Pacific
islanders) following the adoption of a westernized lifestyle, with good public health facilities
and ready access to an assured food supply, in comparison with the prevalence in their
aboriginal state. The suggestion is that their genotype evolved to maximize the storage of
ingested energy as fat, to provide protection against famine, but that a continuous food supply
leads to obesity and insulin intolerance (the 'thrifty genotype' hypothesis). There is also a
'thrifty phenotype' hypothesis, based on the observation that low birthweight is associated
with an increased risk of later development of type 2 DM, the putative mechanism being β-
cell dysfunction induced by fetal malnutrition.
Type 2 DM is a progressive condition. Although there is evidence that it can be prevented in
susceptible individuals by diet and exercise, by the time it presents clinically it will often have
been present for several years. Aggressive treatment may slow its progression, but the
tendency is for continuing loss of β-cell function and increasing insulin deficiency.
Glucose and other nutrients regulate insulin secretion by the pancreatic beta cell. Glucose is transported
by the GLUT2; subsequent glucose metabolism by the β cell alters ion channel activity, leading to
insulin secretion. The SUR (Sulfonylurea receptor) receptor is the binding site for drugs that act as
insulin secretagogues. Mutations in the events or proteins underlined are a cause of MODY or other
forms of diabetes.
I. II. III. IV.
Type 1:
β cell
destruction
Type 2:
Insulin
resistance
with def.
Genetic
defects of β
cells
Genetic
defects in
insulin action
Diseases of
pancreas
Endocrinopathies Drug-
induced
Infections Other
associated
conditions
Gestational
C
A
U
S
E
S
Immune
mediated
Idiopathic
Insulin
resistance
Impaired
secretion
MODY1 –
mutated
HNF 4α
MODY 2 –
glucokinase
MODY 3 –
HNF 1α
MODY 4 –
IPF-1
MODY 5 –
HNF 1β
MODY 6 –
NeuroD1
Type A insulin
resistance
Leprechaunism
RMS
LS
Pancreatitis
Pancreatectomy
Neoplasia
CF
Hemochromatosis
Acromegaly
Cushing's
Glucagonoma
Pheochromocytoma
Somatostatinoma
Aldosteronoma
Thyrotoxicosis
Pentamidine
Nicotinic
acid
IFNα
Phenytoin
Clozapine
Thiazides
Rubella
CMV
Coxsackie
Down's sy.
Turner sy.
Klinefelter's
sy.
4% of
pregnant
women.
30-60%
will
develop
DM later
in life
MODY – maturity onset of diabetes of the young
HNF – hepatocyte nuclear transcription factor
IPF – Insulin promoter factor
Leprechaunism - (also known as Donohue syndrome) is an extremely rare and severe genetic disorder. Leprechaunism derives its name from the fact that
those afflicted with the disease often have elfin features and are smaller than usual. Affected individuals have an insulin receptor with greatly impaired
functionality.
RMS – Rabson-Mendenhall syndrome - rare insulin receptor disorder characterized by severe insulin resistance, developmental abnormalities, and
acanthosis nigricans.
LS – Lipodystrophy syndrome - a disturbance of lipid metabolism that involves the partial or total absence of fat and often the abnormal deposition and
distribution of fat in the body. Insulin resistance is a feature of this syndrome.
Topic 31 Lab diagnosis and management of diabetes mellitus The aim of lab tests regarding DM is to diagnose DM, to monitor the therapy, to predict late
complications, to diagnose metabolic complications and to manage them.
The diagnosis of diabetes mellitus depends upon the demonstration of hyperglycaemia. In a
patient with classic symptoms and signs, this may be inferred from the presence of glycosuria
but glycosuria is not diagnostic of diabetes, even in the presence of classic clinical
features. Therefore, urine samples should be collected (collected or random) as well as
capillary/venous blood (random or in fasting). Note the reference range is somewhat different
for capillary blood and venous blood. The diagnosis is made by detection of fasting plasma
glucose (FPG) and Oral Glucose Tolerance Test (OGTT). The WHO has issued diagnostic
criteria for DM based on 2 principles:
1. The spectrum of FPG and the response to OGTT varies among individuals, and
2. DM is defined as the level of glycemia at which diabetes-specific complications occur
rather than on deviations from a population-based mean.
The results may have some errors: samples stored at 20ºC will show a decrease of 0.4 mmol/L
in glucose levels per hour of storage, while those stored at 4ºC will have an error of 0.1
mmol/L per hour of storage. The sampling may clearly be inappropriate if taken during
glucose infusion.
Apart from determination of glucose levels, antibodies can also be detected to determine the
cause/type of diabetes: antibodies against Islet cell, antibodies against insulin, or antibodies
against glutamic acid decarboxylase (GAD).
These values taken from Harrison do not correspond with those of the department. Here are
the values of the department.
Fasting plasma glucose cut-off values:
Normal: < 6.1 mmol/L (if venous/capillary blood: < 5.6 mmol/L)
Impaired fasting glycemia (IFG): 6.1-7 mmol/L (if venous/capillary blood: 5.6-6.1
mmol/L)
DM: > 7 mmol/L (if venous/capillary blood: > 6.1 mmol/L)
In a patient with typical features of DM (polyuria, polydipsia…) a single measurement is
sufficient:
Fasting plasma glucose: > 7 mmol/L
Random venous plasma: > 10 mmol/L
Random capillary blood: > 11.1 mmol/L
In the absence of symptoms, any of these limits must be exceeded on more than one occasion
for the diagnosis to be made. Individuals who have fasting blood glucose concentrations that
are elevated but not in the diabetic range have impaired fasting glycaemia (IFG). Their
Avi Sayag Clinical Biochemistry
response to a glucose load should be tested to determine whether they have diabetes. Other
indications include unexplained glycosuria7 particularly in pregnancy, the presence of clinical
symptoms with normal fasting glucose, and diagnosis of acromegaly. The OGTT is performed
as follows:
The patient should eat normally for 3 days, with at least 250 grams of sugars;
The patient fasts overnight
Basal blood sample is taken for glucose determination
75g glucose are given in 250-300ml water over the course of 5 minutes (children are
given 1.75g/kg, but it should not exceed 75g!)
The patient then rests for 2 hours. Smoking is not allowed. He/she is allowed to drink
water
Blood sample is taken after 2 hours for glucose determination.
Interpretation of results
DM is diagnosed if the patient's glucose level exceeds 11.1 mmol/L.
Impaired glucose tolerance: 7.8-11.1 mmol/L
Impaired fasting glycemia: 6.1- 7.8 mmol/L
Normal result: < 6.1 mmol/L
Note again that the OGTT is not performed routinely!
Management
Education of patients is vital along with dietary control with or without oral hypoglycaemic
agents in patients with type 2 DM (at least initially; insulin is often required later in the course
of the condition), and with diet and insulin in patients with type 1 DM.
Regular follow-up is essential to monitor treatment and detect early signs of complications,
particularly retinopathy, which can, in many cases, be treated successfully, and nephropathy,
since treatment may slow its progression.
The aims of treatment are two-fold: to alleviate symptoms and prevent the acute metabolic
complications of diabetes, as well as to prevent long-term complications.
In type 1 DM, there is an increasing tendency to use 'basal-bolus' regimens of insulin
injections, whereby a long acting insulin is given at night (to mimic the basal insulin secretion
that occurs even during fasting) with boluses of short-acting insulin at meal times. Continuous
subcutaneous insulin infusion is also being used, but is demanding for the patient. Hitherto,
insulin has had to be given by injection, as it is degraded in the gut. Alternative routes of
administration, for example inhaled, are now being developed and introduced.
In patients with type 2 DM, improved control over that achieved with oral hypoglycaemics
alone can often be achieved by giving a single injection of a long-acting insulin at night,
continuing with the oral agents during the day, but standard insulin regimens may be required
for some patients.
Drugs used in the treatment of type 2 DM include metformin (a biguanide, decreases
glycogenolysis); sulphonylureas (enhances insulin secretion by acting on SUR);
thiazolidinediones (glitazones, agonists of the peroxisome proliferator-activated receptor γ
(PPARγ), which enhance the actions of insulin by increasing the sensitivity to it), and
meglitinides (rapidly and short acting insulin secretagogues by stimulating the smaller subunit
of the K+ channel).
Monitoring The aim of monitoring DM is to control the metabolic balance and to predict complications. 4
tests are used for these purposes:
1. Blood glucose: monitored daily for children and adolescents with DM. Blood glucose
should be more than 3 mmol/L, fasting blood glucose should be less than 7 mmol/L,
and after a meal it can rise up to 10 mmol/L;
2. Urinary glucose: same as for blood glucose; During infections and poor metabolic
control, daily urine tests for ketonuria should be performed.
7 Glycosuria is detected in the urine by dipstick (insensitive but specific). It occurs when plasma
glucose is > 11 mmol/L and the GFR is normal, or when the maximal tubular reabsorption capacity for
glucose is decreased (renal glycosuria).
Avi Sayag Clinical Biochemistry
3. Glycated Hb (HbA1c): should be checked at least every 3 months. The levels should
be between the mean of normal range and up to 1-2% above the range; The
International Diabetes Federation and American College of Endocrinology
recommend that HbA1c values be below 6.5%, while the American Diabetes
Association recommends that the Hb A1c be below 7.0% for most patients.
4. Fructosamine: monitors the level of blood glucose in the past 3 weeks. There is no
standard reference range available for this test. The reference values depend
upon the factors of patient age, gender, sample population, and test method.
Hence each laboratory report will include their specific reference range for the
test. Fructosamine = (HbA1c – 1.61) x 58.82
The control of DM can be made at home (as mentioned in "Management"). If the metabolic
control is good, then DM should be controlled 4 times a year for fasting plasma glucose (for
type II DM), fructosamine and HbA1c, and once a year for renal functions and lipid
parameters.
The complications of DM are:
1. Diabetic retinopathy;
2. Diabetic neuropathy;
3. Diabetic nephropathy; In the initial stage, this condition is predicted by the presence
of microalbuminuria, and in later stage by macroalbuminuria and creatinine.
Microalbuminuria is screened and diagnosed when morning albumin is greater than
20 mg/L and the albumin/creatinine ratio is over 2.5 mg/mmol. In the control of
microalbuminuria urine is collected over 24 hours. If more than 20 microgram/min
(or 30 mg/day) are collected, microalbuminuria is diagnosed (control should be
performed every 6 months). Note that microalbuminuria means the presence of small
amount of albumin in the urine.
4. Cataract
5. Infections
6. Complications of pregnancy: pregnancy is NOT recommended if HbA1c is greater
than 10%. The target values in pregnancy should be pursued:
a. Fasting plasma glucose: < 5.6 mmol/L
b. Post-prandial plasma glucose: < 8 mmol/L
c. HbA1c/fructosamine: up to upper limit of normal range
7. Premature coronary artery disease;
8. Cerebral and peripheral vascular diseases: these are predicted and diagnosed by the
determination of cholesterol, triglycerides, HDL, LDL, VLDV, chylomicrons.
9. Impaired wound healing;
10. The prediction of acute metabolic complications is made by the detection of ketone
bodies in the urine.
Screening:
1. All individuals above 45 should be screened for DM once in 3 years if the results are
normal.
2. More frequent screening in the following cases:
a. Obesity;
b. A family member with DM;
c. Ethnic groups of high risk;
d. Women who had GDM or delivered a baby over 4500g;
e. Hypertensive patients;
f. HDL < 0.9 mmol/L, Tg > 2.8 mmol/L
g. If previous results suggested IGT or IFG
Avi Sayag Clinical Biochemistry
Determination of glucose in serum; point of care tests
(practical topic 17) Glucose in the serum can be detected using lab assays and quick tests.
Lab assays: there are 4 assays available:
1. Glucose oxidase (GOD) assay;
2. Oxygen consumption in GOD reaction
3. Hexokinase assay
4. Glucose dehydrogenase assay
1. Glucose oxidase assay
In this reaction D-glucose + O2 ------> gluconic acid + H2O2 (carried by GOD)
Then, H2O2 +para-amino-phenazone + m-cresol -------> H2O + oxidized product (carried by
peroxidase)
GOD is highly specific for D-glucose, but the second step involving the peroxidase is less
specific.
The intensity of the color product is proportional to the glucose concentration up to 30
mmol/L. Above the linearity limit (30mmol/L) the sample has to be diluted and the result
should be multiplied by the degree of dilution.
After the reagent is added to the sample, it is mixed and kept at room temperature for 30
minutes. The absorbance is determined at 520nm against the reagent blank.
Glucose (mmol/L) = (Abs of sample/Abs of standard) X 7.9
Disadvantages:
Many compounds in the serum/urine (e.g. bilirubin, ascorbic acid, uric acid) can be
oxidized by H2O2 leading to a negative bias (falsely low glucose value)
Some compounds may react by oxidizing the indicator dye leading to a positive bias
(falsely high glucose values).
2. O2 consumption
The amount of O2 consumed in the GOD reaction can be measured by an oxygen sensitive
electrode. This method is highly specific for glucose.
3. Hexokinase assay
Glucose + ATP ------> G-6-P + ADP (carried by hexokinase)
G-6-P + NADP ------> 6-phosphogluconate + NADPH + H+ (carried by G6PDH)
4. Glucose dehydrogenase (GDH)
Glucose + NAD --------> D-gluconolactone + NADH + H+
Reference range: 3.6-6 mmol/L
Falsely low values may be obtained due to glycolysis by blood cells. Glucose level may
decrease if the plasma/serum is not separated from the cellular elements of blood within 1.5-2
hours following blood sampling. Glycolysis in whole blood causes a 5%/hour decrease in
glucose level. This value may be higher in samples with high WBC count or in case of
bacterial contamination. Samples may be stored for prolonged periods if glycolysis is
prevented (by drawing blood into tubes containing NaF).
Falsely high values may be obtained if:
The absorbance of the blank of lipemic serum is not subtracted from the absorbance
of the sample.
The sample is drawn during glucose infusion or right after it.
If fasting samples are taken after meal (inappropriate timing).
Quick tests: using home monitoring devices, glucose values obtained from whole blood are
about 12% lower than plasma values due to difference in water content (glucose is equally
distributed between cellular water and plasma water, but the water content of RBCs is lower
than that of the plasma. The device is generally a strip impregnated by a mixture of GOD and
catalase that ensure the color reaction. The color is evaluated either by a color scale or by a
reflectance photometer.
Avi Sayag Clinical Biochemistry
Point of care tests (POCT)
These tests can be carried out in an inpatient setting, outpatient clinics and by the
patients themselves.
The values measured by the POCT device should be compared with that of the central
lab every 4 months.
There are no strict professional or personnel requirements for POCT.
If laboratory automatization and robotization reduce the turnaround time for a given
test below 30 minutes, then POCT indication is questionable. Thus, for example,
some professional guidelines suggest that if the test results required for MI diagnosis
(CK-MB, troponin, IMA) cannot be delivered within 30 minutes, a POCT should be
used.
Advantages:
o The test can be performed at the site of care.
o A negligible amount of sample is needed (10-15µL)
o Treatment can be started immediately and its efficacy can be followed at the
site of care.
o The devices are compact and cheap.
o They are movable, and usually do not require external power supply.
o The greatest advantage is the very short turnaround time (1-15 minutes).
Disadvantages:
o The customized POCT is quite expensive compared to the serial
measurements carried out in the central lab.
o The accuracy of the POCT does not match that of the tests performed at the
central lab.
o As many people use the device, it leaves more room for errors.
o The older POCT devices cannot always be connected to the central lab's
information system.
The most frequent POCT is the glucose test.
The first POCT used 56 years ago is the pregnancy test.
Other POCTs: cholesterol tests and triglyceride tests. The instrument measures the
amount of light reflected by the dye and translates it into the analyte concentration.
Avi Sayag Clinical Biochemistry
Topic 32 Acute metabolic complications of DM The complications that might accompany diabetes mellitus are:
Diabetic ketoacidosis with or without hyper- and/or hypokalemia (more frequently
associated with type 1 DM)
Non-ketotic hyperglycemia (hyperosmolal non-ketotic coma; more frequently
associated with type 2 DM)
Hypoglycemia
CVA
Lactic acidosis
Diabetic nephropathy
Lipoprotein metabolism in DM
The first 4 can lead to coma.
The first 2 are the 2 main acute complications.
Diabetic ketoacidosis
Coma can be induced by acidosis and hypovolemia. As the intracellular glucose level
decreases, and in the absence of insulin to facilitate glucose intake into the cells (DM type 1),
the cell starts to utilize fatty acids for energy production. Ketone bodies are formed and
accumulate in the plasma (ketonemia). This directly can cause coma, and indirectly through
hyperventilation and vomiting, can cause loss of water, Na+ and K
+, which results in
hypovolemia (that in turn leads to coma). The other pathway leading to coma is through
glycogenolysis, gluconeogenesis and decreased glucose intake into the cells, all are the
consequence of decreased insulin levels. These will cause hyperglycemia, which will induce
osmotic diuresis and a loss of water, Na+ and K
+. As in the previous pathway, this loss will
lead to hypovolemia and coma. As a result of the hypovolemia, the GFR reduces and urea
accumulates in the blood. Proteolysis will also lead to accumulation of amino acids in the
plasma, which in turn will increase urea synthesis and its accumulation in the plasma
(uremia). Thus, ketoacidosis may be the presenting feature of type 1 DM, or may develop in a
patient known to be diabetic who omits to take his insulin or whose insulin dosage becomes
inadequate because of an increased requirement, for example as a result of infection, any
acute illness such as myocardial infarction, trauma or emotional disturbance. The 2 main
ketone bodies responsible for the ketoacidosis are acetoacetate and β-hydroxybutyrate.
Diabetic ketoacidosis is a medical emergency.
Following notification to the lab, two primary tests should be performed in diabetic
ketoacidosis:
1. Blood test: test for glucose, Na+, K
+, HCO3
-, pH and pCO2.
2. Urine test: test for glucose and ketone bodies.
If the potassium levels are low in DM, it can be due to:
1. Osmotic diuresis;
2. Enhanced aldosterone secretion (water and sodium loss)
3. Vomiting.
If the potassium levels are high in DKA, it can be due to:
1. Metabolic acidosis;
2. Disorders of intermediary metabolism;
3. Insulin deficiency;
4. Enhanced muscle catabolism;
5. ICF-to-ECF shift;
6. Renal insufficiency.
A ketotic state is not necessarily caused by DM, but can also be caused by fasting state and
vomiting. Whereas glucose levels are normal or slightly decreased in fasting and vomiting,
they are elevated in DKA.
Avi Sayag Clinical Biochemistry
Detection of ketone bodies can be performed by sampling urine and blood:
1. Urine: Legal test/Ketostix8
2. Blood: Acetest and/or enzymatic detection of β-hydroxybutyrate
Note that most tests do not detect β-hydroxybutyrate, and ketonemia can appear without
ketonuria. This occurs when the GFR is low.
Summary of overall lab findings in ketoacidosis:
Plasma: hyperglycemia, acidosis (low bicarbonate), hyperkalemia, elevated amylase levels,
hyperphosphatemia, presence of ketone bodies, hemoconcentration and mild uremia (note that
some parameters are included in the primary tests while some are not).
Urine: ketone bodies, glycosuria and low pH.
Note that if the patient is comatose due to ketoacidosis, the lab tests that confirm the source of
coma are elevated plasma glucose, low plasma bicarbonate levels, elevated urine glucose
and elevated urine ketone bodies.
Non ketotic hyperglycemia
Not all patients with uncontrolled diabetes develop ketoacidosis. In type 2 DM, severe
hyperglycemia can develop (blood glucose concentration >50 mmol/L) with extreme
dehydration and a very high plasma osmolality, but with no ketosis and minimal acidosis.
This complication is often referred to as hyperosmolar non-ketotic hyperglycemia, but
patients with ketoacidosis usually also have increased plasma osmolality, although not to the
same extent.
Why is it non-ketotic? There is sufficient insulin secretion to prevent the excessive lipolysis
and to oppose the ketogenic action of glucagon that are essential for the generation of
ketoacidosis.
Thus, if the patient is comatose due to hyperosmolal non-ketotic hyperglycemia, the lab tests
that confirm the source of coma are elevated plasma glucose levels with normal (or slightly
depressed) bicarbonate level and elevated urine glucose level.
The issue of hypoglycemia is addressed in the next topic.
8 Ketostix is Bayer's brand name for test strips to measure the level of ketones in the urine. A strip
consists of a thin piece of plastic film slightly larger than a matchstick, with a reagent pad on one end
that is either dipped into a urine sample or passed through the stream while the user is voiding. The pad
is allowed to react for a short time, then its resulting color is compared to a graded shade chart
indicating a detection range from negative presence of ketones up to a significant quantity.
Avi Sayag Clinical Biochemistry
Summary and presentation of the topic:
Pathogenesis of DKA:
Intracellular glucose ↓ + no insulin
FA are utilized/lipolysis
Ketone bodies formed
Ketonemia
Coma hyperventilation
and vomiting
loss of water, Na, K
Uremia GFR ↓ hypovolemia
Coma
Urea
Proteolysis
Insulin ↓
Insulin ↓
Glycogenolysis + gluconeogenesis
Hyperglycemia
Osmotic diuresis
Loss of water, Na, K
Hypovolemia
Coma
Lab: blood test (glucose, Na, K, pH, HCO3-, pCO2); urine test (glucose and ketone bodies)
Reasons for decreased K levels and increased K levels
Non-ketotic hyperglycemia
High FPG + high urine glucose + normal HCO3 � non-ketotic hyperglycemia
High FPG + high urine glucose + low HCO3 � ketoacidosis
Avi Sayag Clinical Biochemistry
Topic 33 Hypoglycemia Hypoglycemia is defined as blood glucose concentration of less than 2.2 mmol/L. Suspicion
of hypoglycemia should arise when the patient is unconscious, behaves strangely or presents
with an altered mental state. Hypoglycemia is more frequently due to decreased hepatic
glucose production than due to increased tissue uptake.
The causes of hypoglycemia can be divided into those causing a low blood glucose
concentration during fasting (fasting hypoglycaemia – at night or early morning, after
physical exercise, or in a severe organic disease) and those in which it follows a stimulus
(reactive hypoglycaemia), including a meal (post-prandial hypoglycaemia), drugs (the
commonest causes are insulin and hypoglycaemic drugs) and alcohol consumption.
The clinical features of hypoglycemia are the result of dysfunction of the nervous system
(neuroglycopenia):
Acute symptoms due to neuroglycopenia include tiredness, confusion, lack of concentration,
ataxia, dizziness, paraesthesiae, hemiparesis, coma, vertigo and diplopia.
Acute symptoms due to sympathetic stimulation are trembling, pallor, tachycardia and
sweating.
There are other non-specific signs in the acute phase which are hunger, weakness and blurred
vision.
In chronic neuroglycopenia the patient presents with personality changes, memory loss,
psychosis and dementia.
Diagnosis
There are two stages in the diagnosis of hypoglycaemia: confirmation of the low blood
glucose concentration and elucidation of the cause. In children and young adults, symptoms
will usually be present only with a concentration of less than 2.2 mmol/L. The elderly tend to
be more sensitive to low blood glucose concentrations; neonates, however, often develop
features only when the blood glucose is <1.5 mmol/L. Although reagent strips and
glucometers can be used to confirm a clinical suspicion of hypoglycaemia, they are
insufficiently accurate at low blood glucose concentrations to provide a definitive diagnosis
and formal laboratory measurements should be used. Blood must be collected into a container
with sodium fluoride, to inhibit glycolysis. Glucose, insulin and C-peptide should be tested
for.
Reactive hypoglycemia Reactive hypoglycemia can be caused by drugs (insulin, sulphonylurea), after a meal
(essential or following gastric surgery), alcohol and others (galactose, fructose and leucine).
Drug-induced hypoglycemia
Most patients with type 1 diabetes experience occasional episodes of hypoglycemia. Attempts
to attain optimum glycemic control increase the risk of hypoglycemia. The secretion of
glucagon becomes impaired in established type 1 diabetes, and the lack of this important
counter-regulatory hormone impairs the body's natural defenses against hypoglycemia.
Insulin: disturbed patients sometimes deliberately administer excessive insulin to
attract attention.
Sulphonylureas: in patients with type 2 diabetes, hypoglycemia can complicate
treatment with sulphonylureas. Chlorpropamide is most frequently implicated. It has a
long plasma half-life and, since it is renally excreted, tends to accumulate in patients
with impaired renal function.
β-Adrenergic blockers: occasionally cause hypoglycemia, but only when other factors
such as starvation, severe exercise or liver disease are involved. β2 receptor
stimulation increases glycogenolysis in the liver. Thus, blocking these receptors
decreases the breakdown of glycogen and facilitates hypoglycemia.
Salicylates: children, but not adults, poisoned with salicylates may develop severe
hypoglycemia. The mechanism of the enhanced insulin secretion due to salicylic acid
appears to be mediated by prostaglandin synthesis inhibition.
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Paracetamol: patients who have taken overdoses of paracetamol (it is probably related
to the severe liver damage that this drug can cause).
Post-prandial hypoglycemia
In patients who have undergone gastric surgery involving either a gastrointestinal anastomosis
or a pyloroplasty, hypoglycemia, developing 90-150 min after a meal, particularly a meal rich
in sugar, is common. There is rapid passage of glucose into the small intestine and release of
hormones that stimulate insulin secretion. The insulin response is excessive and
hypoglycemia ensues as glucose absorption from the gut falls off rapidly, rather than slowly
as it does when gastric emptying is normal.
Symptoms suggestive of hypoglycemia following meals may be described by people who
have not undergone surgery (essential or idiopathic post-prandial hypoglycemia).
Alcohol and reactive hypoglycemia
Insulin- and drug-induced reactive hypoglycemia are potentiated by alcohol. Alcohol also
increases insulin release in response to an oral glucose load and this may enhance any
tendency to post-prandial reactive hypoglycemia.
Other causes of reactive hypoglycemia
Various inherited metabolic diseases have reactive hypoglycemia as a feature.
Sudden cessation of a hypertonic dextrose infusion being given as part of a parenteral feeding
regimen can precipitate hypoglycemia, especially when insulin has been given concomitantly.
Hypoglycemia can also occur after dialysis against a glucose-rich dialysate.
Fasting hypoglycemia 1. High endogenous insulin:
a. Insulinomas: are tumors of the insulin-secreting β-cells of the pancreatic
islets. The presence of an insulin-secreting tumor can be inferred from the
presence of an inappropriately high plasma insulin concentration (>20
pmol/L) at a time when the blood glucose concentration is low (<2.2
mmol/L). C-peptide should also be measured. Although secreted in equimolar
amounts with insulin, C-peptide is cleared from the circulation more slowly,
so it may be a more reliable marker of endogenous insulin secretion than
insulin itself. It should be noted that glucose tolerance tests have no role in
the investigation of possible insulinomas.
b. Nesidioblastosis: hyperinsulinemic hypoglycemia attributed to excessive
function of pancreatic beta cells with an abnormal microscopic appearance.
2. Glucocorticoid deficiency: lack of cortisol can be due either to primary adrenal failure
or secondary to panhypopituitarism. Mild hypoglycemia can occur with isolated
deficiency of ACTH or growth hormone, but in the latter condition, it is never
symptomatic.
3. Liver disease/intoxication: although the liver is central to glucose homoeostasis, its
functional reserve is so great that hypoglycemia is a rare feature of hepatic disease. It
may occur, however, with the rapid, massive hepatocellular destruction that can
follow poisoning with paracetamol and other toxins. The kidneys are the only organs
other than the liver capable of gluconeogenesis; they are also responsible for insulin
degradation. These facts may in part explain the severe hypoglycemia that is
occasionally a feature of end-stage renal disease.
4. Non-pancreatic neoplasms: hypoglycemia can also occur in association with non-
pancreatic neoplasms, including hepatocellular and adrenal carcinomas, carcinoid
tumors and large mesenchymal tumors such as retroperitoneal sarcomas. Patients are
usually not ketotic and, except with some carcinoid tumors, plasma insulin
concentrations are not increased. It has been suggested that increased glucose uptake
by the tumor may be a factor but this is unlikely ever to be the sole cause. Hepatic
glucose output is often reduced although there is a normal glucogenic response to
glucagon. It is probable that most such tumor-related hypoglycemia is related to the
secretion of insulin-like growth factors (IGFs). Plasma IGF-1 concentrations are
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consistently low in such patients but IGF-2 (particularly in the non-protein-bound
form) is often increased, and the ratio IGF-1:IGF-2 is decreased.
5. Alcohol-induced fasting hypoglycemia: hypoglycemia can be induced by ethanol,
which inhibits gluconeogenesis and potentiates the action of insulin.
6. Sepsis: hypoglycemia sometimes develops in patients with septicemia. It is thought to
be a result of the release of cytokines, which may stimulate insulin secretion or have a
direct effect on hepatic glucose production. Renal impairment may also be
contributory.
7. Inherited metabolic disease: fasting hypoglycemia is an important feature of glycogen
storage disease type I.
Hypoglycemia in childhood
Neonatal hypoglycemia: hypoglycemia can develop among infants of diabetic mothers (they
have islet-cell hyperplasia), erythroblastosis fetalis, in cases of intrauterine malnutrition, and
among preterm or small-for-date infants (because they are born with low hepatic glycogen
stores and are more likely to have feeding problems. Extensive physiological changes occur at
birth and, in terms of glucose metabolism, there is a sudden interruption of the maternal
glucose supply, so that glycogenolysis must span the period until feeding becomes
established).
Hypoglycemia in infancy: in infancy, ketotic hypoglycemia can occur (can be idiopathic, or
due to relative carbohydrate deficiency). Hyperinsulinism is another cause (due to
nesidioblastosis, islet-cell hyperplasia or insulinomas). Lastly, inborn errors of metabolism
may lead to hypoglycemia (e.g. galactosemia, fructose intolerance, disorders of β-oxidation of
fatty acids and glycogen storage diseases).
Avi Sayag Clinical Biochemistry
Topic 34 Disorders and laboratory diagnosis of lipid metabolism Lipoproteins are classified on the basis of their densities as demonstrated by their
ultracentrifugal separation. Density increases from chylomicrons through VLDL,
intermediate density (IDL) and LDL to HDL. HDL can be separated, on the basis of density,
into two metabolically distinct subtypes, HDL2 (density 1.064-1.125) and HDL3 (density
1.126-1.21). Distinct subtypes of LDL (LDL-I, II and III, in increasing order of density) are
also recognized. IDL are normally present in the blood-stream in only small amounts but can
accumulate in pathological disturbances of lipoprotein metabolism. However, it is important
to appreciate that the composition of the circulating lipoproteins is not static. They are in a
dynamic state with continuous exchange of components between the various types.
Lipoprotein Density (g/mL) Source Main function
CM < 0.95 seen after 1h of
centrifugation
Intestine in response to a
fatty meal
Transport of exo. TG
from gut to tissue + chol.
from gut to liver
VLDL 0.96-1.006 seen after
20h of centrifugation
Liver – in response to a
high CH meal
Transport of endo. TG
from tissue to liver and
back
IDL 1.007-1.019 Catabolism of VLDL Precursor of LDL
LDL 1.02-1.063 seen after
24h of centrifugation
Catabolism of VLDL via
IDL in peripheral tissues
when VLDL gives up TG
Chol. transport from
liver to tissues
HDL 1.064-1.21 seen after
36h of centrifugation
Liver, intestine;
catabolism of CM and
VLDL
Chol. Transport from
tissues to liver
Lipoproteins are composed of:
1. A polar lipid surface layer (phospholipids, cholesterol, apolipoproteins);
2. A non-polar lipid core (TG, cholesteryl ester)
3. Enzymes (LCAT, phospholipase A2, paraoxonase)
The proteins (the apolipoproteins) are very weakly associated with a specific lipoprotein, and
are easily transferred to another lipoprotein of a different class. They have a structural role
and a binding site for receptors. There are several apolipoproteins: B-100, B-48, A-I, A-II, A-
IV, C-I, C-II, C-III and E.
The major differences between different lipoproteins (CM, VLDL, IDL, LDL, HDL) are in:
1. The size of the neutral lipid core (the non-polar core)
2. The lipid composition in the core
3. The apolipoproteins in the core
CM have the highest percentage of lipids and TG, followed by VLDL.
LDL has the highest percentage of cholesterol, and HDL has the highest amount of proteins
(LDL has more lipids % than HDL).
5 major complications are associated with hypertriglyceridemia:
1. Risk for atheroma
2. Acute pancreatitis (and recurrent abdominal pain as a result of it)
3. Pseudohyponatremia
4. Focal neurological syndromes
5. Rheumatological manifestations
High levels of TG can be observed in the following settings:
1. Alcohol comsumption
2. Estrogen (for contraception or post-menopausal treatment)
3. Use of vitamin A derivatives
4. Thiazide diuretics (in high doses)
5. non-selective β-blockers (without intrinsic sympathomimetic activity)
The increased levels of TG are associated with:
1. Increased LDL and small dense LDL particles;
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2. Reduced HDL
3. Reduced apoA-I (only)
4. DM is frequently found
5. Obesity may contribute
6. Glucose intolerance with insulin resistance and hyperinsulinemia
The disorders of lipid metabolism are based on the levels of the various lipids as well as on
electrophoresis or ultracentrifugation of lipoproteins. Thus, before addressing the disorders,
the lab diagnosis is described.
Cholesterol, HDL, LDL assays (practical topic 19) Cholesterol The sample is serum obtained from whole blood, centrifuged at 1500g for 10 minutes at 4ºC.
Samples should be collected after 12-14 hour fasting.
The determination of cholesterol has 3 steps:
1. Cholesterol-ester is hydrolyzed by cholesterol esterase (Ch-ester�Ch)
2. Cholesterol is oxydized by cholesterol oxydase (Ch + O2 � Ch + H2O2)
3. H2O2 + 4aminoantipirin + phenol � chinoimine (by peroxidase)
Chinoimine is a color product. Its absorbance is measured, and the cholesterol levels are
calculated.
A reference range for cholesterol does not exist, as the cholesterol level exceeds the desired
value in the majority of the population. Rather, the risk limit is used. The risk limit is 5.2
mmol/L. Above 6.8 mmol/L has to be reduced by diet or drugs. If total cholesterol is 5.2-6.8
mmol/L, it is necessary to determine HDL and LDL levels as well as other risk factors
(smoking, etc.). A 3-minute venous compression can increase cholesterol values by up to 10%
(as well as in blood collected during standing). Also, vitamin C reacts with H2O2 and thus
lowers the values of cholesterol.
HDL In the presence of divalent cations (Mg
+2 for example), lipoproteins can be selectively
precipitated with polyanions. By using precipitating reagents in the appropriate
concentrations, the β-, preβ-lipoproteins and chylomicrons can be precipitated, and the α-
lipoprotein (HDL) that remains in the supernatant can be determined (for α, β and preβ bands,
see next practical topic – 20). The most frequently used precipitating agent is Mg+2
-phospho-
wolframate. Results obtained with this method correlate well with the HDL-C values
measured from the HDL fraction after ultracentrifugation.
LDL LDL is measured by selective precipitation, direct immunoseparation or by using the
following formula:
LDL-C(mmol/L) = total cholesterol – HDL-C – TG/2.2
This formula holds when Tg levels do not exceed 4.5 mmol/L and if type III
hyperlipoproteinemia can be excluded.
Some current assays contain different detergents and other chemicals, which allow specific
blocking or solubilization of lipoprotein classes in order to achieve specificity for LDL. The
LDL is measured enzymatically in the same cuvette with HDL.
It is desirable to keep the LDL below 3.4 mmol/L, and start treatment above 4.8 mmol/L.
The ratio LDL/HDL is diagnostically informative of the risk to develop atherosclerotic
vascular disease (e.g. a ratio of 3.6 for women represents an average risk.
Triglyceride assay, visual test, lipoprotein electrophoresis (practical
topic 20) Visual test The appearance of the plasma in the lab may provide the first clue that a patient has
hyperlipidemia. In health, in the fasting state, plasma is clear. Following a meal, it often
becomes opalescent owing to the light-scattering properties of chylomicrons and VLDL. At
triglyceride concentrations above about 4 mmol/L, the plasma becomes increasingly turbid;
with severe hypertriglyceridemia, it appears milky (lipemic). If plasma is left undisturbed,
chylomicrons float to the surface, leaving a clear infranatant layer; VLDL remain in
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suspension. LDL do not scatter light and even at high plasma cholesterol concentrations the
plasma remains clear9.
Blood for lipid studies should be drawn after an overnight fast (12-14 h), when chylomicrons,
derived from dietary fat, should have normally been cleared; a pathological disturbance may
thus be inferred if they are present. The patient should have kept to his or her own normal diet
for two weeks before the blood is taken. Alcohol should not have been taken on the evening
before blood sampling. Alcohol is a common cause of hypertriglyceridemia even in patients
who have otherwise fasted. When lipid studies are done on a patient who has had a
myocardial infarction or stroke, blood should either be taken within 24 h or after an interval
of three months, since the metabolism of lipoproteins is disturbed during the convalescent.
By keeping serum or plasma at 4ºC for 18-24 hours, chylomicrons are separated and form a
creamy layer on top of the sample, while VLDL remain in the infranatant and in high
concentration cause turbidity (these 2 are the main carriers of Tg). LDL and HDL (that mostly
contain cholesterol) have no effect on the visual appearance of the sample. If the sample if
clear, the Tg level is < 2.5 mmol/L in most cases. Above 2.5 mmol/L the serum is opalescent,
and above 6.5 mmol/L it is very turbid.
Tryglycerides Tg is measured by enzymatic methods only (4 steps):
1. Tg � glycerol + fatty acids (lipase, esterase)
2. Glycerol + ATP � G-3-P + ADP (glycerol kinase)
3. G-3-P + O2 � dihydroxyacetone-P + H2O2
4. H2O2 + 4aminophenazone + p-chlorphenol � chinoimine + H2O + HCl
(peroxidase)
Reference values are age-dependent:
<29 y/o : 1.35 mmol/L
<39 y/o : 1.68 mmol/L
>40 y/o : 1.76 mmol/L
Values above these refer to hypertriglyceridemia.
Lipoprotein electrophoresis Electrophoresis (mostly on agarose or cellulose acetate) separate lipoproteins according to
their charges. Quantitation of the lipoprotein fractions is carried out by densitometric
scanning of the electrophotogram. On agarose gel, lipoproteins are separated from the cathode
to the anode in the following order: chylomicrons, β, preβ and α bands.
The α band which migrates the furthest corresponds to the HDL
The β band which migrates the shortest corresponds to the LDL
The preβ which migrates to the middle corresponds to the VLDL
Chylomicrons do not enter the pores of the gel due to their large size.
Ultracentrifugation separates lipoproteins according to their density:
HDL>LDL>VLDL>chylomicrons.
The disorders associated with lipid metabolism are: hyperlipoproteinemia,
hypolipoproteinemia, hypertriglyceridemia and atherosclerosis.
Hyperlipoproteinemia There are 5 types according to the WHO: I, II (A and B), III, IV, and V.
Type I – increased CM
Type IIA – increased LDL
Type IIB – increased LDL and VLDL
Type III – increased IDL or β-VLDL
Type IV – increased VLDL
Type V – increased VLDL and CM
9 HDL and LDL do not contribute to the visual examination. Only CM and VLDL do.
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Type Primary Secondary Signs
I Lipoprotein lipase (LPL)
deficiency (AR)
apoCII deficiency (because it
is a cofactor for LPL)
Uncontrolled DM,
SLE, hypothyrosis,
pancreatitis, oral
contraceptives,
abnormal plasma
globulins
Elevated Tg and normal-to-
elevated cholesterol.
Clear visual appearnace.
IIA AD – block in LDL
synthesis/transport/ uptake/
metabolism
apoB-100 deficiency (most
cases of type II)
IIB AD (polygenic)
Hypothyrosis,
mixedema, nephrosis,
obstructive biliary
disease, acute
intermittent porphyria
Elevated cholesterol, and
nornal Tg.
Clear visual appearnace.
Slightly turbid serum
III AR – IDL metabolism is
altered;
Abnormal apoE;
Absence of hepatic lipase
Hypothyrosis, DM,
SLE, primary biliary
cirrhosis
Elevated cholesterol, and
elevated Tg.
Turbid, slim chylomicron
layer.
IV Familial
hypertriglyceridemia (AD)
and familial combined
hyperlipidemia (AD)
Hypothyrosis,
nephrosis, obesity,
alcoholism, DM, oral
contraceptives and
steroid therapy
Elevated Tg, normal-to-
elevated chylomicrons.
Turbic appearance.
V Familial
hypertriglyceridemia (AD)
Nephrosis, obesity,
alcoholism, DM,
myeloma, pancreatitis,
hyperuricemia
Elevated Tg, normal-to-
elevated chylomicrons.
Turbic appearance.
Hypolipoproteinemia There are 2 categories to this disorder:
1. Low cholesterol with normal/low HDL
2. Low HDL alone
1. Low cholesterol with normal or low HDL 3 different diseases can lead to this condition:
1. Abetalipoproteinemia: this is an AR disorder. In abetalipoproteinaemia, there is a defect in
the synthesis of apo B; CM, VLDL and LDL are absent from the plasma. Clinically, there is
malabsorption of fat, vitamins A, E and K, acanthocytosis (abnormal star-shaped red blood
cells), retinitis pigmentosa and an cerebellar ataxia.
2. Hypobetalipoproteinemia: in this AD condition there is a partial deficiency of apoB due to
a mutation in the apoB gene. LDL and cholesterol are decreased, but the level of TG and
VLDL remain normal.
3. Chylomicron retention disease: in this condition there is a defect in apoB-48. The condition
manifests in fat malabsorption and low circulating lipids.
2. Isolated low HDL 5 different diseases can lead to this condition:
1. Familial hypoalfalipoproteinemia: this AD condition is caused by a gene defect of hepatic
lipase and apoA-I, IV/C-III. Low HDL levels are the manifestation with increasing risk for
early CHD.
2. ApoA-I and apoC-III deficiency: this AR condition is caused by a mutation in one or more
of these genes (including apoA-IV gene). Low HDL, cornea disease and an increased risk for
early CHD are the features of this condition.
3. ApoA-I variants: this AR disease is caused by a specific mutation in the apoA-I gene,
which leads to increased catabolism of this protein. Low HDL, cornea disease, increased risk
for CHD and xanthomas are the main features.
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4. Tangier disease: In Tangier disease (AR), plasma HDL concentrations are reduced;
clinically, the condition is characterized by hyperplastic, orange tonsils, the accumulation of
cholesteryl esters in other reticuloendothelial tissues (hepatosplenomegaly), early CHD and
low HDL. The condition is due to a loss of function mutation in the gene that codes for the
protein ABCA1, which normally stimulates the uptake of cholesterol into HDL, thus leading
to increased degradation of HDL.
5. LCAT deficiency (fish-eye disease): this AR disease is the result of a mutation in the
LCAT (lecithin:cholesterol acetyltransferase) gene. Low HDL, glomeruloscelorosis, anemia,
cornea disease and early CHD are the main features. Note: ApoA1 is a cofactor of LCAT. LCAT converts free cholesterol into cholesteryl ester (a
more hydrophobic form), which is then sequenstered into the core of the lipoprotein
eventually making the newly synthesized HDL spherical, and forcing the reaction to be
unidirectional, since the cholesterol particles are removed from the surface. This enzyme is
bound to HDL and LDL in the plasma.
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Topic 35 Risk factors of atherosclerosis
1. Gender: estrogen increases cholesterol removal by the liver, and the progression of
atherosclerosis is less rapid in premenopausal women than in men. In addition,
epidemiologic evidence shows that estrogen replacement therapy protects the
cardiovascular system in postmenopausal women. On the other hand, large doses of
estrogen increase the incidence of blood clots, and even small doses produce a slight
increase in clotting. In addition, in several studies, estrogen treatment of
postmenopausal women failed to prevent second heart attacks. The reason for the
discrepancies between the epidemiologic and experimental data is currently unsettled.
2. The effect of increased plasma levels of homocysteine and related molecules such as
homocystine and homocysteine thiolactone, a condition sometimes called
hyperhomocystinemia, is associated with accelerated atherosclerosis. Markedly
elevated levels resulting from documented mutations of relevant genes are rare, but
mild elevations occur in 7% of the general population. The mechanism responsible
for the accelerated vascular damage is unsettled, but homocysteine is a significant
source of H2O2 and other reactive forms of oxygen, and this may accelerate the
oxidation of LDL. Homocysteine is an intermediate in the synthesis of methionine. It
is metabolized by enzymes that are dependent on vitamin B6, vitamin B12, and folic
acid. Supplementation of the diet with these vitamins reduces plasma homocysteine,
usually to normal.
3. Cholesterol, HDL and Tg: lowering plasma cholesterol and triglyceride levels and
increasing plasma HDL levels slows, and in some cases reverses, the atherosclerotic
process. The desired decrease in lipids can sometimes be achieved with dietary
restriction of cholesterol and saturated fat alone, even though dietary restriction
initiates a compensatory increase in cholesterol synthesis in the body. When dietary
treatment is not adequate, reducing conversion of mevalonate to cholesterol with
statins, drugs that inhibit hepatic 3-methylglutaryl coenzyme A (HMG-CoA)
reductase, the enzyme which catalyzes this reaction, is beneficial (an example of an
available HMG-CoA reductase inhibitor is simvastatin).
4. In cases in which there is severe hypercholesterolemia because of congenitally
defective LDL receptors, gene therapy has been tried with promising preliminary
results. (Antioxidant treatment with agents such as vitamin E, and β-carotene has
been used to inhibit oxidation of LDL, and this reduces the incidence of
atherosclerotic changes in experimental animals. However, the results of antioxidant
treatment in humans have generally been disappointing or negative).
5. Men who smoke a pack of cigarettes a day have a 70% increase in death rate from
ischemic heart disease compared with nonsmokers, and there is also an increase in
women. Deleterious effects of smoking include endothelial damage caused by carbon
monoxide-induced hypoxia.
6. Because of the increased shear stress imposed on the endothelium by an elevated
blood pressure, hypertension is another important modifiable risk factor for
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atherosclerosis. Lowering blood pressure has its greatest effect in reducing the
incidence of stroke, but there are beneficial effects on ischemic heart disease as well.
With modern methods of treatment, blood pressure in hypertensives can generally be
reduced to normal or near-normal values.
7. In diabetics, there are microvascular complications and macrovascular complications,
which are primarily related to atherosclerosis. There is a twofold increase in the
incidence of myocardial infarction compared with nondiabetics; severe circulatory
deficiency in the legs with gangrene is relatively common; there are more thrombotic
strokes, and renal failure is a serious problem. It is interesting in this regard that
rigorous control of blood pressure in diabetics has been shown to be more efficacious
in reducing cardiovascular complications than rigorous control of blood glucose.
8. The nephrotic syndrome: also accelerates the progression of atherosclerosis. In the
nephrotic syndrome there is increased hepatic production of lipids and lipoprotein(a).
9. Lp(a): consists of an LDL-like particle and a specific apolipoprotein a, which is
covalently bound to ApoB of the LDL-like particle. Apo(a) is expressed by
hepatocytes and the assembly of Apo(a) and the LDL-like particle seems to take place
at the outer hepatocyte surface. Lp(a) is highly homologous to plasminogen and to
tPA, and it competes with plasminogen for its binding site, leading to reduced
fibrinolysis. Also, because Lp(a) stimulates secretion of PAI-1, it leads to
thrombogenesis. In addition, because of the LDL-like component, Lp(a) contributes
to atherosclerosis.
10. Obesity: obesity is associated with type 2 diabetes, hypertriglyceridemia,
hypercholesterolemia, and hypertension, all of which are risk factors in their own
merit.
11. Family history of ischemic heart disease, stroke: probably multiple genetic
mechanisms.
12. Hypothyroidism: decreased formation of LDL receptors in the liver. The thyroid
hormone regulates the synthesis of LDL receptor at the gene level (gene expression).
Avi Sayag Clinical Biochemistry
Topic 36 Disturbances of uric acid metabolism Gout is a disorder caused by the tissue accumulation of excessive amounts of uric acid, an end
product of purine metabolism. It is marked by recurrent episodes of acute arthritis, sometimes
accompanied by the formation of large crystalline aggregates called tophi, and chronic joint
deformity. All of these result from precipitation of monosodium urate crystals from
supersaturated body fluids. Although an elevated level of uric acid is an essential component
of gout, not all such individuals develop gout, indicating that influences besides
hyperuricemia contribute to the pathogenesis. Gout is divided into primary (90%) and
secondary forms (10%). Elevated uric acid levels can result from overproduction of uric acid,
reduced excretion, or both. Most cases of gout are characterized by a primary overproduction
of uric acid.
Uric acid synthesis: after the synthesis of purine nucleotides (guanosine monophosphate,
adenosine monophosphate and inosine monophosphate) the catabolism produces uric acid. It
should be noted that the pool of purine nucleotides is derived from dietary nucleic acids
(300mg) and from endogenous synthesis (400mg – de novo synthesis and tissue breakdown).
The urate pool is mainly secreted by the kidney (75%) and partly by the GI (25%). In the
kidney, 100% is filtered, then almost everything is reabsorbed in the proximal tubule, just to
be secreted in the distal part of the proximal tubule (50%). Then, in the distal tubule 38-44%
is reabsorbed, so the net urinary excretion is 6-12% of the amount filtered.
Note that adenine does not form uric acid.
Although the cause of excessive uric acid biosynthesis in primary gout is unknown in most
cases, rare patients have identifiable enzymatic defects. For example, complete lack of
HGPRT (hypoxanthine guanine phosphorybosyl-transferase) gives rise to the Lesch-Nyhan
syndrome (complete loss of the enzyme – secondary gout). This X-linked genetic condition is
characterized by excessive excretion of uric acid, severe neurologic disease with mental
retardation, and self-mutilation (but interestingly, little in the way of gout!). Because of the
almost complete absence of HGPRT, purine nucleotide synthesis via the salvage pathway is
blocked. This has two effects: an accumulation of PRPP (phosphoribosylpyrophosphate), a
key substrate for the de novo pathway, and increased activity of amido-PRT (due to elevated
PRPP and reduced feedback inhibition from purine nucleotides). As a consequence, de novo
pathway purine biosynthesis is augmented, resulting eventually in excess production of the
uric acid end product. Less severe deficiencies of HGPRT (partial deficiency – primary gout)
cause clinically severe gouty arthritis, occasionally associated with mild neurologic disease.
In primary hyperuricemia, 10% is due to overproduction and the remaining 90% is due to
underexcretion.
In secondary gout, hyperuricemia can be caused by increased urate production (e.g., rapid cell
lysis during chemotherapy for lymphoma or leukemia, increased purine intake) or decreased
excretion (due to chronic renal insufficiency or administration of thiazides), or both.
Hypothyroidism and hyperparathyroidism can also lead to renal retention.
The risk for developing gout increases significantly when plasma urate is above 0.42
mmol/L in males and above 0.36 mmol/L in women. Above 0.54 mmol/L the risk is 90% for
males. The precipitation of uric acid in the joints triggers a chain of events that lead to joint injury.
Uric acid is less soluble in pH less than 5.75. The precipitated crystals are directly
chemotactic, and can also activate complement to generate chemotactic C3a and C5a
fragments. This leads to a local accumulation of neutrophils and macrophages in the joints
and synovial membranes; in attempting to phagocytize the crystals, these cells become
activated, leading to the release of additional mediators including chemokines, toxic free
radicals, and leukotrienes, particularly leukotriene B4. The activated neutrophils also liberate
destructive lysosomal enzymes. Macrophages participate in joint injury by secreting a variety
of proinflammatory mediators such as IL-1, IL-6, and TNF. While intensifying the
inflammatory response, these cytokines can also directly activate synovial cells and cartilage
cells to release proteases (e.g., collagenase) that cause tissue injury. The resulting acute
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arthritis typically remits in days to weeks, even if untreated. Repeated bouts, however, can
lead to the permanent damage seen in chronic tophaceous arthritis.
Clinical Features Gout is more common in men than in women; it does not usually cause symptoms before the
age of 30. Four stages are classically described:
(1) asymptomatic hyperuricemia,
(2) acute gouty arthritis,
(3) "intercritical" gout, and
(4) chronic tophaceous gout.
Asymptomatic hyperuricemia appears around puberty in males and after menopause in
women. In asymptomatic hyperuricemias, quatitation of urinary uric acid excretion is carried
out. If it is less than 600 mg/day, the patient is an "underexcretor" and uricosuric drugs are
administered (such as probenicid, sulfynpyrazone that block renal reabsorption). However, if
the patient excretes more than 600 mg/day, then he is an "overproducer" and allopurinol is
administered (inhibits xanthine oxidase). After a long interval of years, acute arthritis appears
in the form of sudden onset of excruciating joint pain associated with localized erythema and
warmth; constitutional symptoms are uncommon, except for possibly mild fever. The vast
majority of first attacks are monarticular; 50% occur in the first metatarsophalangeal joint ,
and 90% in the instep, ankle, heel, or wrist. Untreated, acute gouty arthritis may last for hours
to weeks, but it gradually completely resolves and the patient enters an asymptomatic
intercritical period. Although some individuals never have another attack, most experience a
second episode within months to a few years. In the absence of appropriate therapy, the
attacks recur at shorter intervals and frequently become polyarticular. Eventually, after a
decade or so, symptoms fail to resolve completely after each attack, and the disease
progresses to chronic tophaceous gout. At this stage, radiographs show characteristic juxta-
articular bone erosion caused by the crystal deposits and loss of the joint space. Progression
leads to severe crippling disease.
Renal manifestations of gout can appear as renal colic associated with the passage of gravel
and stones, and can evolve into chronic gouty nephropathy. About 20% of individuals with
chronic gout die of renal failure.
Numerous drugs are available to abort or prevent acute attacks of arthritis and mobilize
tophaceous deposits. Their use is important because many aspects of gout are related to the
duration and severity of hyperuricemia.
The diagnosis of gout is primarily clinical. Hyperuricemia can be demonstrated, and
confirmation is made by demonstrating tophi or monosodium urate in synovial fluid. It should
be differentially diagnosed with rheumatoid arthritis and pseudogout.
Hypouricemia – less than 0.12 mM (less common). It can be secondary to severe
hepatocellular disease (purine synthesis or xantine oxidase defect), to defective renal tubular
reabsorption of uric acid, and to overtreatment with allopurinol. Hypouricemia can be caused
by congenital deficiency of xantine oxidase (xantinuria).
Avi Sayag Clinical Biochemistry
Topic 37 Lab diagnostics of AMI (practical topic 21 is included) There are 2 interrelated types of MI, with different morphology, pathogenesis and clinical
significance:
Transmural infarct is an MI involving the full thickness of the ventricular wall; it is
usually caused by severe coronary atherosclerosis, with acute plaque rapture and
superimposed occlusive thrombosis.
Subendocardial infarct is typically limited to the inner one third of the ventricular
wall; it is caused by increased cardiac demand in the setting of limiting supply due to
fixed atherosclerotic disease; alternatively, subendocardial infarction can occur in an
evolving transmural infarct when the coronary obstruction is relieved in sufficient
time to prevent transmural necrosis.
The warning signs are pain in the jaw, neck, arms, shoulders or back, chest pressure,
squeezing or pain, shortness of breath, nausea, sweating or feeling faint.
The average time from onset of symptoms to presentation is 2.6 hours.
The average time from presentation to treatment is 1.7 hours, while the recommended time is
30-60 minutes! Thus, shortening the time from onset of symptoms to therapy is critical.
Pathogenesis
Transmural infarcts
Transmural infarcts are largely a consequence of coronary atherosclerosis and one (or more)
disrupted plaques. Significant plaques typically occur in the proximal 2 cm of the LAD and
left circumflex coronary arteries and in the proximal and distal thirds of the right coronary
artery. In a few cases, vasospasm and platelet aggregation cause MIs without atherosclerotic
stenosis. With sufficient collateral blood flow, even complete vessel occlusion will not
necessarily result in MI.
The initial event in most transmural MIs is erosion, ulceration, fissuring, rupture or
hemorrhagic expansion (collectively called acute plaque change or disruption) of an
atherosclerotic plaque.
Plaques involved in coronary events typically have a large lipid pool, a thin fibrous
cap, and a macrophage-rich inflammation; plaques with such features are considered
susceptible to rupture, and are termed vulnerable. Patients at risk of cardiovascular
events may have multiple vulnerable plaques.
If the patient survives, thrombi may either lyse spontaneously or after fibrinolysis;
alternatively, vasospasm may relax. In both cases, flow is reestablished and some
myocardium is spared from necrosis.
Reperfusion of precariously injured cells may restore viability but leave the cells
poorly contractile (stunned) for 1-2 days.
Nearly all transmural MIs affect the left ventricle; 15% simultaneously involve the
right ventricle, particularly in posterior-inferior ventricle infarcts. Isolated right
ventricle infarction occurs in 1-3% of cases.
Subendocardial infarcts
Subendocardial infarcts are usually caused by:
Diffuse coronary atherosclerosis and global borderline perfusion made transiently
critical by increased demand, vasospasm, or hypotension but without superimposed
thrombosis.
Plaque disruption with overlying thrombus that spontaneously lyses (or is removed by
therapeutic intervention), thereby limiting the extent of myocardial injury.
Myocardial injury is usually less than in a transmural infarct and often multifocal.
Clinical features
About 25% of patients experience sudden death after MI (presumably secondary to a
fatal arrhythmia), most before reaching a hospital.
Avi Sayag Clinical Biochemistry
Of those surviving, the risk of death within 1 month after MI is 7-10%, and 80-90%
will develop complications.
Early restoration of flow (thrombolysis or balloon angioplasty) through occluded
vessels responsible for the infarction yields a better prognosis.
Complications
Complications of an MI depend on the size and the location of injury, as well as functional
myocardial reserves. Overall mortality rate in the first year after MI is 30% and thereafter 5%
to 10% per year. Typical complications include:
Arrhythmias
CHF
Cardiogenic shock (usually seen when >40% of the left ventricle is infracted).
Ventricular rapture
Rarely, papillary muscle infarction with or without rupture can cause severe mitral
regurgitation.
Fibrinous pericarditis is common 2-3 days after MI (but is not usually clinically
significant).
Mural thrombosis is next to a noncontractile area, with risk of peripheral
embolization.
Stretching of a large area of transmural infarction (expansion) that may heal into a
ventricular aneurysm; both are prone to mural thrombosis.
Repetitive infarction (extension).
Diagnosis Molecules released from damaged cells during MI:
Ions and metabolites are released to the blood stream, while macromolecules are released to
the lymphatic system.
Cardiac proteins most commonly used in lab tests are myoglobin, cardiac troponin T
and cardiac troponin I.
Enzymes most commonly used: CK, CK-MB. Formerly, LDH, LDH isoenzymes and
GOT (glutamate oxalacetate transaminase, aka aspartate aminotransferase, ASAT).
Determination of more than one enzyme should be made, evaluation of the time curve
of changes, and the use of an isoenzyme specific to the organ should be ensured.
Future tests will include ischemia modified albumin, hsCRP, NT pro BNP and
sCD40L and IL-6.
Determination of creatine kinase – CK Considering these 3 reactions, the activity of CK can be determined:
Creatine-phosphate + ADP � creatine + ATP (enzymatic reaction carried by CK)
ATP + glucose � glucose-6-P + ADP (carried by hexokinase)
G-6-P + NADP+ � 6-phosphogluconate + NADPH + H
+ (carried by G6PDH)
There is a linear correlation between the increase in the amount of NADPH and CK activity.
NADPH has a maximum absorbance at 340nm.
The test is carried out using serum sample, and a reagent is added. The reagent contains
hexokinase, glucose, NADP+ and G6PDH. Hemolysis interferes with the determination of
CK, because RBCs release adenylate cyclase (not CK!). AC converts 2 ADPs to ATP + AMP,
and the ATP carries the second reaction, leading to overestimation of CK levels. The activity
of CK is given by U/L – one unit is the enzyme activity that converts 1 µmol substrate in 1
minute.
The reference interval measured at 37ºC: 24-195 U/L.
CK can be elevated in the following settings:
o IM injection
o Damage to skeletal muscles (or exercise)
o Hypo- and hyperthermia
o Intoxication
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o CVA
o Old age
CK has 3 isoenzymes: CK-MM, CK-MB and CK-BB (there is also mitochondrial CK
– CK-MiMi). These are dimeric combinations of the brain (B) subunit and the muscle
(M) subunit.
The main source of CK-MB is cardiac muscle. It is released into the circulation upon
tissue injury. In the heart, the total CK is 187 U/g. 40% is given by CK-MB and 60%
by CK-MM (in the skeletal muscle, the total CK is 1093 U/g. 97% is given by CK-
MM and 3% by CK-MB).
Normally, CK-MB is less than 6% in the serum (and the rest is CK-MM).
CK-MB can be determined using the following methods:
o Electrophoresis;
o Immunoinhibition – the M subunit is inhibited by a specific Ab and the
residual activity is measured. Thus, after the inhibition of the M subunit in
the MM and MB isoenzymes, only the activity of the B subunit of the CK-
MB will be measured. However, macroCK can occur in the serum leading to
overestimation of the CK-MB level. Since the M subunit is inhibited, the
measured CK activity is due to the B subunit of the CK-MB and thus the
activity has to be doubled. This method is very sensitive and quick;
o Determination of CK-MB mass concentration by immunoassay – an
expensive method, which is unable to give the result as a percentage of the
total CK activity. However, it is very specific and precise, and thus it is the
recommended method.
Reference interval measured at 37ºC: CK-MB <6% of total CK, < 24 U/L CK enzyme also has isoforms, which are the result of post-translational modification. In the
circulation, the C-terminal lysine is cleaved off by a plasmatic carboxypeptidase. Thus, 3
isoforms of CK-MM are present and 2 isoforms of CK-MB. CK-MM1 – both M subunits lack
the C-terminal lysine. CK-MM2 – one M subunit lacks the C-terminal lysine. CK-MM3 – both
subunits retain their C-terminal lysine. CK-MB1 – the M subunit lacks the c-terminal lysine.
CK-MB2 – the M subunit retains its C-terminal lysine. CK-MM3 and CK-MB2 are
characteristic of AMI, because the post-translational modification cannot occur in the freshly
released enzymes. The electrophoresis method is suitable for the detection of CK isoforms,
but is not commonly used in routine diagnostics.
macroCKs are large compounds of CK that are of two types:
o type I: these are complexes of a CK isoenzyme (most frequently CK-BB)
with immunoglobulins. They frequently occur among women older 50 years
of age. They also occur in severe GI disease, vascular disease, adenomas and
carcinomas.
o Type II: these are oligomeric mitochondrial CK. It occurs mostly in severe
malignancies or liver diseases.
o macroCKs interfere with immunoinhibition methods of CK-MB
determination.
Determination of troponins Troponins are very useful in the diagnosis and prognosis of acute coronary syndrome.
Normally, cTnT is not detectable in the blood of healthy persons. The 2 troponins
(cTnI and cTnT) can be elevated even if CK-MB is not. This implies minor
myocardial damage. Troponins are predictive of death when they are elevated,
regardless whether CK-MB is elevated or not.
Both troponin I and T have 3 isoforms: in slow twitch fibers and in fast twitch fibers
of skeletal muscles, and in cardiac muscle.
Most troponins in the heart are in their insoluble form, and only 4-5% are in the
cytoplasm in their soluble form. It is probably the soluble form that is responsible for
the fast elevation of troponins after AMI, while the insoluble form accounts for the
sustained troponin increase.
Avi Sayag Clinical Biochemistry
cTnT is an early marker of MI (but not the earliest, though). It remains elevated
longer than CK-MB (troponin reaches its peak after 24 hours, it then decreases a bit
in the next 24 hours, and remains elevated for additional 48 hours before it gradually
decreases in the course of couple of days. CK-MB, on the other hand, reaches its peak
after 24 hours from injury, to a lesser extent than troponin, and sharply decreases in
the next 24 hours). cTnT is a sensitive marker, and it can be used for monitoring
reperfusion during thrombolysis. Also, it can be used for sizing MI. It is more
sensitive than CK-MB (troponin's level is elevated in cases of myocardial damage
that is not defined as AMI by the WHO). The upper reference limit (URL) for
troponin is 0.04 µg/L. Values between 0.04-0.1 µg/L suggest myocardial damage,
whereas values above 0.1 µg/L suggest AMI.
cTn false positivity can be obtained in the following cases:
o Heterophilic antibodies
o HAMA (Human Anti-Mouse Antibodies)
o Rheumatoid factor
o Fibrin clot
o Microparticles
o Hemolysis
o Lipemia
o Icterus
o Immunocomplex formation
o Technical errors
Troponin level is determined by immunochemical assays.
Myoglobin lacks cardiac specificity as a marker. That is, if the myoglobin is elevated
4-8 hours following the onset of pain, but the ECG shows no sign of cardiac
condition, then more cardiac-specific markers should be sought for. However, if
myoglobin is not elevated 4-8 hours following the onset of pain, myocardial necrosis
can be excluded. That is, myoglobin is sensitive. It is the only early marker measured
(2-6 hours after the onset of AMI and it decays within 10-16 hours), despite its
specificity.
Troponin level is determined by immunochemical assays.
Determination of LDH (Lactate Dehydrogenase):
Avi Sayag Clinical Biochemistry
LDH catalyses the reaction: pyruvate + NADH + H+ � lactate + NAD
+ (it also
catalyses the reverse conversion).
LDH activity correlates with the NADH consumption, which is monitored by
measuring the rate of absorbance decrease at 340 nm. Hemolysed serum should not
be used, since RBCs contain 150 times more LDH activity than serum!
5 isoenzymes –
o LDH-1 (4H) - in the heart and RBCs
o LDH-2 (3H1M) - in the heart and RBCs
o LDH-3 (2H2M) - in the lungs
o LDH-4 (1H3M) - in the kidneys
o LDH-5 (4M) - in the liver and striated muscle
The relative amount of LDH isoenzymes in the serum:
LDH2>LDH1>LDH3>LDH4>LDH5
Usually LDH-2 is the predominant form in the serum. A LDH-1 level higher than the
LDH-2 level (a "flipped pattern"), suggests myocardial infarction (the normal ration
LDH1/LDH2 is < 0.8). Following a myocardial infarction, levels of LDH peak at 3-4
days and remain elevated for up to 10 days. In this way, elevated levels of LDH can
be useful for determining if a patient has had a myocardial infarction if they come to
doctors several days after an episode of chest pain. Tissue breakdown elevates levels
of LDH, and therefore a measure of it indicates e.g. hemolysis. Other disorders
indicated by elevated LDH include cancer, meningitis, encephalitis, acute pancreatitis
and HIV.
It should be noted that the use of LDH (as well as GOT and total CK) is outdated.
IMA – Ischemia Modified Albumin: during ischemia, the N-terminus of albumin is
altered (copper is released by localized ischemia, and in the presence of vitamin C
they carry a fenton reaction yielding free radicals that alter the N-terminus of
albumin) such that it can no longer bind metal, such as cobalt. When albumin
circulating in the blood comes in contact with an ischemic tissue in the heart, some of
the albumin molecules are converted to IMA. The levels of IMA are elevated among
ischemic patients; it is continuously produced during ischemia; it rises very quickly
and remains elevated during the ischemic event.
o In the Albumin Cobalt Binding (ACB) test, cobalt is added to the serum
sample. Cobalt normally binds to albumin, but not to IMA. The ACB test
measures the unbound cobalt. Higher levels of unbound cobalt indicate
greater concentrations of IMA
o IMA rises rapidly from the end of balloon inflation (around 10 minutes). The
National Academy of Clinical Biochemistry recommends that 60 minutes
elapse from sampling to lab results.
o Some other conditions lead to elevated IMA: some cancers, cirrhosis, and
acute infections (free radicals are produced in these cases) as well as in brain
ischemia (stroke) and end-stage renal disease.
Recommended time from presentation to treatment of AMI is 30-60 minutes.
Avi Sayag Clinical Biochemistry
Topic 38 Hypervitaminosis and hypovitaminosis Factors leading to vitamin deficiency:
1. inadequate intake (with normal requirement)
2. impaired absorption (general malnutrition or malabsorption)
3. impaired vitamin metabolism (if metabolism is necessary for function)
4. increased requirement
5. increased loss
6. therapy (e.g. hemodialysis or total parenteral nutrition)
The classic deficiency syndromes are the end result of a process in which
deficiency of a vitamin leads first to mobilization of body stores, then to
tissue depletion, biochemical impairment (subclinical deficiency) and
eventually to frank deficiency.
The functions of vitamins are almost entirely intracellular, and their plasma
concentrations do not necessarily reflect intracellular concentrations and thus
functional availability.
These are the most frequently used tests:
i. B1: transketolase activity
ii. B2: glutathione reductase
iii. B6: aspartate aminotransferase activity
iv. C: leukocyte ascorbate concentration; ascorbate in urine after vitamin
C load.
Vitamin B1 (thiamine)
Thiamine pyrophosphate (TPP) is a cofactor in the metabolism of pyruvate (oxidative
decarboxylation), carbohydrate metabolism, it's a coenzyme for active aldehydes (Mg is
required), and in a reaction of the pentose shunt pathway catalyzed by the enzyme
transketolase. In the nervous system, it participates in the hydrolysis of TPP at axonal
membranes. The body contains only about 30 times the daily requirement of this vitamin.
Diets high in carbohydrate require more thiamine for their assimilation than diets high in fat
and so, for example, subclinical thiamine deficiency may be unmasked in malnourished
patients given glucose intravenously. The source of the vitamin is cereals, liver, heart, kidney
and pork meat.
Deficiency of vitamin B1 causes beriberi, in which there are cardiac symptoms and
neurological symptoms.
The cardiac symptoms include
o Peripheral vasodilation;
o Retention of sodium and water (� edema); and
o Myocardial failure.
The neurological symptoms include
o Peripheral neuropathy
Avi Sayag Clinical Biochemistry
o Wernicke's encephalopathy, characterized by ophthalmoplegia and ataxia
and which may progress rapidly to stupor and death, and
o Korsakoff's psychosis, of which memory loss is usually the most obvious
feature.
One method involves administration of a glucose load and measurement of the plasma
pyruvate concentration. An excessive rise is seen in thiamin deficiency because the vitamin is
a cofactor for the conversion of pyruvate to acetyl CoA. However, the most sensitive method,
which will detect subclinical deficiency, is measurement of transketolase in a red cell
haemolysate, the enzyme activity being measured both with and without the addition of
thiamin pyrophosphate to the reaction mixture. Enzyme activity may be normal in subclinical
deficiency but is increased by the addition of the coenzyme. If the deficiency is clinically
obvious, the basal enzyme activity will be low.
Vitamin B2 (Riboflavin)
Riboflavin, when combined with 1 ATP yields FMN (flavin mononucleotide) and
when combined with 2 ATPs it yields FAD (flavin adenine dinucleotide).
It functions as a cofactor in redox reactions and as a prosthetic group of some
enzymes.
The source of the vitamin is meat, milk, eggs, liver, kidney, heart and vegetables.
Deficiencies are mostly seen in alcoholic patients due to low intake of the vitamin.
Clinically, deficiency manifests as glossitis, angular stomatitis (an inflammation of
the mucous lining of any of the structures in the mouth), photophobic dermatitis,
anemia (normochromic and normocytic) and burning sensation of the skin and eyes.
Assessing riboflavin status involves measurement of the red cell enzyme glutathione
reductase with and without the vitamin.
Nicotinic acid (Niacin)
Nicotinic acid is the precursor of nicotinamide. This is a constituent of the coenzymes
nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP), which are
essential to glycolysis and oxidative phosphorylation.
Part of the body's nicotinic acid requirement is met by endogenous synthesis from
tryptophan.
The dietary source is yeast, liver, poultry, milk, canned salmon, wheat and leafy
vegetables.
Deficiency causes pellagra or pellagra-like syndrome (carcinoid syndrome in which
there is increased metabolism of tryptophan to hydroxyindoles which is consequently
less available for nicotinic acid synthesis) that manifests as
o weight loss
o anemia
o dementia
o diarrhea
o photosensitive dermatitis and
o mental changes (apathy).
Nicotinic acid status can be assessed by measurement of its urinary metabolites,
although this is rarely necessary.
Vitamin B6 (Pyridoxine)
2 active derivatives: pyridoxal phosphate and pyridoxamine phosphate
The vitamin participates in
o Amino acid metabolism (as a prosthetic group of aminotransferases and in
decarboxylation of amino acids)
o Synthesis of heme precursor delta-aminolevulinic acid (glycine + succinyl
CoA); and
o Synthesis of sphingomyelin precursor (serine + palmytoyl CoA).
The source of the vitamin is meat, poultry, fish, yeasts and seeds.
Avi Sayag Clinical Biochemistry
Deficiency in children leads to
o Dermatitis
o Glossitis
o Nausea and vomiting
o Epileptiform convulsions
It should be noted that vitamin B6 is an antagonist of many drugs, including INH and
penicillamines.
Excess of vitamin B6 leads to
o Peripheral neuropathy
o Ataxia
o Paresthesias
o Muscle weakness
Lab test: activity of aspartate aminotransferase in RBCs.
Vitamin B12 (cobalamine)
The vitamin has many functions including nucleic acid synthesis, methylation of
homocystein (to yield methionine) and participation in synthesis of myelin sheath in
the nervous system.
The source of the vitamin is solely dietary (animal products).
2 molecules are necessary for its absorption: IF and TCII.
Clinical conditions leading to deficiency:
o Autoimmune destruction of parietal cells (no IF)
o Gastrectomy (no parietal cells � no IF)
o Small intestinal disease (Crohn's disease, surgical resection)
o Deficiency among veggies
Deficiency causes:
o Megaloblastic anemia
o Subacute combined degeneration of the spinal cord (demyelination)
Lab test: plasma B12 immunoassay.
(B12 and folic acid are greatly elaborated in other topics, including pathology).
Folic acid
Folic acid functions as a carrier of hydroxymethyl and formyl groups, it is essential
for synthesis of purines and pyrimidines and intimately related to B12 in replication
of cellular genes and in maturation of RBCs.
The source of folic acid is fruits and vegetables.
Deficiency leads to megaloblastic anemia.
Lab: plasma immunoassay (RBC is better, as the concentration in red cells reflects the
body's folate reserves, while plasma concentrations reflect recent dietary intake).
Vitamin C (Ascorbic acid)
Ascorbic acid is essential for the hydroxylation of proline residues in collagen and
thus for the normal structure and function of this protein. It acts by maintaining the
iron in the hydroxylating enzyme in the reduced (Fe2+
) state, that is, acting as an
antioxidant.
It also facilitates the intestinal absorption of dietary non-heme iron by keeping it in
the Fe2+
state.
Its source is citrus fruits and vegetables. Most animals can synthesize vitamin C from
glucose.
Deficiency of vitamin C leads to defective and weak collagen fibers, and the
intercellular substance thus cannot withstand stress.
Scurvy manifests in
o Petechiae and purpura
o Cutaneous bleeding and
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o Intracranial hemorrhages among infants
o Poor wound healing
o Salivary and lacrimal gland dryness
o Anemia
o Osteoporosis
The concentration of ascorbate in plasma reflects recent dietary intake and is a poor
index of tissue stores of the vitamin. These are better assessed by determination of
ascorbate concentration in leukocytes. In practice, this is seldom necessary, since
ascorbic acid is cheap and non-toxic, so a therapeutic trial of vitamin supplementation
is the simplest procedure to confirm suspected vitamin C deficiency.
Also, the vitamin can be measured in the urine after vitamin C loading.
Vitamin A (Retinol)
Its active derivatives are retinol, retinal and retinoic acid.
This vitamin is a constituent of the retinal pigment rhodopsin. It is also essential for
the normal synthesis of mucopolysaccharides and growth of epithelial tissue.
Vitamin A is present in the diet and can also be synthesized from dietary carotenes. It
is stored in the liver for 5-10 months. The normal liver contains considerable stores of
the vitamin and deficiency is rarely seen in affluent societies.
Deficiency causes
o night blindness, while in more severe cases, degenerative changes in the eye
may lead to complete loss of vision.
o Dry conjunctiva (xerophthalmia)
o Keratomalacia (ulcus cornea)
o Keratinization in the skin
Excess causes:
o Dizziness
o Headache
o Abdominal pain
o Vomiting
o Skin desquamation within few days
o If chronic: anorexia and hepatomegaly
It can be measured in plasma, in which it is transported bound to prealbumin and a
specific retinol-binding globulin. A low binding protein concentration can cause the
plasma concentration of vitamin A to be low and impair its delivery to tissues even
when hepatic stores of the vitamin are adequate.
Vitamin D (Cholecalciferol – D3)
Its main function is in regulation of calcium homeostasis.
Vitamin D is obtained from endogenous synthesis, by the action of ultraviolet light on
7-dehydrocholesterol in the skin to form cholecalciferol (vitamin D3), and from the
diet. Dietary vitamin D is largely vitamin D2 (ergocalciferol); the only important
dietary sources are fish and some margarines, which are artificially fortified with
vitamin D. Vitamins D2 and D3 undergo the same metabolic changes in the body and
have identical physiological actions.
It is hydroxylated first in the liver to 25-hydroxycholecalciferol (25-HCC, calcidiol)
and then in the kidney to 1,25-dihydroxycholecalciferol, and 24,25-
dihydroxycholecalciferol. The 24,25-Vit-D has no biological function, and this is the
mechanism of the kidney to keep the levels of active vitamin D within acceptable
limits. Recent evidence shows that 1α-hydroxilation can also occur in target cells,
bypassing the kidney for activation of the vitamin directly from the liver.
Deficiency:
o Rickets in children
o Osteomalacia in adults
Excess:
Avi Sayag Clinical Biochemistry
o Hypercalcemia
o Hypercalcuria (predisposition to renal stones)
Vitamin E (Tocopherol)
Vitamin E (tocopherol) is an important antioxidant, particularly in cell membranes,
protecting unsaturated fatty acid residues against free radical attack.
Its source is vegetable oils and plants.
Clinical deficiency may occur in severe malabsorption, particularly in infants.
Manifestations include hemolytic anemia and neurological dysfunction. In addition,
there is a decrease in unsaturated fat level in cells, and abnormal structure and
function of cellular organelles (mitochondria, lysosomes and cell membrane).
Vitamin K
Vitamin K is required for the γ-carboxylation of glutamate residues in coagulation
factors II (prothrombin), VII, IX and X. This process confers physiological activity
by permitting the binding of calcium to the proteins.
Its source is vegetables and it is synthesized by bacteria in the colon.
Vitamin K deficiency results in an increase in the prothrombin time, a functional
assay of relevant coagulation factor activity. These factors are synthesized in the liver
and the prothrombin time is also used as a test of liver function. Its most frequent use
is in the monitoring of patients on anticoagulant treatment with antagonists of vitamin
K (e.g. warfarin).
Causes of deficiency:
o Fat malabsorption
o Antibiotics
Avi Sayag Clinical Biochemistry
Topic 39 Lab diagnosis of hepatocellular damage; evaluation of liver function Terminology:
ALT – alanine transaminase (also called serum glutamic pyruvic transaminase (S-
GPT) or alanine aminotransferase (ALAT): significantly elevated levels of ALT often
suggest other medical problems such as viral hepatitis, CHF, liver damage, biliary
duct problems, infectious mononucleosis, or myopathy. For this reason, ALT is
commonly used as a way of screening for liver problems. However, elevated levels of
ALT do not automatically mean that medical problems exist. Fluctuation of ALT
levels is normal over the course of the day, and ALT levels can also increase in
response to strenuous physical exercise. When elevated ALT levels are found in the
blood, the possible underlying causes can be further narrowed down by measuring
other enzymes. For example, elevated ALT levels due to liver-cell damage can be
distinguished from biliary duct problems by measuring alkaline phosphatase. Also,
myopathy-related ALT levels can be ruled out by measuring creatine kinase enzymes.
Several drugs elevate the ALT levels. For example, Zileuton, the lipoxygenase
inhibitor used in the treatment of acute asthma exacerbation. ALT is found only in the
cytoplasm. Reference values: < 40 U/L.
AST - Aspartate transaminase (also called serum glutamic oxaloacetic transaminase
(S-GOT) or aspartate aminotransferase (ASAT): it is raised in acute liver damage, but
is also present in red blood cells, and cardiac and skeletal muscle and is therefore not
specific to the liver. The ratio of AST to ALT (GOT/GPT) is called deRitis ratio. In
most primary liver diseases, the ALT and AST levels are elevated in roughly a 1:1
(because they are both present in the cytoplasma). The AST:ALT ratio is generally
highest in alcoholic liver disease and lowest in acute and chronic viral hepatitis when
the ratio may be less than 1:1. For patients with chronic alcohol-induced liver
damage, the ratio is often greater than 2:1 (in cases of severe hepatocellular damage,
both cytoplasmic and mitochondrial GOT are released, so the relative increase of
GOT is higher than that of GPT). However, differences in laboratory methods limit
the usefulness of the ratio. Another rule of thumb: if the ALT (GPT) is high (greater
than 400 U/L), alcoholic liver disease is unlikely, regardless of the aminotransferase
ratio. Elevated AST levels are not specific for liver damage, and AST has also been
used as a cardiac marker. AST is found in the mitochondria and in the cytoplasm.
ALP - Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts
of the liver. It works best at pH=10. ALP levels in plasma will rise with large bile
duct obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. ALP is
also present in bones, intestines and placental tissue, as well as in tumors where it
retains its activity even at 65ºC! As it is present in bones, it is higher in growing
children (as their bones are being remodelled) and elderly patients with Paget's
disease. Reference interval: 100-280 U/L.
GGT - Gamma glutamyl transpeptidase: reasonably specific to the liver and a more
sensitive marker for cholestatic damage than ALP, GGP may be elevated with even
minor, sub-clinical levels of liver dysfunction (in biliary obstruction it can be elevated
30-fold). GGT is raised in alcohol toxicity (acute and chronic). It is also found in the
cells lining the biliary ducts. Reference interval: 7-50 U/L.
LDH – Lactate dehydrogenase: LDH5 is the most abundant LDH isoenzyme in liver
cells. Therefore, LDH5 activity and ratio are significantly increased in hepatocellular
damage.
Avi Sayag Clinical Biochemistry
Lab tests used for the diagnosis and monitoring of liver diseases and for evaluation of liver
functions can be divided into the following groups:
1. Substances synthesized by the liver:
i. Plasma proteins (albumin, haptoglobin, AFP)
ii. Cholinesterase activity
iii. Clotting factors activity
2. Substances metabolized by the liver:
i. Drugs
ii. Bilirubin metabolism and excretion
iii. Cholesterol and triglycerides
3. Substances released from damaged hepatic tissue:
i. Enzymes from damaged hepatocytes (GOT, GPT, LDH5)
ii. Increased synthesis/enhanced excretion of other enzymes (GGT,
ALP)
4. Substances in the plasma excreted by the liver:
i. Endogenous metabolites (bile acids, bilirubin, ammonia)
ii. Exogenous materials (lidocain, coffein, galactose)
TESTS
1. Tests of cellular injury GPT, GOT and LDH5 are elevated.
- In slight inflammation (minor injury) the deRitis ratio is reduced (i.e. GOT<GPT)
since the mitochondria remains intact.
- In more severe injuries the ratio increases as the mitochondria is injured as well
(note again the location of the various enzymes).
2. Tests of liver function 1. Serum bilirubin: the 300 mg/day production of bilirubin are derived from Hb
breakdown (80%) and from myoglobin breakdown (20%). Bilirubin can be
unconjugated (bound non-covalently to albumin) or conjugated to glucoronic acid
(10% to monoglucoronide and 90% to diglucoronide. During persistent conjugated
hyperbilirubinemia, when the excretory function of the liver is impaired, a covalently-
bound conjugated bilirubin fraction is found – delta bilirubin). Bilirubin is water
soluble when it is conjugated and in the cis form (and it is water insoluble in the trans
form and when it is unconjugated). Reference range - < 17 µmol/L (mainly
unconjugated). Conjugation occurs in the ER.
2. Bromosulfophthalein tests (BSP test): a very sensitive and specific test of liver
excretory function. 5 mg/Kg are administered IV. After 45 minutes, the percent of the
BSP that remains in the blood is measured. Reference range: 0-5% - normal; 5-25% -
moderate dysfunction; 25-75% - severe; >75% very severe. This test is particularly
useful in the setting of anicteric hepatitis, residual change after recovery from
hepatitis, in cirrhosis and all stages of chronic hepatitis, and when fatty liver is
suspected or when toxic liver damage is suspected.
3. Urobilinogen
4. Albumin concentration: reference range – 30-60 g/L.
5. Clotting factors due to parenchymal damage: in moderately severe cases PT is
elevated, and in severe cases PT, APTT and TT are elevated while the fibrinogen is
lowered. In the setting of obstructive icterus PT and APTT are elevated.
6. Pseudocholinesterase activity: this enzyme rapidly metabolizes succinylcholine,
thereby controlling its duration of action. This enzyme may be lowered in liver
disease (hepatitis, cirrhosis).
7. Galactose tolerance test: this test is based on the ability of the liver to convert
galactose to glycogen. It is measured by the rate of excretion of galactose following
ingestion/IV injection of a known amount. Normally, less than 3 grams appear in the
urine within 5 hours after the ingestion of 40 grams.
8. Lipid parameters
Avi Sayag Clinical Biochemistry
9. Urea/Ammonia: hyperammonemia can be inherited (due to deficiency of urea-cycle
enzymes) or acquired (due to renal failure or liver disease. Cirrhosis, hepatitis and
Reye's syndrome may lead to this condition).
10. Bile acids: normal reference range – 2-4 grams. The function of the bile acids is to
facilitate cholesterol excretion, solubilize lipids (thus facilitating their absorption) and
to activate pancreatic enzymes. Most of the bile acid is primary bile acid (cholic acid
and chenodeoxycholic acid). Around 30% is secondary bile acids (deoxycholate and
lithocholate). Prior to the secretion of primary bile acids, they are conjugated with
glycine or taurine to increase their solubility in water. The endogenous microflora
converts the primary acids into the secondary form. 95% of the bile pool is recycled
(that is, it is reabsorbed in the distal ileum by active transport, and in the jejunum and
colon by diffusion). It is decreased when bile acid synthesis is defective, and in cases
of resection/inflammation/bypass of the ileum. It is elevated (by 50%) in fasting,
following a meal, and in cholestasis.
11. Serum iron and copper: the liver synthesizes transferrin and stores iron in
ferritin/hemosiderin. In hepatic icterus the iron is elevated and in obstructive icterus
its levels are normal. The liver also incorporates copper into ceruloplasmin (the
excess is excreted in the bile). In hepatic icterus the copper is normal and in
obstructive icterus its levels are elevated.
Avi Sayag Clinical Biochemistry
Assay of serum bilirubin. Detection of bilirubin and UBG in the urine
(practical topic 22) Assays of serum bilirubin:
There are 4 methods for the determination of serum bilirubin:
1. Direct spectrophotometry: bilirubin concentration is proportional to the absorbance of
the sample measured at 455nm. If we substract the absorbance measured at 575nm,
we will eliminate the interference of HbO2 (HbO2 has about the same absorbance at
both wavelengths, while bilirubin absorbs only at 455nm). This method can be used
only in newborn, since they do not have any interfering compounds.
2. Jendrassik-Grof method: the principle is based on the photometric measurement of a
color product. This method can be automated and is widely used. Bilirubin reacting
with diazo-sulfanylic acid is transformed into a color product called azobilirubin.
There is a linear correlation between the absorbance of the color product at 580nm
and the concentration of bilirubin. (The conjugated bilirubin reacts with the diazo-
sulfanylic acid, while the unconjugated form reacts with it only in the presence of
coffein). Reference interval: <20 µmol/L.
3. HPLC: can be used only as a reference method (time consuming).
4. Dry chemistry method: for this method we use slides with 3 layers – the top layer
consists of Na-benzoate to dissociate the albumin from bilirubin. The middle layer
contains gelatine to bind serum proteins, and the bottom layer contains a cationic
polymer that binds bilirubin. The amount of both forms of bilirubin can be measured
by spectrophotometry.
Bilirubin in the urine:
No bilirubin can be found in the urine of healthy individuals. In diseases, conjugated bilirubin
can be found in the urine. There are 2 principal tests:
1. Rosin test: we put 1% alcoholic iodine solution on the urine sample. If bilirubin is
present, a green ring is visible on the border of the 2 liquids.
2. EL-U-TEST test strip: the detection is based on the reaction of bilirubin with
diazonium salt. 1 minute after immersing the strip in the urine sample, we compare
the color of the strip to that on the box.
UBG in the urine:
There are 2 principal tests:
1. Ehrlich's reaction: UBG and stercobilinogen form a red color when reacting with
Ehrlich's reagent. The sample should be fresh and cooled to room temperature. The
color should be pink. Red color implies increased levels.
2. EL-U-TEST: same as for bilirubin. The color of the scale turns from light pink to red,
depending on the concentration of UBG. Normal values are above 1.7 µmol/L, the
borderline value is higher than 17µmol/L, and the result is definitely pathological
above 68µmol/L. False negative results can be obtained if the sample is stored for a
long time (especially when exposed to light) due to the oxidation of UBG.
Avi Sayag Clinical Biochemistry
Topic 40 Lab diagnosis of cholestasis and liver cirrhosis High pressure in the bile duct and bile tree induces enzyme synthesis. Since the bile secretion
is blocked, the enzymes flow back into the systemic circulation. The tests performed in
cholestasis are:
1. ALP – the optimal pH for the enzyme is 9-10.5. It has several isoenzymes: tissue non-
specific (liver, bone and kidney), intestinal, placental, germ-cell and Regan (in some
tumors). In cholestasis, special forms of ALP may appear (high molecular weight
ALP, ALP complexed to lipoprotein X and intestinal ALP). The reference interval is
100-280 U/L.
2. GGT – this enzyme is a good indicator of cholestasis. It may also be elevated in
alcohol abuse, when anticonvulsive drugs are taken, and when rifampicin is taken.
Reference interval: 7-50 U/L.
3. Lipoprotein X – this lipoprotein is detected in cholestasis with a sensitivity of 0.57. It
is composed of high amount of free cholesterol (30%) and phosphatidylcholine
(60%). TG and cholesteryl-ester are low (2-5%). Albumin, APO-C and APO-E are
also present, and its density resembles LDL. In agar gel electrophoresis it migrates to
the cathode, unless LCAT and APO-A1 are added, which reduce its size and cause it
to move toward the anode. Since the composition is similar to the bile, LpX
represents bile extravasated to the plasma secondary to cholestasis.
4. Bilirubin is elevated (see previous topic)
5. Bile acids are elevated (see previous topic)
In liver cirrhosis:
Reduced: albumin, cholesterol synthesis and cholinesterase.
Elevated: PT, ALP, GGT, AST, ALT, LDH5, ammonia, bilirubin, bile acids.
deRitis ratio is elevated.
The cause of liver cirrhosis should be elucidated:
In 40% of cases, the cause is alcoholism: in acute alcohol abuse, ethanol can be
measured in body fluids (very rapidly cleared). In chronic alcohol abuse, GGT, GOT,
and GPT are elevated. These markers, though organ-specific, tell little about the
etiology of the disease. They are not sensitive, and detect alcohol abuse when it
results in organ damage. Another marker is carbohydrate transferrin (sialo-Tf). Most
of the Tf is in the isoform of tetrasialo-Tf or trisialo-Tf (98%). The rest is in the
asialo-Tf, monosialo-Tf or disialo-Tf. The level of these Tf is elevated in chronic
excessive alcohol intake (60g for 30 days), and there is a strong correlation between
the levels measured and the amount of alcohol intake. The levels return to normal
after 10-18 days.
In 30% of cases, the cause is hepatitis B infection: transaminases are elevated, PT is
elevated and albumin is low. Hepatitis B is diagnosed by serology to detect HbsAg
and core Ab (HbcAb). Detection of HbsAg present for more than 6 months implies
chronic carrier state.
Drugs and toxins: examples are paracetamol, tetracyclins, methotrexate, rifampicin,
methyl-dopa, isoniazid.
Wilson's disease: the problem lies in copper deposition. Thus, ceruloplasmin is < 300
mg/L and serum copper is elevated.
Hemochromatosis: can be primary or secondary. Serum Fe is elevated, Tf saturation
is 80-100%, ferritin levels are high, and definitive diagnosis can be made with
molecular biology.
Primary biliary cirrhosis: elevated ALP and transaminases, as well as the presence of
autoantibodies (antimitochondrial, antinuclear, smooth muscle autoAb).
Avi Sayag Clinical Biochemistry
Topic 41 Pathobiochemistry and lab diagnosis of the GI tract 6 main pathologies should be considered:
1. Peptic ulcer disease
a. Chronic duodenal ulcer
b. Chronic benign gastric ulcer
c. Zollinger-Ellison syndrome (hereinafter "Z-E syndrome")
2. Gastritis
a. Erosive gastritis
b. Non-erosive gastritis
3. Gastric Cancer
The main diagnostic tools are endoscopy and contrast radiography (but not for Z-E
syndrome).
3 main lab tests should be carried out:
1. Gastric acid secretion
The acid in fluid aspirated through a nasogastric tube is measured in the resting state to
determine the basal stimulation and after the administration of pentagastrin (an analogue of
gastrin) to determine the maximal secretion.
Reference values: for males, the basal value should be less than 10 mmol/h and the maximal
value should be less than 45 mmol/h. For women, the basal value should be less than 6
mmol/h and the maximal one should be less than 35 mmol/h.
3 pathologies can be inferred from these values:
1. Achlorhydria: in this condition the basal value is low (and sometimes even undetected), and
there is no response to pentagastrin. But when the gastric antrum is intact in achlorhydria or
in renal failure, the gastrin concentration is normal, because the G cells are intact (they secrete gastrin). Several conditions may lead to achlorhydria: autoimmune disorders where
there is antibody production against parietal cells which normally produce gastric acid; the
use of antacids or drugs that decrease gastric acid production (such as H2-receptor
antagonists) or transport (such as proton pump inhibitors, e.g. omeprazole); a symptom of rare
diseases such as mucolipidosis (type IV; a symptom of Helicobacter pylori infection which
neutralizes and decreases secretion of gastric acid to aid its survival in the stomach; a
symptom of pernicious anemia, atrophic gastritis, VIPomas (see next topic) or of stomach
cancer; radiation thrapy involving the stomach; and gastric bypass procedures such a
Duodenal Switch, where the largest acid producing parts of the stomach are either removed,
or blinded. Thus, in this condition it is obvious that nothing will cause the stimulation of the
parietal cells).
2. Chronic duodenal ulcer: the peak acid output is increased in this condition.
3. Chronic benign gastric ulcer: the peak gastric output is normal in this condition.
2. Determination of gastrin level This test is good for the diagnosis of Z-E syndrome. Z-E syndrome is a disorder where
increased level of gastrin is produced, causing the stomach to produce excess hydrochloric
acid. Often the cause is a tumor (gastrinoma) of the duodenum, pancreas or stomach (a triade)
producing more gastrin. Gastrin then causes an excessive production of acid which can lead to
peptic ulcers (in almost 95% of patients). The diagnosis is made by these lab tests: secretin
stimulation test, which measures gastrin levels in response to secretin, fasting gastrin levels
and antral acidity. High serum gastrin levels are diagnostic of Z-E syndrome (normal plasma
gastrin is less than 50 ng/L. In Z-E syndrome it exceeds 200 ng/L). In uncertain cases, the
secretin provocation test10
is applied.
10 A test that measures the ability of the pancreas to respond to secretin. Secretin is a hormone secreted by the
small intestines in the presence of partially digested food from the stomach and stimulates the pancreas to secrete
bicarbonate to neutralize stomach acidity. A tube is passed through the nose into the duodenum. Secretin is
administered and the contents of the duodenal secretions are aspirated and analyzed over a period of about 2 hours.
Avi Sayag Clinical Biochemistry
60% of all cases of Z-E are malignant, and 30% are MEN I (Multiple Endocrine Neoplams).
The symptoms of the disease are ulcers (due to hypersecretion of acid), diarrhea, epigastric
pain, steatorrhea (due to inactivation of lipases, as the low pH in the small intestine
inactivates these enzymes), and B12 is not absorbed (pernicious anemia).
3. Detection of H. pylori infection
a. C13
-urea breath test If H. pylori is suspected, this test is performed. The patient arrives after an overnight fasting,
and his exhaled air is collected in a tube to determine the basal value of CO2 in his breath.
Patients swallow 75 mg urea labeled with an uncommon isotope in 150 ml apple juice (either
radioactive C14
or non-radioactive C13
). In the subsequent 10-30 minutes, the detection of
isotope-labeled CO2 in exhaled breathe indicates that the urea was split; this indicates that
urease (the enzyme that H. pylori uses to metabolize urea) is present in the stomach, and
hence that H. pylori bacteria are present. The 14
CO2/13
CO2/12
CO2 is analyzed using GC/MS
(HPLC).
Diagnosis of H. pylori is highly important, as is it associated with chronic active gastritis,
peptic ulcer, MALT lymphoma and stomach carcinoma.
Diseases of the intestinal tract 1. Malabsorption
Triolein breath test If fat malabsorption is suspected, this test is performed. The patient fasts overnight. A basal
sample of expired CO2 is collected. Then, the patient is given isotopically labeled triolein in a
30-gram fat meal, and samples of expired CO2 are collected hourly for 7 hours. The isotope is
measured in the CO2 samples.
2. Diarrhea: excess water in the feces due to long exposure to water that exceeds the
reabsorption capacity of the colon (3.8L/day), intake of osmotically active substances that are
not absorbable, deranged motility, and exudation of mucus, blood and protein due to
inflammation of the intestines.
3. Ulcerative colitis and Crohn's disease
4. Celiac disease:
In this condition, there is damage to the mucosa of the small intestine resulting in
malabsorption. Symptoms include weight loss, bloating, flatulance, diarrhea, and the feces is
large in volume, pale, watery and malodorous. Since malabsorption is a feature of the disease,
some biochemical alterations may occur in folic acid, B12, iron (these 3 lead to anemia),
calcium and vitamin D (leading to osteomalacia, osteoporosis and secondary
Avi Sayag Clinical Biochemistry
hyperparathyroidosis) and fat-soluble vitamins (leading to neurological problems). Lab tests
performed include:
Anti-gliadin Ab (IgA and IgG in ELISA)
Anti-reticulin Ab
Anti-endomisium Ab
Anti-transglutaminase Ab
IgA
Gut biopsy
Molecular genetic examination (HLA DQ type)
Rate of γδ T cells.
(The problem in this condition is sensitivity to gluten. The damage is immune-mediated
against the mucosa of the small intestine).
Diagnosis of intestinal disorders:
1. General screening tests should include albumin, Ca+2
, B12 and peripheral blood smear.
2. Xylose absorption test: a variety of tests involving the ingestion of carbohydrates and the
measurement of their plasma concentrations or urinary excretion have been developed for the
investigation of small intestinal function. The best known is the xylose absorption test, which
involves the administration of a test dose of d-xylose, a plant sugar. This is absorbed from the
jejunum without prior digestion. It is only partly metabolized in the body, mainly excreted
unchanged in the urine, where it can be measured. Accurately timed urine collection is
essential. Misleadingly low results are obtained if the glomerular filtration rate is decreased,
as occurs in renal failure and many normal elderly people. Other factors that can produce
misleading results include delayed gastric emptying, edema and obesity. An alternative
approach is to measure serum xylose concentration 60 min after administering xylose.
3. Disaccharaide test: suspected intestinal disaccharidase deficiency can be investigated by
administering the appropriate disaccharide (50 g) orally and measuring the blood glucose
response. If the result is abnormal, specificity can be improved by comparing the result with
that obtained following administration of the equivalent quantities (25 g each) of the
constituent monosaccharides. The test is unphysiological because of the large oral load of, for
example, lactose, although patients who do not develop symptoms during the test are not
lactose intolerant.
4. Breath Hydrogen test: a more reliable test is to measure breath H2 after giving the
disaccharide: because it is not absorbed, the disaccharide reaches the colon where one of the
products of bacterial fermentation is H2. The definitive investigation for disaccharidase
deficiencies is measurement of the appropriate enzyme in a biopsy sample. 5. Triolein breath test (see above)
6. Fecal fat test
7. Fecal amino acid measurement
8. Gut biopsy
9. Specific tests such as occult blood (colon cancer), CEA, Schilling test
10. Radiological examination
Avi Sayag Clinical Biochemistry
Topic 42 Lab diagnosis of acute pancreatitis There are 2 forms of acute pancreatitis: the interstitial (edematous) form, which is the milder
form with minimal organ damage and usual full recovery, and the hemorrhagic form
(necrotizing), which is more serious with organ failure and local complications (pseudocyst
formation, ascites, pleural effusion and pancreatic/peripancreatic abscesses).
Acute pancreatitis usually occurs in middle-aged people as a sudden acute abdominal pain.
The pain usually occurs after the ingestion of a large meal, or the consumption of alcohol, and
is referred to the back. Due to electrolyte imbalance, bradykinin and prostaglandins are
released, which may lead to hypotension and circulatory shock. Toxic psychosis and fever are
also features of the disease. Mild jaundice may accompany the condition, as the inflamed
pancreas compresses the bile duct.
Causes:
1. Metabolic causes: alcohol, drugs, hypercalcemia, hyperlipoproteinemia and genetics.
2. Mechanical: gallstones, ERCP (endoscopic retrograde cholangiopancreatography),
abdominal injury, perioperative injury.
3. Infections: mumps, coxackievirus and Mycoplasma pneumonia
4. Vascular: atheroembolism and polyarteritis nodosa
Alcohol, viruses, drugs, ischemia and trauma cause direct acinar cell injury.
Duodenal reflux leads to accumulation of lecithin in duodenal content. Lecithin is converted
into lysolecithin which causes direct acinal cell injury as well.
Cholelithiasis, chronic alcoholism and obstructing lesions lead to duct obstruction.
Metabolic causes, alcohol and duct obstruction cause deranged intracellular transport.
Pathomechanism:
Intercellular leak of enzymes, the release of intracellular enzymes and activation of enzymes
be lysosomal hydrolases all activate enzymes. The activated enzymes cause inflammation,
proteolysis of pancreatic substances (by proteases), fat necrosis (by lipases) and necrosis of
vessels with hemorrhages (by elastases). All these manifest as acute pancreatitis.
The lab tests performed:
1. Serum α-amylase
2. Urine amylase
3. Serum lipase – can also appear in the urine, but only traces (insignificant).
4. Pancreatic isoamylase – in cases of acute pancreatitis in patients with renal failure,
and for the diagnosis of the coexistence of mumps and acute pancreatitis.
5. Immunoreactive trypsin
6. Serum elastase
α-amylase is not specific for the pancreas, and it also occurs in the salivary glands, in the
gonads, in the Fellopian tube, in the small intestine, in striated muscles, in adipose tissue, and
in the lungs. In the serum, amylase P and S are found (2 isoenzymes: P for pancreatic form,
and S for salivary gland form).
α-amylase is also present in the urine, in ascites and in pleural effusion. Its function is to
digest starch (hydrolyzes the α-1,4 glycosidic bond), and it is cleared through the kidney.
Amylase activity is increased in the following conditions:
1. In acute pancreatitis, its levels are elevated to more than 10 times the upper range
limit (reference range: 21-101 U/L)
2. In perforated peptic ulcer, tumors of the lung and ovaries and ruptured ectopic
pregnancy, its levels are elevated to more than 5 times the URL.
3. In acute abdominal disorders (such as acute cholecystitis, intestinal obstruction,
mesenteric infarction and peritonitis), acute alcoholic intoxication, salivary gland
disorders, neoplastic hyperamylasemia, severe glomerular impairment, opiate
administration, diabetic ketoacidosis and macroamylasemia, its levels are elevated but
rarely more the 5 times the URL.
Avi Sayag Clinical Biochemistry
In acute pancreatitis, the ratio between the clearance of amylase and creatinine is also
elevated (normally, it is 2-5%). In renal insufficiency, though, the ratio is normal (because
both Cam and Ccr are low proportionally).
Serum amylase starts to rise 2-12 hours after onset of acute pancreatitis, it peaks after 12-48
hours and returns to normal after 3-4 days.
Urine amylase starts to rise 2-12 hours after onset of acute pancreatitis, it peaks after 24-72
hours and returns to normal after 5-7 days.
Serum lipase starts to rise 4-8 hours after onset of acute pancreatitis, it peaks after 24-48
hours and returns to normal after 7-14 days.
The sensitivity of α-amylase alone in the diagnosis of acute pancreatitis is 80%, but when the
test is combined with lipase the sensitivity rises to 94%.
Lipase is produced in the pancreas and hydrolizes emulsified triglycerides (it cleaves the fatty
acids from the glycerol). Pancreatic colipase is a coenzymes of lipase. It is mainly found in
the serum, but traces can be also found in the urine. The RES probably clears this enzyme.
The reference range is 0-190 U/L.
Further lab findings in acute pancreatitis include:
Coagulation disorders (DIC)
Hypocalcemia
Hypoglycemia and hyperglycemia
Glycosuria
Hypoproteinuria
Albuminuria
Increased serum urea concentration
Avi Sayag Clinical Biochemistry
Topic 43 Clinical biochemistry of hypothalamus and hypophysis For details about the hormones and functional structure of the pituitary and hypothalamus
please consult Histology – Avi's notes, page 1 (or you can use Ross directly, but obviously I
recommend mine ☺ ).
Growth Hormone (STH) This hormone promotes growth. Therefore, it
Increases protein synthesis;
Increases synthesis of glucose by the liver
Increases lypolysis and
Decreases uptake of glucose by the tissues
Remember that this hormone counteracts the actions of insulin.
Growth hormone releasing hormone (GHRH) is secreted from the hypothalamus and
stimulates the secretion of GH from the adenohypophysis.
Somatostatin inhibits its production (also secreted from the hypothalamus). Somatostatin has
many other actions both within the hypothalamo-pituitary axis and elsewhere. For example, it
inhibits the release of thyroid-stimulating hormone (TSH) in response to thyrotrophin-
releasing hormone (TRH) and it is present in the gut and pancreatic islets, where it inhibits the
secretion of many gastrointestinal hormones including gastrin, insulin and glucagon.
GH stimulates the liver to produce insulin-like growth factor-1 (IGF-1), also known as
somatomedin-C. IGF-1 has homology with insulin and shares some of the actions of this
hormone. IGF-1 exerts negative feedback at the level of the pituitary, where it modulates the
actions of GHRH, and at the level of the hypothalamus where, together with GH itself, it
stimulates the release of somatostatin (that inhibits the release of GH). The concentration of
GH in the blood varies widely through the day. Physiological secretion occurs in sporadic
bursts, lasting for 1-2 h, mainly during deep sleep. The rate of secretion increases from birth
to early childhood and then remains stable until puberty, when a massive increase occurs,
stimulated by testosterone in males and estrogens in females; thereafter the rate of secretion
declines to a steady level before falling to low levels in old age.
Secretion can be stimulated by stress, exercise, a fall in blood glucose concentration, fasting
and ingestion of certain amino acids.
Diagnosis: stimuli can be used in provocative tests for diagnosing GH deficiency. GH
secretion is inhibited by a rise in blood glucose and this effect provides the rationale for the
use of the oral glucose tolerance test (OGTT) in the diagnosis of excessive GH secretion (a
suppressive test). Insulin hypoglycemia test (see Marshall page 138 for protocol) is a
stimulating test.
Pathology: excessive secretion (usually due to a pituitary tumor) causes gigantism in children
and acromegaly in adults; 95% of cases are due to pituitary tumor/MEN1 and 5% are due to
ectopic GHRH secretion. As IGF-I is also elevated, it causes abnormal glucose tolerance,
DM, CHF (due to hypertension) and osteoporosis. Deficiency causes growth retardation in
children and can cause fatigue, loss of muscle strength, impaired psychological wellbeing and
an adverse cardiovascular risk profile (elevated plasma total and LDL-cholesterol
concentrations and hyperfibrinogenaemia) in adults, and hypoglycemia (as insulin will work
unopposed). Tests include: random GH, GH after OGTT, IGF-1 and prolactin level.
Prolactin Functions
To initiate and sustain lactation
A role in breast development
Inhibits the synthesis of GnRH (at high concentrations, therefore it inhibits ovulation
and spermatogenesis).
Control: dopamine secreted from the hypothalamus inhibits the release of prolactin.
Thyroid releasing hormone (TRH) and VIP (vasoactive intestinal polypeptide
secreted from the GI) stimulate secretion of prolactin. Physiologically, pregnancy and
breast-feeding stimulate its secretion, as well as stress and sleep. Its secretion
Avi Sayag Clinical Biochemistry
increases during pregnancy, but concentrations fall to normal within approximately
seven days after birth if a woman does not breastfeed. With breast-feeding,
concentrations start to decline after about three months, even if breast-feeding is
continued beyond this time.
Pathology: causes include
o Physiological: stress, sleep, pregnancy and suckling
o Drugs: dopaminergic receptor blockers, dopamine antagonists, pituitary
tumors, trauma.
o Clinical features: females: oligomenorrhea, amenorrhea, infertility,
galactorrhea11
; males: impotence, infertility, gynacomastia.
Thyroid stimulating hormone (TSH)
Thyroid-stimulating hormone (TSH) is composed of an α- and a β-subunit; the amino
acid composition of the α-subunit is common to TSH, the pituitary gonadotrophins
and human chorionic gonadotrophin (hCG), but the β-subunit is unique to TSH.
The release of TSH is regulated at the level of the hypothalamus and by the thyroid
gland itself: TRH from the hypothalamus stimulates its secretion, while somatostatin
and dopamine inhibit its secretion. If plasma concentrations of thyroid hormones
decrease (T3 and T4), TSH secretion increases, stimulating thyroid hormone
synthesis; if they increase, TSH secretion is suppressed (negative feedback).
Pathology: in primary hypothyroidism, TSH secretion is increased; in
hyperthyroidism it is decreased. TSH deficiency can cause hypothyroidism but
hyperthyroidism due to TSH-secreting tumours is rare. Further details in topic 44.
Gonadotrophins (FSH and LH) FSH and LH both consist of two subunits: the β-subunits are unique to each hormone
but the amino acid sequence of the α-subunits is the same, and is found in both TSH
and hCG.
The synthesis and release of both hormones are stimulated by the hypothalamic
decapeptide, gonadotrophin-releasing hormone (GnRH), and their effects are
modulated by circulating gonadal steroids. GnRH is secreted episodically, resulting in
pulsatile secretion of gonadotrophins with peaks in plasma concentration occurring at
approximately 90-min intervals.
In males, LH stimulates testosterone secretion by Leydig cells in the testes: both
testosterone and estradiol, derived from the Leydig cells themselves and from the
metabolism of testosterone, feedback to block the action of GnRH on LH secretion.
FSH, in concert with high intratesticular testosterone concentrations, stimulates
spermatogenesis; its secretion is inhibited by inhibin, a hormone produced during
spermatogenesis.
In females, the relationships are more complex. Estrogen (mainly estradiol) secretion
by the ovaries is stimulated primarily by FSH in the first part of the menstrual cycle;
both hormones are necessary for the development of Graafian follicles. As estrogen
concentrations in the blood rise, FSH secretion declines until estrogens trigger a
positive feedback mechanism, causing an explosive release of LH and, to a lesser
extent, FSH. The increase in LH stimulates ovulation and development of the corpus
luteum, but rising concentrations of estrogens and progesterone then inhibit FSH and
LH secretion; inhibin from the ovaries also appears to inhibit FSH secretion. If
conception does not occur, declining concentrations of estrogens and progesterone
from the regressing corpus luteum trigger menstruation and LH and FSH release,
initiating the maturation of further follicles in a new cycle. Before puberty, plasma
concentrations of LH and FSH are very low and unresponsive to exogenous GnRH.
With the approach of puberty, FSH secretion increases before that of LH.
11 A spontaneous flow of milk from the breast associated with breastfeeding.
Avi Sayag Clinical Biochemistry
Adrenocorticotrophic hormone (ACTH) ACTH stimulates adrenal glucocorticoid (and mineralocorticoids to a lesser extent)
secretion. ACTH is a fragment of a much larger precursor, pro-opiomelanocortin
(POMC), which is the precursor not only of ACTH but also of β-lipotrophin, itself the
precursor of endogenous opioid peptides (endorphins). ACTH release is controlled by
CRH.
ACTH secretion is pulsatile and also shows diurnal variation, the plasma
concentration being highest at approximately 0800 h and lowest at midnight.
Secretion is greatly increased by stress and is inhibited by cortisol. Thus, cortisol
secretion by the adrenal cortex is controlled by negative feedback, but this and the
circadian variation can be overcome by the effects of stress.
Diagnosis: dexamethason suppresses ACTH and insulin stimulates its secretion.
Pathology: increased secretion of ACTH by the pituitary is seen with pituitary tumors
(Cushing's disease) and in primary adrenal failure (Addison's disease). The hormone
may also be secreted ectopically by non-pituitary tumors (mainly lung cancer).
Excessive ACTH synthesis is associated with increased pigmentation, owing to the
melanocyte-stimulating action of ACTH and other POMC-derived peptides.
Decreased secretion of ACTH may be an isolated phenomenon, but is more
commonly associated with generalized pituitary failure.
There 4 conditions that should be differentially diagnosed from pituitary tumor. In
pituitary tumor, the basal cortisol is elevated, dexamethason test is positive, ACTH is
elevated and CRH is positive. This combination is unique to pituitary tumor (CRH is
only positive in pituitary tumor).
Problems that arise in determining the reference intervals for these hormones stem from
differences in levels among males and females, from circadian changes, from cycles of these
hormones and from the methods used to define the reference range.
In hypopituitarism, the decrease in the synthesis of hormones is in the following order:
GH>FSH/LH>ACTH> TSH (G-FLAT). A combined decrease is more frequent than an
isolated decrease.
The order of frequency with which hormone secretion occurs in patients with pituitary tumors
is prolactin>GH>ACTH>FSH/LH>TSH
The posterior pituitary gland secretes ADH and oxytocin, synthesized in the hypothalamus in
the supraoptic nucleus and in the paraventricular nucleus.
ADH (aka vasopressin) Uncontrolled ADH secretion leads to SIADH (see topic 27).
Decreased ADH secretion leads to diabetes insipidus that can be either:
Cranial diabetes insipidus (CDI): due to tumor, trauma, meningitis, encephalitis, and
familial CDI.
Nephrogenic diabetes insipidus (NDI): due to metabolic hypokalemia, hypercalcemia,
drugs, chronic renal disease and familial NDI.
Avi Sayag Clinical Biochemistry
Diabetes insipidus is diagnosed by the fluid deprivation test. The patient is allowed a light
breakfast with no fluid, and no smoking is permitted. The patient is then weighted. Fluids are
deprived for 8 hours. Every hour the patient is weighted, the osmolality and volume of urine
are measured, and the plasma osmolality is measured. After 8 hours, the patient is allowed to
drink and desmopressin (a synthetic analogue of ADH) is administered intranasally
(according to the book, I.M). Finally, the urine osmolality is measured during 4 hours. In a
normal subject, the urine becomes concentrated in response to fluid deprivation and plasma
osmolality does not exceed 295 mmol/kg. In diabetes insipidus, the urine does not become
concentrated and plasma osmolality rises. In patients who are water overloaded before the test
is started, the urine may not become concentrated: plasma osmolality is usually low and may
remain so since ADH secretion is only stimulated if it rises above 285 mmol/kg. Thus, the
urine becomes concentrated only if the plasma osmolality exceeds this level. If the results of a
fluid deprivation test are equivocal (as in practice they often are), the plasma ADH response
to hypertonic saline infusion can be assessed. The response is normal in patients with NDI or
primary polydipsia, but decreased in patients with CDI. The former two conditions can be
distinguished by comparing plasma ADH concentration with urine osmolality after a period of
fluid deprivation. In NDI, plasma ADH is much higher than normal.
Avi Sayag Clinical Biochemistry
Topic 44 Pathobiochemistry and laboratory diagnosis of hypothyroidism and
hyperthyroidism For the histology of the thyroid gland, please consult previous notes (Avi's Histology Notes,
Thyroid Gland, page 10).
The 2 hormones, T3 and T4 diffuse passively through the membrane of the target cell, where
they bind to intracellular nuclear receptors12
. The association with the receptor leads to
dissociation of heat shock proteins, and the complex is thus in its activated conformation. The
complex is translocated to the nucleus (in case the receptor is in the cytoplasm). It then binds
to the DNA on non-palindromic sequences. Steroid receptors often form dimers. In the
nucleus, the complex acts as a transcription factor, augmenting or suppressing transcription of
particular genes by its action on the DNA (for general knowledge, the receptor for the thyroid
hormones belong to the RXR receptors (for more details, you may consult Avi's Biochemistry
Notes, page 16).
There are 3 thyroid hormones: T4 (thyroxine), T3 (triiodothyronin) and calcitonin, which is
produced in the parafollicular cells (C cells) and not in the follicular cells.
Thyroid hormones have diverse actions:
1. Calorigenic effect on tissues: they stimulate the basal metabolic rate, oxygen
consumption and heat production (through actions that include stimulating the Na+/K
+
pump and increasing the availability of energy substrates).
2. Stimulating protein synthesis, and thus
3. Growth development
4. Increase the sensitivity of the cardiovascular and nervous system to catecholamines
(β-adrenergic receptors), and thus
5. Stimulate heart rate and contraction
6. Stimulate carbohydrate metabolism
7. Increase synthesis and catabolism of cholesterol and TG.
The overall effect of thyroid hormones is to increase net catabolism.
Synthesis of thyroid hormones:
Thyroid hormones are synthesized by first trapping serum iodine. Then, iodination processes
of tyrosine residues on the thyroglobulin molecule occurs. When one iodine binds to the
tyrosine it forms monoiodotyrosine – MIT, and when 2 iodine atoms bind to the tyrosine they
form diiodotyrosine – DIT. When 2 DITs undergo a coupling reaction, they form T4, and
when one MIT and one DIT undergo a coupling reaction, they form T3. Finally, the
thyroglobulin is proteolytically cleaved to release the hormones. The cleavage occurs in the
follicular cells, and the thyroglobulin is stored in the follicular colloid.
Whereas the thyroid is the only organ that secretes T4, T3 can be produced by the kidney, the
heart and the liver by conversion from T4. In the periphery, most T4 is de-iodinized to T3,
which has a 10-fold greater affinity to the nuclear receptors than T4, and is thus 4-5 times
more effective (actually, 40% of T4 is converted to T3 and 45% to rT3). It follows that we
can regard T4 as a prehormone.
Thyroid hormones in the blood
The normal plasma concentration of T4 is 60-150 nmol/L, and T3 – 1-2.9 nmol/L (T4 is > 50
times T3). Almost all is bound to thyroxine (T4) binding globulin (TBG), and to a lesser
extent to thyroxine-binding prealbumin and to albumin (99.98% of T4 and 99.66% of T3 are
bound). However, the free T4 is only 2-3 times the free T3 – measured in pmol/L).
TBG is 1/3 saturated.
The binding proteins serve to maintain the free T3 and T4 concentration within narrow limits,
while ensuring the hormones are readily available to the tissues. Only the free thyroid
hormones are physiologically active.
12 NOTE: it is generally agreed today that the receptors for the thyroid hormones are located in the
nucleus (and there they heterodimerize), while other receptors for glucocorticoids, aldosterone,
testosterone and estradiol are located in the cytoplasm (where they homodimerize).
Avi Sayag Clinical Biochemistry
The increase in the concentration of the binding proteins (or in their affinity) is determined by
4 main factors:
1. Increased TBG concentration:
a. Genetic factors
b. Non-thyroidal illnesses (HIV infection, infectious and chronic active
hepatitis, estrogen-producing tumors)
c. Physiological conditions (pregnancy, newborns)
d. Drug use (oral contraceptive, estrogens, tamoxifen, methadone)
2. Increased prealbumin concentration
3. Increased binding to albumin
4. T4 binding to autoantibodies.
In the initial steady state, TBG is 1/3 saturated with T4. If TBG concentration increases, it
will cause more T4 to be bound, thus reducing the fT4 concentration. This stimulates TSH
secretion which leads to an increase in the release of T4 from the thyroid. T4 becomes
redistributed between the bound and the free states, leading to a new steady state with the
same fT4 concentration but an increased total T4.
A decrease in the concentration of the binding proteins (or in their affinity) is determined by 3
main factors:
1. Decreased TBG concentration:
a. Genetic factors (those who have a deficiency in the binding proteins show no
clinical abnormality, though)
b. Non-thyroidal illnesses (major illness or surgical stress, nephrotic syndrome,
malnutrition, malabsorption)
c. Drug use (androgens, anabolic steroids, large doses of glucocorticoids)
2. Decreased TBG binding capacity (drugs such as salicylates and phenytoin displace
thyroid hormones from their binding proteins, thus reducing total, but not free,
hormone concentration once a steady state is reached).
3. Decreased prealbumin concentration
There are 3 main tests performed for thyroid function:
1. TSH This is the first hormone measured (first line) as its sensitivity is the highest. That is, TSH
levels are decreased even at the earliest stages of hyperthyroidism, when the disease may still
be subclinical. However, it is best to measure TSH in combination with fT4 (for
hypothyroidism) and fT3 (for hyperthyroidism).
In primary hypothyroidism, TSH levels are greatly increased (if there is too little thyroid
hormones there is no suppression of the secretion of TSH in a negative-feedback mechanism),
while in borderline cases the increase is smaller (but still, there is an increase). TSH is also
elevated in other diseases which are not primary: TSH secreting tumors and during recovery
from severe illnesses (e.g. sick euthyroid state).
TSH is decreased in hyperthyroidism (too much thyroid hormones suppress the secretion of
TSH), even if the hyperthyroidism is subclinical (as it is very sensitive). It is also decreased in
secondary hyperthyroidism and in severe general illnesses.
2. fT3 and fT4 The measurement of free hormone concentrations poses major technical problems since the
binding of free hormones in an assay, usually by an antibody, will disturb the equilibrium
between bound and free hormone and cause release of hormone from binding proteins. This
problem has been solved (I don't know how and it's not interesting).
Low levels are measured in various forms of hypothyroidism. fT3 is not an adequate test in
this case as it can be normal (especially in mild cases). Measurement of plasma total T4 (tT4)
concentration has the major disadvantage in that it is dependent on binding protein
concentration as well as thyroid activity. For example, a slightly elevated plasma tT4
concentration, compatible with mild hyperthyroidism, can occur with normal thyroid function
if there is an increase in plasma binding protein concentrations (as in pregnancy for example,
due to increased synthesis caused by estrogens). In addition, the values of the total thyroid
Avi Sayag Clinical Biochemistry
hormones has a wide reference range that overlaps with values measured in hypo- and
hyperthyroidism.
In primary hypothyroidism, TSH levels are elevated and fT4 levels are reduced.
In secondary hypothyroidism, TSH levels are reduced and fT4 levels are reduced. (TSH levels
are reduced because of a secondary cause such as hypopituitarism, and the low levels of fT4
are due to the low levels of TSH).
Primary hypothyroidism can be due to the followings:
1. Autoimmune hypothyroidism (Hashimoto's thyroditis)
2. Idiopathic atrophic hypothyroidism
3. Thyroidectomy, radioactive iodine, anti-thyroid drugs to correct for hyperthyroidism
(carbimazole), goiter (in Hashimoto's disease or in iodine deficiency) or cancer.
4. Primary or secondary congenital hypothyroidism
5. Dyshormonogenic hypothyroidism (impaired hormone synthesis due to an enzyme
defect)
6. Secondary acquired hypothyroidism (pituitary tumor or hypothalamic tumor, vascular
insufficiency, trauma and infection)
7. Iodine deficiency (less iodine for the synthesis of thyroid hormones).
It is important, though, to distinguish between patients who have a frank thyroid disease, and
patients hospitalized for other non-thyroid diseases. In hospitalized patients, the results of lab
tests occasionally resemble hypothyroidism, even though their thyroid is fine. The diagnosis
of hypothyroidism that coincides with other diseases they are hospitalized for is quite
difficult. Lastly, dopamine and corticosteroid (in many times given to hospitalized people)
decrease TSH levels.
The occurrence of abnormalities of thyroid function tests in patients with non-thyroidal illness
has been termed the 'sick euthyroid syndrome'. Typically, during the acute phase of an
illness, fT3 concentration and, less often, fT4 concentration is decreased. TSH is usually
normal or low. During recovery, TSH may rise transiently into the hypothyroid range as
thyroid hormone concentrations return to normal.
In investigating the etiology of primary hypothyroidism, anamnesis should be taken (has the
patient received any radioactive iodine treatment? Has he had thyroidectomy? Does he have a
family history of hypothyroidism? Has he been taking anti-thyroid drugs?). Then, antibodies
should be suspected (anti-TPO13
, anti-thyroglobulin).
In screening for congenital hypothyroidism, serum TSH levels should be measured 6-8 days
after delivery.
In investigating the etiology of secondary hypothyroidism (and hyperthyroidism) the third test
should be performed:
3. TRH-test In this test, plasma TSH is measured immediately before, and 20 and 60 min after, giving the
patient 200 µg of TRH i.v. The normal response is an increase in TSH concentration of 2-20
mU/L in 20 min, with reversion towards the basal value at 60 min.
13 TPO – Tyrosine Peroxidae, the enzyme that carries out the addition of iodine to tyrosine residues.
Avi Sayag Clinical Biochemistry
Hyperthyroidism can be primary, secondary or thyroiditis.
Primary hyperthyroidism can be the result of:
1. Toxic multinodular goiter;
2. Toxic thyroid adenoma
3. Thyroid carcinoma (rare)
4. Ectopic thyroid tissue (for example in struma ovarii).
Secondary hyperthyroidism can due to endogenous or exogenous reasons:
1. Exogenous: iodine or iodine-containing drugs can stimulate synthesis of thyroid
hormones.
2. Endogenous: increased levels of TSH or other substances that stimulate thyroid
hormones. These conditions can be seen in
a. Grave's disease (Ab to TSH receptors that stimulate them)
b. Neonatal hyperthyroidism (due to maternal IgG – those of a mother sick with
Grave's disease – that cross the placenta and stimulate the TSH receptors of
the baby). This condition is temporary as these Ab will be cleared from the
baby's blood eventually.
c. TSH-secreting pituitary tumors (rare)
d. Trophoblastic tumors (hCG has a weak stimulatory effect on thyroid
secretion). This can be the cause of hydatiform moles, choriocarcinoma and
embryonal carcinoma of the testes.
Regarding Grave's disease, following partial thyroidectomy, serum fT3 and TSH levels
change. Before surgery, the patient is rendered euthyroid with anti-thyroid drugs. Initially,
TSH secretion remains suppressed, but eventually it rises in response to low fT3. Normal
thyroid hormone secretion by the remaining thyroid tissue is maintained by increased TSH
stimulation, but eventually, the patient becomes hypothyroid. Hypothyroidism can also
develop in patients treated with anti-thyroid drugs or radioiodine.
Thyroditis can be subacute (early infection), due to Hashimoto's thyroditis (though
hypothyroidism is more common in this condition), after delivery (5-10% of women suffer
from thyroditis after pregnancy) and following radiation therapy (radiation thyroditis).
Lab of hyperthyroidism: TSH levels are greatly reduced (< 0.1 mU/L. Normal range is 0.3-4
mU/L), while fT3 and fT4 are elevated (fT3 is more appropriate to measure in
hyperthyroidism).
In mild hyperthyroidism, or when TSH secretion is inhibited by drugs, THS levels are
reduced, and fT3 and fT4 are normal.
In TSH-secreting pituitary tumors, TSH levels may be normal or elevated, while fT3 and fT4
are elevated too.
In summary, these autoantibodies might be found in thyroid diseases:
1. Anti-TPO and anti-thyroglobulin antibodies: in Hashimoto's thyroiditis, in post-
partum thyroiditis, in Grave's disease and in subacute thyroiditis.
2. Thyroid stimulating immunoglobulin (TSI): in Grave's disease
3. Anti-TSH receptor Ab: in Grave's disease.
Avi Sayag Clinical Biochemistry
Off-Topic – Introduction to Hypo- and Hypercalcaemia Plasma calcium concentration is regulated by 3 hormones: PTH, calcitriol (vitamin D) and
calcitonin. Most calcium in the body is in the bone (99% of body calcium, 25,000 mmol,
which is 1 kg). Calcium in the plasma is found in 3 forms: bound to albumin (46%), bound to
anions (7%, to bicarbonate, citrate, lactate, phosphate) and as free calcium (ionized calcium,
47%). The bound form renders calcium non-diffusible. Total calcium in the plasma measures
2.1-2.6 mmol/L.
PTH PTH is a 84-amino acid polypeptide, formed in the parathyroid gland as a pre-pro-PTH with
115 amino acids. It is cleaved twice: first to form pro-PTH (after cleavage of 25 AA) and then
intact PTH (after cleavage of the remaining 6 AA). The N-terminal, with 34 AA, is the active
part of the hormone. In the plasma it is found as an intact hormone (with all 84 AA), as a
cleaved hormone (with just the 34 AA of the N-terminal) and as C-terminal fragments
(various lengths of amino acids). Only the intact PTH and the N-terminal fragment are the
active forms of the molecule, with half life of 3-4 minutes. The C-terminal fragments are
inactive, with half life of 2-3 hours. PTH (and calcitriol) act to increase plasma calcium
levels, and their secretion is triggered by hypocalcaemia.
PTH acts on several target cells:
1. On the bone: it acts rapidly on osteocytes to absorb calcium, which then passes to
osteoblasts via canaliculi, where calcium is secreted from them facilitated by vitamin
D. It also acts on osteoclasts (in the long term) and stimulates their proliferation (thus
more calcium is resorbed from the bone to the plasma).
2. On the kidney: it acts on the distal tubules to increase calcium and Mg+2
reabsorption,
and on the proximal tubules to decrease phosphate and bicarbonate reabsorption and
to induce 1α-hydroxylation (to form calcitriol).
PTH binds to target cell receptors (Mg+2
-dependent) and activates adenylate cyclase, which
forms cAMP. This leads to influx of calcium into the cell to carry the desired signal
transduction pathway. High levels of plasma calcium and calcitriol (also severe
hypomagnesemia) inhibit PTH secretion in a negative-feedback mechanism.
Calcitriol 7-dehydrocholesterol is hydroxylated first in the liver to 25-hydroxycholecalciferol (25-HCC,
calcidiol) and then in the kidney to 1,25-dihydroxycholecalciferol, and 24,25-
dihydroxycholecalciferol. The 24,25-Vit-D has no biological function, and this is the
mechanism of the kidney to keep the levels of active vitamin D within acceptable limits.
Recent evidence shows that 1α-hydroxylation can also occur in target cells, bypassing the
kidney for activation of the vitamin directly from the liver. Calcitriol acts on the gut to
stimulate calcium and phosphate absorption, and on the osteoblasts in the bone to promote
secretion of calcium to the ECM, and thus promotes mineralization. In high concentrations,
however, it acts on the bone to stimulate osteoclastic resorption. Calcitriol inhibits its own
formation in the kidney. Low levels of PTH also inhibit its 1α-hydroxylation in the kidney
(because the kidney reads it as "there is enough Ca+ in the blood and there is no need for
vitamin D"). It should be noted that vitamin D also participates in cellular differentiation, and
thus has a role in malignancies. It also stimulates production of cytokines, and thus has an
immunomodulatory effect.
Calcitonin This hormone is secreted from the C cells in the thyroid when plasma calcium concentration
rises, but its physiological activity is unknown. After total thyroidectomy, when no calcitonin
is secreted, there are NO clinical syndromes that can be ascribed to its deficiency, and
calcium homeostasis is normal. Its levels are elevated during pregnancy and lactation. The
hormone has also been detected in the GI and CNS (as a neurotransmitter). It can be shown
experimentally to inhibit osteoclast activity, and thus bone resorption. Also, it serves as a
screening marker for medullary thyroid cancer (see topic 8 for more details).
Avi Sayag Clinical Biochemistry
Topic 45 Hypocalcaemia Causes of hypocalcaemia:
1. Decreased protein-bound calcium – the methods used for determining plasma calcium
concentration measure total calcium. So, if the protein-bound calcium is decreased,
then the measure will show low plasma calcium. This can be caused by overhydration
with calcium-free fluids and hypoalbuminemia (as in the nephrotic syndrome). Recall
that 46% of calcium is bound to albumin in the plasma).
2. Decreased PTH secretion:
a. Primary hypoparathyroidism
b. Autoimmune hypoparathyroidism
c. Pseudohypoparathyroidism (in fact, the PTH levels are high, but the body
responds as if the levels were low).
3. PTH secretion is appropriate:
a. Secondary hyperparathyroidism
b. Vitamin D deficiency
c. Impaired metabolism of vitamin D
d. Renal disease
e. Increased inactivation
f. Drugs
Blood collected into EDTA tube can artefactually yield hypocalcaemia (EDTA binds Ca+2
).
Hypoparathyroidism
This can be congenital or acquired. The congenital form may be associated with DiGeorge
syndrome: a genetic anomaly in which the 3rd
and 4th pharyngeal pouches fail to develop,
leading to many abnormalities, the most important being aplasia of the thymus
(immunodeficiency develops) and the parathyroid gland (hypoparathyroidism). The
congenital form also includes pseudohypoparathyroidism: a condition that superficially
resembles hypoparathyroidism, but plasma concentrations of PTH are elevated. The two types
are both hereditary. The effects of PTH are mediated through the formation of cAMP.
In type 1, the activation of adenylate cyclase is defective, and cAMP is not formed in
response to the binding of PTH to its receptor. This manifests as rounded face, skeletal
abnormalities and learning difficulties.
In type 2, cAMP is formed, but the responses to it are blocked. The two types can be
distinguished by measuring urinary cAMP after administration of PTH. In normal individuals,
and in patients with type 2 pseudohypoparathyroidism, there is an increase of urinary cAMP,
while in type 1 there is none.
The acquired form includes:
1. Surgery: thyroidectomy (total/partial), laryngectomy or parathyroidectomy (the
parathyroid is removed in these operations).
2. Autoimmune disorders: patients with hypoparathyreosis have antibodies against
parathyroid cells and calcium-sensing receptors14
in the parathyroid gland.
3. Hemochromatosis: accumulation of iron in the gland may lead to dysfunction.
4. Infiltrative states.
Secondary hyperparathyroidism
Plasma PTH concentrations are also raised in many patients with chronic renal disease, liver
diseases and vitamin D deficiencies. All these conditions are associated with decreased
synthesis of calcitriol, which causes hypocalcemia. The diagnosis of secondary
hyperparathyroidism in blood test: serum Ca+2
is low or normal, Pi can be low or elevated,
ALP is elevated and PTH is elevated (most important: high PTH + low Ca+2
).
14 These sensor initiate secretion of PTH when no Ca
+2 is bound to them. If these are dysregulated,
their set-point changes, they sense that the Ca+2
concentration is adequate (when it's not) and signal not
to secrete PTH even though it is required.
Avi Sayag Clinical Biochemistry
The causes of secondary hyperparathyroidism can be classified into 2 groups: with
osteomalacia and rickets, and without them.
1. With osteomalacia or rickets:
a. Due to a decrease of calcium and vitamin D uptake, a decrease in calcium
absorption, diet low in vitamin D, steatorrhea or malabsorption.
b. Decrease in the production of vitamin D: this can be due to chronic renal
failure, chronic liver disease and lack of 1α-hydroxylation.
c. Increased inactivation of vitamin D: drugs given in anticonvulsive therapy
can inactivate vitamin D.
d. Lack of 25-hydroxycholecalciferol
e. Vitamin D receptor defect.
2. Without osteomalacia and rickets:
a. Acute pancreatitis: when the pancreas is damaged, free fatty acids are
generated by the action of pancreatic lipases. Insoluble calcium salts form in
the pancreas, and the free fatty acids chelate the salts, resulting in calcium
deposition in the retroperitoneum (i.e. saponification of fatty acids).
b. Neonatal hypocalcaemia: the problem lies in the parathyroid glands that do
not yet function adequately; and in infants of mothers with diabetes or
hyperparathyroidism, because these women have higher-than-normal ionized
Ca+2
levels during pregnancy.
Clinically, hypocalcaemia may cause increased neuromuscular excitability, muscle spasm,
convulsions and tetany. If the condition lasts, cataracts may occur.
In order to detect the etiology of hypocalcaemia, albumin levels should be measured first
(normally – 30-60 g/L). If albumin levels are low, there are 2 options: the Ca+2
level is either
normal or low. If it is normal, then the problem is due to low albumin levels
(hypoalbuminemia) or overhydration (i.v of fluids low in calcium). If the Ca+2
levels are low
(or if the albumin level is normal), the phosphate level should be checked (remember that
high Pi levels inhibit activation of vitamin D). If the levels of Pi are high, it might be because
of renal failure that fails to excrete Pi. Therefore, we have to check the urea level or creatinine
level to check renal functions. If creatinine/urea levels are high, then the kidney is to blame,
and the hypocalcaemia is due to secondary hyperparathyroidism with renal failure (usually
end-stage renal failure). But if the creatinine/urea levels are normal, the cause can be either
hypoparathyroidism or pseudohypoparathyroidism. We have to measure PTH for that: if it's
low, then it's hypoparathyroidism, and if it's high, it's because of pseudohypoparathyroidism.
But if Pi levels are low to begin with, then the reason for the hypocalcaemia is secondary
hypoparathyroidism without renal failure.
To sum it up, these tests should be performed in calcium metabolism:
Serum total calcium with albumin, serum Ca+2
, Pi (in fasting), ALP, total CO2 levels,
creatinine/urea and PTH. In the urine: Ca+2
, Pi and cAMP.
Why measure total CO2 levels? Alkalosis and acidosis (also due to hyper- or hypoventilation)
can lead to hypo- or hypercalcaemia: in alkalosis, H+ dissociates from albumin and Ca
+2
binding to albumin increases. There is also an increase in Ca+2
complex formation. Thus, the
concentration of Ca+2
falls, and this may produce clinical hypocalcaemia, although total
plasma Ca+2
is unchanged.
Avi Sayag Clinical Biochemistry
Topic 46 Hypercalcaemia Causes of hypercalcaemia include:
1. Increased protein-bound Ca+2
: for the same reason mentioned in the previous topic.
This can result from dehydration and prolonged venous stasis.
2. Increased PTH secretion: this can be due to
a. Primary and tertiary hyperparathyroidism (NOT secondary!). Occasionally,
patients with end-stage renal failure become hypercalcaemic, owing to the
development of autonomous PTH secretion, probably as a result of the
prolonged hypocalcaemic stimulus. Such hypercalcaemia may manifest for
the first time in a patient given a renal transplant, who becomes able to
normally metabolize vitamin D. This is termed tertiary
hyperparathyroidism. The diagnosis of primary hyperparathyroidism in
blood test: serum Ca+2
is elevated, Pi is low, ALP is normal or elevated, as
well as PTH levels. Tertiary hyperparathyroidism is diagnosed in blood tests:
elevated: serum Ca+2
,ALP, PTH (highly), Pi is low or elevated.
b. Ectopic PTH production
c. Familial benign hypercalcaemia: a syndrome of lifelong hypercalcemia
inherited as an autosomal dominant trait, and caused by a mutation in the
calcium sensor gene leading to constitutive PTH secretion. The diagnosis is
made by detecting hypercalcemia after parathyroidectomy, and low rate of
calcium secretion in the urine.
3. Conditions related to appropriate PTH secretion:
a. Vitamin D excess
b. Sarcoidosis: the mechanism of action in sarcoidosis most commonly thought
to cause hypercalcemia is dysregulated production of vitamin D by activated
macrophages, that is, as a result of 1α-hydroxylation of 25-
hydroxycholecalciferol by macrophages in the sarcoid granulomas. Similarly,
TB can cause hypercalcaemia.
c. Milk-alkali syndrome: hypercalcaemia is associated with the ingestion of
milk and antacids for the control of dyspeptic symptoms. Alkali increases the
renal reabsorption of filtered calcium but the precise mechanism is unknown.
This syndrome is uncommon, and becoming more so since the introduction of
drugs that inhibit gastric acid secretion for the treatment and prevention of
peptic ulceration. It should also be remembered that dyspepsia itself may be a
feature of hyperparathyroidism since calcium stimulates gastrin release.
d. Malignancies: parathyroid hormone related peptide (PTH-rP) is elevated in
50-90% of patients with hypercalcemia associated with malignancy. Its N-
terminal shows homology to PTH, which can bind to PTH receptors and
mimic its biological action. Its gene is located in chromosome 12 (PTH gene
is located on chromosome 11). In addition, bone metastases cause direct
calcium resorption from the bone. Lastly, cytokines (IL-1 and TNF) or
prostaglandins cause osteoclast activation, and thus bone resorption.
e. Hyperthyroidism (rare)
f. Drugs: Thiazide diuretics sometimes cause mild hypercalcaemia, owing to an
effect on renal calcium excretion. Chronic lithium therapy can cause
increased PTH secretion and is an occasional cause of hypercalcaemia. The clinical features of hypercalcemia are:
1. Renal failure, renal tubular damage and nephrocalcinosis;
2. Decreased neuromuscular excitability (muscle weakness, tiredness, impaired
concentration and other mental changes);
3. Direct heart effect – total calcium level above 3.75 mmol/L leads to cardiac arrest,
cardiac arrhythmia and hypertension.
Avi Sayag Clinical Biochemistry
Off-Topic
Introduction to Topics 47, 48 The adrenal gland is composed of the medulla, which is part of the sympathetic system and is
not essential for life, and the cortex, which has 3 layers: zona glumerulosa (secretes
aldosterone), zona fasciculata (cortisol) and zona reticulata (secretes androgens).
All in the green frame takes place in the zona glumerulosa, and all in the black frame – in the
z. fasciculata and z. reticulata. Note the enzymes common to all 3 zones: cholesterol
hydroxylase, 17α-hydroxylase and 3β-hydroxydehydrogenase. Note also the enzyme that are
unique to the zona glumerulosa: corticosterone methyloxidase I and II, and to the zona
reticulata: desmolase.
ACTH is needed to convert cholesterol to pregnenolone. Therefore, for the production of
aldosterone, ACTH is needed (but is not a must), ATII, and probably factor X (the production
is also induced by hyperkalemia).
Cortisol inhibits the secretion of ACTH and CRH in the hypothalamus (long loop), and
ACTH inhibits CRH in a negative feedback mechanism (short loop).
95% of cortisol in the blood is bound to transcortin (cortisol-binding globulin). 5% of cortisol
is free in the plasma, and can thus be freely excreted in the urine. Transcortin is fully
saturated at normal cortisol concentrations. Because of this, if cortisol production increases,
the concentration present in the plasma in the free form, and thus the amount that is excreted,
increases proportionately.
Avi Sayag Clinical Biochemistry
Functions of glucocorticoids:
1. Increase protein catabolism;
2. Increase glycogenolysis;
3. Increase hepatic gluconeogenesis;
4. Inhibit ACTH secretion;
5. Sensitize arterioles to the actions of noradrenaline, hence involved in the maintenance
of blood pressure.
The adrenal medulla has 2 cell types:
1. Chromaffin cells (so named because of the brown-black color after staining with
potassium- dichromate)
2. Neural cells
The adrenal medulla is strongly related to the sympathetic nervous system embryologically
(derived from neural crest cells) and functionally (it produces catecholamines and has
sympathetic innervation).
Pathological conditions of the adrenal medulla are associated with catecholamine
hypersecretion.
In the adrenal medulla there is synthesis of catecholamines:
Tyrosine � DOPA � dopamine � norepinephrine � epinephrine
Tyrosine is found in the plasma, and the formed DOPA is located in the mitochondria.
Dopamine is released to the cytoplasm and stored in granules, and the formed norepinephrine
is stored in granules. Epinephrine is found in the cytoplasm and stored in granules.
Apart from the adrenal medulla, catecholamine synthesis occurs in extramedullary chromaffin
cells (paraganglions) and in sympathetic neurons. It follows that storage of catecholamines
occurs in the granules of the chromaffin cells as well as in the granules of sympathetic nerve
endings. They are released by exocytosis during sress, physical activity, hypoxia,
hypoglycemia and hypovolemia.
Dopamine is mainly secreted in the CNS and less in the adrenal medulla and sympathetic
system. Norepinephrine is secreted mainly in the CNS and sympathetic system and less in the
adrenal medulla (just a little bit less, though). Epinephrine is secreted exclusively in the
adrenal medulla.
Catecholamines can be reuptaken by storage granules in nerve endings, excreted in their free
form (1-3%), excreted in their conjugated form, or undergo metabolic inactivation. The
metabolic products can be intermediate products (metanephrine) or endproducts (VMA from
E and NE, and HVA from dopamine).
Avi Sayag Clinical Biochemistry
Topic 47 Clinical biochemistry of disturbances of the adrenal cortex Disorders of the adrenal cortex can be either adrenal hypofunction (Addison's disease) or
hyperfunction (Cushing's syndrome, Conn's syndrome and congenital adrenal hyperplasia).
Cushing's syndrome Definition: overproduction of glucocorticoids (mineralocorticoids and androgens can also be
excessive, depending on the mechanisms and their relation to the biosynthesis of cortisol).
Causes:
1. Cushing's disease: adrenal hyperfunction secondary to a pituitary corticotroph
adenoma. This accounts for 60-70% of spontaneous Cushing's syndrome.
2. Adrenal adenoma and carcinoma
3. Ectopic ACTH production (e.g. carcinoma of the bronchus and carcinoid tumors).
4. Corticosteroid or ACTH treatment.
Clinical features:
1. Weight gain, central obesity, the growth of fat pads along the collar bone and on the
back of the neck (buffalo hump) and a "moon face".
2. Thinning of the skin (which causes easy bruising and dryness), purple or red striae
(the weight gain in Cushing's syndrome stretches the skin, which is thin and
weakened, causing it to hemorrhage)
3. Proximal muscle weakness (hips, shoulders), and hirsutism (facial male-pattern hair
growth due to elevations in androgens).
4. The excess cortisol may also affect other endocrine systems and cause, for example,
insomnia, reduced libido, impotence, amenorrhoea and infertility.
5. Psychological disturbances, ranging from euphoria to psychosis. Depression and
anxiety are also common.
6. Persistent hypertension and hypokalemia (due to cortisol's enhancement of
epinephrine's vasoconstrictive effect, and due to the fact that cortisol precursors and
cortisol itself have some mineralocorticoid activity, thus causing Na+ retention and
hypertension).
7. Insulin resistance (especially common in ectopic ACTH production), leading to
hyperglycemia, which can lead to diabetes mellitus.
8. Due to excess ACTH, it may also result in hyperpigmentation. This is due to MSH
production as a byproduct of ACTH synthesis from Pro-OpioMelanoCortin (POMC).
Pseudo-Cushing's syndrome, in which patients appear cushingoid, can occur in Cushing's
disease, alcoholism and obesity (rare). If alcoholism is the inducer, the symptoms resolve
rapidly upon withdrawal of alcohol.
Diagnosis:
1. 24-hour urinary cortisol excretion: normal 24h cortisol excretion is <300 nmol/L.
2. Dexamethasone suppression test: dexamethasone is a synthetic glucocorticoid that
binds to cortisol receptors in the pituitary and suppresses ACTH release (and thus the
secretion of cortisol by the adrenals). In the overnight test, 1 mg is given at night and
blood is drawn for measurement of cortisol at 9 am the next morning. In normal
individuals, this should be less than 50 nmol/L. A failure of suppression is
characteristic of Cushing's syndrome.
3. Insulin hypoglycemia test: normal increase in plasma cortisol concentration occurs in
response to hypoglycemia, and this response is abolished even in mild Cushing's
syndrome.
4. Loss of diurnal variation of cortisol secretion is an early feature of Cushing's
syndrome (but a problematic test: see Marshall page 163).
5. ACTH measurement
6. CRH measurement: can be useful to differentiate between Cushing's disease and
ectopic ACTH secretion. In Cushing's disease, CRH typically increases plasma
ACTH, whereas with ectopic ACTH secretion or an adrenal tumor there is typically
no response.
Avi Sayag Clinical Biochemistry
Here is the DD of Cushing's syndrome:
Conn's syndrome Definition: excessive production of aldosterone.
Causes:
1. Adrenal adenoma and carcinoma;
2. Bilateral hypertrophy of zona glumerulosa cells;
3. Glucocorticoid-remediable aldosteronism: an autosomal dominant condition in which
aldosterone synthesis is under the control of ACTH (rare).
Hyperaldosteronism can be also secondary to enhanced renin secretion:
1. Hypoalbuminemia (edema � renal hypoperfusion�increased renin secretion)
2. Nephrotic syndrome (causes hypoalbuminemia)
3. CHF (renal hypoperfusion�increased renin secretion)
4. Renal artery stenosis (renal hypoperfusion�increased renin secretion)
5. Renin-secreting tumors (renal juxtaglomerular cell tumor)
Clinical features:
1. Hypokalemia (always) and hypertension;
2. Muscle weakness and parasthesias
3. Polydipsia, polyuria and nocturia
Diagnosis:
Lab diagnosis consists of measuring low K+ and high Na
+ in the blood, and high K
+ in the
urine.
Screening for hyperaldosteronism includes measuring low K+ in the serum, high K
+ in the
urine, renin and aldosterone measurements, and Na+ loading test, in which 200 mmol/L of
Na+ is given over 24 hours, followed by Na
+ and K
+ measurements. Normally, aldosterone
should be inhibited by the increased Na+ reaching the distal tubule, and less Na
+ should be
found in the plasma. Aldosterone secretion is stimulated through the action of renin;
therefore, it is helpful to measure the plasma renin activity at the same time as the
concentration of aldosterone to establish whether aldosterone secretion is autonomous or
under normal control.
Congenital adrenal hyperplasia (CAH) Definition and causes: a group of inherited metabolic disorders of adrenal steroid hormone
biosynthesis. Their clinical features depend upon the position of the defective enzyme in the
synthetic pathway, which determines the pattern of hormones and precursors that is produced.
21-hydroxylase deficiency: accounts for around 95% of all cases of CAH.
The remaining 5% are due to deficiency of 11β-hydroxylase.
The deficiency can be complete or partial.
21-hydroxylase deficiency is often incomplete and adequate cortisol synthesis can be
maintained by increased secretion of ACTH by the pituitary. It is this that causes hyperplasia
of the glands. Because of the metabolic block, the substrate of the enzyme (17α-
hydroxyprogesterone) accumulates and there is increased formation of adrenal androgens.
Clinical features: female infants affected by CAH may be born with ambiguous genitalia, but
when the enzyme deficiency is only partial, the condition may not present until early
adulthood with hirsutism, amenorrhoea or infertility (late onset CAH). Males may present
Avi Sayag Clinical Biochemistry
with pseudoprecocious puberty in their second or third year of life but are not virilized at
birth.
Diagnosis: diagnosis is made by demonstrating an elevated concentration of 17α-
hydroxyprogesterone (17-OHP) in the plasma at least two days after birth (before this time,
maternally derived 17-OHP may still be present in the infant's blood).
Treatment is monitored by measurement of plasma 17-OHP.
Partial 11β-hydroxylase deficiency is also more common than complete deficiency of the
enzyme. Increased androgen production causes virilization (development of male secondary
sexual characteristics), which tends to be more severe than in 21-hydroxylase deficiency (but
again, it is not present in males at birth). Hypertension develops, owing to the accumulation of
11-deoxycorticosterone, a substrate of the defective enzyme that has salt-retaining properties.
The diagnosis rests upon the demonstration of an increased plasma concentration of either 11-
deoxycortisol or its urinary metabolite. Treatment is with cortisol alone: although aldosterone
secretion is defective, 11-deoxycorticosterone provides an adequate mineralocorticoid
activity.
Hypofunction of the adrenal gland
Addison's disease Definition: a rare endocrine disorder in which the adrenal gland does not produce enough
steroid hormones (glucocorticoids and often mineralocorticoids).
Causes:
1. The commonest cause of adrenal hypofunction is suppression of the pituitary-adrenal
axis by glucocorticoids used therapeutically.
2. Autoimmune adrenalitis
3. AIDS
4. TB
5. Amyloidosis
6. Hemochromatosis
7. Adrenalectomy
Symptoms: fatigue, weight loss, weakness, pigmentation (because of high concentrations of
ACTH, which occur because of the loss of negative feedback by cortisol: ACTH has some
melanocytes stimulating activity) and hypotension.
Acute adrenal failure is a medical emergency characterized by severe hypovolemia, shock and
hypoglycemia. It can be precipitated by stress (infection, trauma and surgery). Hemorrhage
into the adrenal glands may occur as a complication of anticoagulant treatment and in
meningococcal septicaemia, and can result in acute adrenal failure.
Adrenal failure can occur secondarily to pituitary failure as a result of decreased stimulation
by ACTH.
Diagnosis:
1. Hyponatraemia can occur, since the lack of cortisol reduces the ability of the kidneys
to excrete the water load, but there is no renal salt wasting since aldosterone secretion
is not dependent upon ACTH.
2. Hyperkalemia
3. Cortisol levels below 50 nmol/L is diagnostic of adrenal failure.
Cortisol levels above 550 nmol/L excludes the diagnosis.
In cases when the levels of cortisol are between 50-550 nmol/L, the short Synacthen test
should be performed (= ACTH stimulation test):
Blood should be taken for ACTH assay before giving ACTH.
Blood is taken at 9am for measurement of cortisol.
250 microgram of soluble ACTH are given i.m (Synacthen)
Blood sampling for cortisol is taken at 9:30 and at 10:00am.
Normal results: plasma cortisol after ACTH – increase of 200 nmol/L with peak of >550
nmol/L.
Avi Sayag Clinical Biochemistry
Topic 48 Clinical biochemistry of disturbances of the adrenal medulla Consult again the introductory section to topics 47 and 48, regarding the adrenal medulla.
There are several conditions associated with increased catecholamine metabolism derived
from chromaffin cells:
1. Phaeochromocytoma (or phaeochromoblastoma)
2. Paraganglioma
The followings are derived from neural cells:
1. Neuroblastoma
2. Ganglioneuroma (or ganglioneuroblastoma)
And:
1. Melanoblastoma
Phaeochromocytoma (phaeochromoblastoma and paraganglioma) These are neoplasms composed of chromaffin cells. Their features follow the "rule of 10":
1. 10% are malignant;
2. 10% are familial, or are associated with familial syndromes (MEN type 2a and 2b,
neurofibromatosis type 1 and Hippel-Lindau disease); Some familial forms are
without any other abnormalities or associated with islet cell tumors.
3. 10% are bilateral; and
4. 10% are extra-adrenal (paraganglioma)
Clinical symptoms:
1. Hypertension: in attacks or sustained hypertension (in 5% there is no hypertension)
2. Tachycardia, headache, sweating 3. Abdominal pain
4. Nausea and vomiting
5. Visual disturbances
6. Nervousness, irritability
7. Increased appetite but loss of weight (hypermetabolic condition)
8. Dyspnea
9. Angina and cardiac hypertrophy
If you suspect that your patient might have phaeochromocytoma, you should screen for it.
Why suspect? The patient present with characteristic symptoms, among which hypertension
resistant to therapy. Also, patients with neurofibromatosis, and those who experienced
hypertensive episodes during anesthesia or delivery of a baby.
Diagnosis:
We should remember that foods and drugs and other factors may influence plasma level and
excretion of catecholamines, and that catecholamines show diurnal variation (therefore a 24h
urine collection is useful). So, when preparing the patient for catecholamine determination,
the patient should avoid taking drugs that interfere with catecholamine determination for at
least 8 days prior to the test (methyl-DOPA, levo-DOPA, α-adrenergic antagonists, etc), as
well as certain foods (spices) that should be avoided for at least 3 days.
Catecholamines in the plasma: the patient should lie for 30 minutes, and a canule should be
introduced 20-30 minutes in advance. This is in order to avoid any stress, physical and
psychological. Blood should be drawn at the same time of the day, and not immediately after
eating. In addition, these tests can be performed:
1. Suppression test with clonidine or pentolinium: pentolinium is a sympathetic
ganglion-blocking drug that reduces catecholamine secretion in normal subjects but
not in patients with phaeochromocytomas; in such patients, secretion is autonomous.
Blood is taken for catecholamine measurement before and 15 minutes after giving 2.5
mg pentolinium by intravenous injection.
2. Provocative test with glucagon
3. Imaging: CT, MRI
Avi Sayag Clinical Biochemistry
Catecholamines in the urine: urine should be collected over 24 hours, and heavy exercise
should be avoided. Urine can be collected spontaneously in neonates and small children,
together with creatinine ratio determination. Metanephrine should be measured in the urine,
VMA (but NOT HVA/HMMA) and catecholamines. Measurement is done with HPLC.
Further lab changes in this condition include:
1. Hyperglycemia and glycosuria during an attack
2. Impaired glucose intolerance
3. Renal disease caused by the hypertension
4. Changes caused by volume depletion
Catecholamines may also rise in these conditions:
Diabetic ketoacidosis, AMI, acute CNS disturbance, post-operative, hypo- and
hyperthyroidism, following heavy exercise, volume depletion, renal disease, heavy alcohol
intake, hypoglycemia, stress and various drugs.
What are the drugs that can interfere with catecholamine determination?
These are grouped into 3:
Drugs interfering with HPLC
assay
Drugs inhibiting
catecholamine production /
release / excretion
Drugs increasing
catecholamine excretion
Methyldopa
Levodopa
Phenothiazine
MAO inhibitors
Clonidine
Reserpine
Bromocriptine
Dexamethasone
Theophylline
Aminophylline
Caffeine
Ethanol
Glucagon
Insulin
Amphetamine
Vasodilators
The treatment can be surgery (if diagnosed and done early enough, full recovery results) with
administration of α-adrenergic receptor blockade (phenoxybenzamine). If surgical removal is
not possible, α-adrenergic receptor blockers or drugs that inhibit the synthesis of
catecholamines (metyrosine) should be administered.
Neuroblastoma This tumor can occur in the neuroblasts of the adrenal medulla, or in the sympathetic trunk
(ganglioneuroma, ganglioneuroblastoma). It's the 3rd
most frequent malignancy in childhood,
and is mostly located in the abdomen (most cases arise in either the adrenal medulla or in the
retroperitoneal sympathetic ganglia).
Symptoms include general symptoms of malignancy with metastases, but hypertension is
usually absent (as opposed to phaeochromocytoma).
Diagnosis:
Urine: NE, normetanephrine, dopamine, VMA, HVA – increased excretion. Excretion of
epinephrine is normal, though.
If a single assay is made, then the sensitivity is around 75%. Therefore, a combination of 2
assays is better: VMA + HVA, or VMA + catecholamine, then sensitivity rises to 95%.
NSE (neuron specific anolase) may increase too.
The higher the increase, the poorer the prognosis.
Avi Sayag Clinical Biochemistry
Topic 49 Clinical biochemistry of the reproductive system (This topic is quite long, but it actually verbalizes the entire lectures on this topic, and aims to
explain the "coded" slides of the department. It is thus longer than the actual recapped version
of the topic).
Male Reproductive System The hypothalamus-pituitary-testis axis:
Testosterone is produced in Leydig cells (also known as "thecal cells"). It is also produced in
the adrenal cortex, but in very small amounts – the majority there is DHEA (recall from the
introductory section for the adrenal cortex).
In the circulation, about 97% of testosterone is bound mainly to sex hormone binding globulin
(SHBG) with high affinity, and to albumin with lower affinity. The free fraction (2-3%) is
readily available to tissues; albumin binds testosterone more loosely than SHBG and albumin-
bound testosterone may be in part available (i.e. active).
In most tissues, testosterone is converted into dihydrotestosterone (DHT) by the aid of 5α-
reductase. Why is it converted to DHT in target tissues? It appears that DHT has a greater
affinity for androgen receptors in target tissues than testosterone (both have receptors,
though), and that the DHT-receptor complex is more effective. Defects in the receptors for
either DHT, testosterone or both lead to various clinical abnormalities (collectively termed
"androgen insensitivity").
If there is a deficiency of 5α-reductase, DHT will not be formed. Male internal genitalia
develop normally (they are not dependent on DHT), but masculinization, which requires
DHT, is incomplete.
In the brain and in adipose tissue, testosterone might be converted into estrogen. This might
be related to libido, sexual behavior and feed-back regulation.
Testosterone has anabolic effects, through the action of GH, which increases protein synthesis
and decreases protein breakdown. It promotes the fusion of epiphyseal plates and promotes
the retention of sodium, potassium, water, sulfates and phosphates.
Together with FSH that acts on Sertoli cells, they promote spermatogenesis in puberty (FSH
acts on Sertoli cells, and there is a paracrine communication between Sertoli and germ cells).
As mentioned, testosterone has a role in the development of secondary male sex features (size
of penis, deeper voice, pubic hair, body configuration, size of muscles, and everything that
makes a man A man, Amen…).
Recall from the introduction to topics 47, 48, that testosterone is formed mainly in the testis
and to a lesser degree in the adrenal cortex; androstendione is formed in the testis and in the
adrenal cortex, and DHEA is, too, formed in the testis and in the adrenal cortex (these are the
3 musketeers of androgens…).
Avi Sayag Clinical Biochemistry
Measurements of testosterone in the plasma:
Testosterone is measured in the plasma using immunoassays. After birth, the plasma
concentration increases for 3 months and returns to baseline by the age of 1 year. It remains in
its baseline until puberty.
Around age 6-7, there is an increase in DHEA and androstendione, and around age 16-19,
adult values of testosterone are reached.
Male reproductive abnormalities can be classified into 3 main groups:
1. Hypogonadotrophic hypogonadism (secondary hypogonadism) a. Panhypopituitarism (congenital or acquired)
b. Hypothalamic syndrome (congenital or acquired)
i. Structural defects (neoplasms, inflammations)
ii. Prader-Willi syndrome (7 genes on paternal chromosome 15 are
missing, and hypogonadism is part of the clinical features)
iii. Laurence-Moon syndrome
c. GnRH deficiency (Kallmann's syndrome - secondary hypogonadism)
d. Hyperprolactinemia (through the inhibition of GnRH by prolactin. It can be
caused by drugs that block dopaminergic receptors, like haloperinol, or
deplete dopamine, like methyldopa and reserpine; it can also be caused by
prolactinomas).
e. Malnutrition and anorexia nervosa
f. Drug-induced suppression of LH, such as androgens, estrogens, tranquilizers,
antidepressants, antihypertensives, barbiturates, cimetidine (an H2-receptor
blocker given to treat stomach acidity, but it increases the activity of
estrogens through a non-interesting mechanism of interference with cyt-P-
450).
2. Hypergonadotrophic hypogonadism (primary hypogonadism) a. Acquired condition: irradiation, mumps (can be complicated with orchitis),
castration and cytotoxic drugs.
b. Chromosome defects:
i. Klinefelter's syndrome (47,XXY)
ii. Autosomal and sex chromosomes, polyploids
iii. True hermaphroditism
c. Defective androgen biosynthesis (look at the chart in topic 47)
i. 20α-hydroxylase (desmolase)
ii. 17,20-lyase deficiency
iii. 3β-hydroxysteroid dehydrogenase deficiency
iv. 17α-hydroxylase deficiency
v. 17β-hydroxysteroid dehydrogenase deficiency
d. Testicular agenesis
e. Selective seminiferous tubular disease
f. Others:
i. Noonan's syndrome (cryptochidism is one of the symptoms)
ii. Streak gonads (hypoplastic and dysfunctioning gonads)
iii. Myotonic dystrophy
iv. Acute and chronic disease
3. Defects in androgen action a. Complete androgen insensitivity (testicular feminization)
b. Incomplete androgen sensitivity:
i. Due to androgen-receptor defects
ii. 5α-reductase deficiency
Diagnosis of hypogonadism should distinguish between primary and secondary
hypogonadism: in both cases, testosterone levels are low. But:
In primary hypogonadism the problem is confined to the testis; thus, low concentrations of
testosterone do not inhibit FSH and LH secretion. The lab results will be: low testosterone and
high FSH and LH. Furthermore, seminiferous tubule defects are associated with high FSH,
Avi Sayag Clinical Biochemistry
and Leydig cell defects with high LH levels. hCG can be used to test Leydig cell function,
because it has an action similar to LH.
In secondary hypogonadism, the problem is extrinsic to the testis. Thus, low (or sometimes
normal) FSH and LH lead to low testosterone levels.
In addition, depending on the basal underlying disease, some other tests should be considered
such as: chromosome analysis, sperm analysis (to detect problems in the SN tubules),
prolactin determination, SHBG measurement and dynamic tests (GnRH stimulation test).
An unwelcome consequence of hypogonadism is infertility that may be caused by 5 main
disorders:
1. Endocrine disorders:
a. Hypothalamic dysfunction (kallmann's syndrome)
b. Pituitary failure (tumor, surgery)
c. Hyperprolactinemia (drug, tumor)
d. Exogenous androgens
e. Thyroid disorders
f. Adrenal hyperplasia
g. Testicular failure
2. Anatomical factors a. Congenital absence of vas deferens
b. Obstructed vas deferens
c. Congenital abnormalities of the ejaculatory system
d. Varicocele
e. Retrograde ejaculation (that's a funny condition in which the man ejaculates
into his urinary bladder and then urinates the sperm. It's not a problem of
infertility anymore, because the sperm can be separated from the urine by
centrifugation. My roommate was curious to know if they can reach orgasm,
so I checked, and they do, but not a strong one though).
3. Abnormal spermatogenesis a. Idiopathic azoospermia
b. Chromosomal abnormalities
c. Mumps complication (orchitis)
d. Cryptochidism
e. Chemical/radiation exposure
4. Abnormal motility a. Absent cilia (Kartagener's syndrome)
b. Antibody formation
5. Psychosocial factors a. Impotence (psychological)
b. Decreased libido
Diagnosis of male infertility differentiates between 4 causes:
1. If LH levels are high, but testosterone is low, the problem is in Leydig cells
2. If LH levels are low, and testosterone is low, the problem lies in the hypothalamus-
pituitary axis.
3. If FSH levels are high, and the sperm count is low, the problem lies in the SN tubules.
4. If FSH levels are low, and the sperm count is low, the problem lies in the
hypothalamus-pituitary axis.
Semen analysis should be performed to check the motility and number of sperm.
Immunological tests should also be performed.
Avi Sayag Clinical Biochemistry
Female Reproductive System Description of the hypothalamus-pituitary-gonad axis:
1. The hypothalamus secretes GnRH (a decapeptide) in a pulsatile manner, with a peak
secretion every 90 minutes. It induces FSH and LH secretion from the pituitary.
2. In the pituitary, LH and FSH are produced in the basophilic cells. They have an α
subunit and a β subunit. The α subunit is identical in these hormones and in TSH and
hCG. The β subunit is unique to each of them, therefore a specific assay is possible to
detect them.
a. FSH causes follicular maturation (granulosa cell appearance), increases the
LH receptors on granulosa cells, together with LH it stimulates estrogen
secretion from granulosa cells, and increases inhibin secretion. It inhibits the
secretion of GnRH in the hypothalamus (short loop).
b. LH increases androgen synthesis in thecal cells, in combination with FSH it
stimulates estrogen secretion, it starts luteinization, and increases
progesterone synthesis. It inhibits the secretion of GnRH in the hypothalamus
(short loop).
c. Prolactin is produced in the acidophilic cells, and it is necessary for lactation
and progesterone secretion. TRH from the hypothalamus stimulates its
secretion (and NOT GnRH) and is inhibited by dopamine.
3. In the ovary, estrogen, progesterone and androgens are produced.
a. Estrogen: an 18-carbon long hormone, which is synthesized in the granulosa
cells of the follicle. It promotes feminization of the genital tract and
secondary sex features, it regulates the proliferative phase of the menstrual
cycle, and together with FSH it increases the number of LH receptors on
granulosa cells. In low or sustained concentrations, it inhibits LH and FSH
secretion (in days 1-13, and 15-28), but on day 14 of the menstrual cycle, its
levels surge abruptly and cause an increase in LH secretion. It also has an
anti-osteoporotic effect, and a protective role against atherosclerosis. Apart
from the follicle, it is also produced in the placenta and in adipose tissues
(like testosterone, it is produced there from androgens). The most effective
form of estrogen is 17β-estradiol. This is converted into estrone. Estriol is the
metabolic product of DHEA in pregnant women. Like testosterone, 97% is
bound to albumin and SHBG, and the remaining is free. The free form, and to
some extent the albumin-bound form, are the active ones. As mentioned,
SHBG binds both estradiol and testosterone, but it has a greater affinity for
testosterone, and the serum concentration in males is lower. Estrogen is
mainly metabolized in the liver (to form estriol), and the conjugated forms
are made soluble and thus excreted in the urine. Estrogen is not specific to the
ovary, as it is also produced in the adrenal cortex. It is present in both males
and females, but the quantities are different. Estrogens inhibit the secretion of
GnRH in the hypothalamus, and of LH and FSH in the pituitary (long loop).
b. Progesterone is a 21-carbon long hormone produced in the corpus luteum in
the ovary. It regulates the secretory phase of the menstrual cycle, prepares the
uterus for implantation and maintains pregnancy. With estradiol, between
days 15-28 of the menstrual cycle, it inhibits LH secretion. Progesterone is
also secreted from the placenta (trophoblast). In the first 4 months, the corpus
luteum secretes progesterone and thus maintains pregnancy. Then, the corpus
luteum regresses, and the placenta takes over the secretion of progesterone
and thus it is now the placenta that maintains pregnancy. Progesterone is
transported in the blood bound to CBG (transcortin, with high affinity and
low amount) and to albumin (90-98%) and the remaining (2-10%) is free and
biologically active. It is metabolized in the liver and the end-products are
excreted in the urine. There are 3 major forms: pregnendione, pregnenalone
and pregnenediol. Like estrogen, it is produced also in the adrenal cortex,
present in both sexes and differs in quantity. Progesterone inhibits the
Avi Sayag Clinical Biochemistry
secretion of GnRH in the hypothalamus, and of LH and FSH in the pituitary
(long loop).
c. 66% of androgens (testosterone, DHEA and androstendione) in the female
are produced in the adrenal cortex, and 34% in the ovaries. They are
necessary for the growth of pubic hair and axillary hair. Here, too, the active
form of testosterone is DHT formed by the assistance of 5α-reductase. The
metabolites (17-oxosteroids) are excreted in the urine.
Before puberty, the pituitary is unresponsive to GnRH; thus, FSH and LH levels are low, and
therefore estradiol levels are low. During the climacteric (menopause), estradiol levels are low
due to ovarian failure, but the levels of FSH and LH are high (not inhibited). First, FSH levels
rise, then LH. In menopause, the only source of estradiol is from the metabolism of adrenal
androgens, in adipose tissues and in the liver. The increase of FSH concentrations is a better
indicator of ovarian failure than the decrease in estradiol levels, which shows considerable
variability.
Diagnosis of hypo- and hypergonadism has to differentiate between primary and secondary
hypo- or hypergonadism.
In primary hypogonadism (hypergonadotrophic hypogonadism), estrogen levels are low, but
LH and FSH are high; whereas in secondary hypogonadism (hypogonadotrophic
hypogonadism) estrogen levels, FSH and LH levels are low.
In primary hypergonadism, estrogen levels are high, but FSH and LH levels are low; whereas
in secondary hypergonadism, estrogen levels, FSH and LH levels are all high.
Female hypogonadism can be symptomatically classified into 3 categories:
1. Amenorrhea;
2. Hirsutism and virilism
3. Infertility and sterility
1. Amenorrhea Primary: menstruation has never occurred until the age of 16. The cause may be in one or
more of the following glands:
- Hypothalamus: weight loss, intensive exercise, anorexia;
- Pituitary: hypopituitarism, prolactinemia
- Gonads (most cases): dysgenesis/agenesis/aplasia of ovaries, the Fallopian tube,
the uterus, the vagina
- Adrenal: congenital adrenal hyperplasia
- Thyroid: hypothyroidism
Secondary: an absence of menstruation for at least 6 months among women who had
previously normal cycles. The causes may be:
- Physiological: pregnancy, menopause
- Hypothalamus (most cases – 80%): weight loss, stress, anorexia, drugs
- Pituitary: acquired hypopituitarism, Sheehan syndrome (post-partum pituitary
necrosis), hyperprolactinemia (leads to galactorrhea-amenorrhea syndrome)
- Ovary: poly-cystic ovary, tumors, inflammation, autoimmune dysfunction
- Uterus: tumor or inflammation (rare)
- Endocrine cause: DM, hypo- and hyperthyroidism, late onset CAH, Cushing's
syndrome
- Post-pill amenorrhea: caused by hypothalamus suppression, increased prolactin
levels and endometrium atrophy.
2 causes are worth further elaboration:
1. Prolactinemia: galactorrhea-amenorrhea syndrome:
Amenorrhea is caused by high concentration of prolactin in the plasma that interferes with the
pulsatility of GnRH.
Causes include prolactinomas, of which 90% are microadenomas (tumor is <1cm) and 10%
are macroadenomas (>1cm in diameter). Obstruction of blood supply to the pituitary gland
leads to less dopamine reaching the pituitary, thus less inhibition of prolactin secretion. Drugs
Avi Sayag Clinical Biochemistry
that block dopamine receptors or deplete dopamine can cause the same effect. Surgery,
ectopic secretion and hypothyroidism can also be the cause.
Symptoms include galactorrhea, amenorrhea and infertility.
Diagnosis should include prolactin determination and TRH stimulation test.
Therapy includes administration of bromocriptin (a dopamine agonist) and surgery.
2. Polycystic ovary syndrome: a condition of hyperandrogenization and chronic anovulation
in the absence of specific underlying adrenal or pituitary disease.
Causes include abnormalities in enzyme synthesis and hypothalamic causes. Elevated
androgens may lead to virilism15
. Inhibition of FSH (due to increased estrogen) leads to
anovulation, and stimulation of LH (due to increased estrogen) increases androgen secretion.
Symptoms include amenorrhea and infertility, virilism, acne, obesity, enlargement of the
ovaries and cysts in the ovaries with hyperplasia of thecal cells.
Diagnosis requires at least 2 of the following features: polycystic ovaries, oligo-ovulation or
anovulation, and clinical/biochemical evidence of androgen excess. Lab diagnosis should
include elevated levels of testosterone, estrogen, and LH; low levels of FSH, and
dexamethasone suppression test to differentiate adrenal origin.
Diagnosis of amenorrhea:
1. Exclude pregnancy: high LH levels may suggest pregnancy before a pregnancy test is
performed: hCG cross-reacts in some assays with LH.
2. Inspection: obvious signs that might require suspicion of congenital syndromes (and
performance of chromosomal analysis).
3. Gynecological examination (US, cytology, biopsy)
4. Measurement of LH, FSH, estradiol:
a. In ovarian failure: LH, FSH are elevated, estradiol is low
b. In hypothalamus/pituitary insufficiency: LH, FSH and estradiol are low
5. Prolactin measurement
6. Androgen concentration
7. Measurement of thyroid hormones, cortisol and ACTH.
There are 6 functional tests that should be considered:
1. Progesterone challenge test: uterine dysfunction is an uncommon cause of
amenorrhoea. It can be excluded, if necessary, by the progestogen challenge test. If
medroxyprogesterone acetate is given orally (10 mg daily for 5 days), the occurrence
of vaginal bleeding 5-7 days later indicates that the uterus was adequately
oestrogenized. If bleeding does not occur, the test is repeated, giving oestrogen
(ethinyloestradiol, 50 mg daily for 21 days, with progestogen on the last 5 days).
Absence of bleeding indicates uterine disease. If bleeding occurs, oestrogen
deficiency is present.
2. Estrogen + progesterone challenge test: performed to differentiate between uterus
abnormality and ovarian abnormality. Estrogen is administered for 21 days and then
progesterone for 7-10 days. Stop medication for 7 days. Interpretation: if no
withdrawal of bleeding occurs, it suggests uterine bleeding outflow obstruction; if
withdrawal of bleeding occurs (but not after progesterone alone), obtain serum FSH
and serum LH – if both are elevated, it suggests hypergonadotrophic hypogonadism,
but if both are low, it suggests hypogonadotrophic hypogonadism
3. Clomifene citrate test: clomifene binds to hypothalamic receptors and blocks the
inhibitory effect of peripheral steroids on GnRH release. Normally, the block of the
negative feedback on GnRH causes the increase in GnRH and thus elevation of LH
and FSH. A positive response refers to the intact function of the hypothalamus-
pituitary axis. This test differentiates between primary and secondary hypogonadism.
4. GnRH test: this test is aimed to differentiate between pituitary abnormality and
hypothalamus abnormality. 100µg of GnRH are administered, and the levels of LH
and FSH are measured after 20 minutes and 60 minutes. Normal response is recorded
15 A female disorder in which there is development of secondary male sexual characteristics
Avi Sayag Clinical Biochemistry
if FSH and LH levels increase, suggesting hypothalamus insufficiency. If the levels of
FSH and LH do not increase, it suggests pituitary insufficiency.
5. Gonadotrophin test: FSH and LH (or hCG) are administered to evaluate the ovarian
functions. Estrogen and progesterone levels should be elevated if the problem lies
outside the ovaries.
6. Dexamethasone suppression test: see Topic 47, "Diagnosis".
Treatment of amenorrhea includes replacement therapy with whatever is missing:
progesterone, estrogen +progesterone, FSH, LH, GnRH, clomifene citrate.
Protocol for investigation of amenorrhea is found in Marshall page 198.
2. Hirsutism and virilism Hirsutism is an increase in body hair. Causes include increased androgen secretion, deceased
SHBG and increased androgen sensitivity.
1. Drugs: androgens and progesterone
2. Adrenal: CAH (where there is a decrease in aldosterone and cortisol production
and an increase in androgen synthesis); acquired adrenal hyperplasia; androgen-
producing tumor; Cushing's syndrome
3. Ovary: polycystic ovary syndrome; androgen-producing tumor; hyperthecosis;
post-menopause
4. Familial hirsutism
5. Idiopathic hirsutism
Symptoms include menstrual irregularity or amenorrhea, obesity, acnes, infertility,
clitoromegaly, deep voice and increased body hair.
Diagnosis:
1. Testosterone measurements: should be performed 3 times at 20-minute intervals,
due to pulsatile secretion.
2. DHEA, androstendione (to determine adrenal origin)
3. SHBG
4. FSH, LH (LH increases in PCOS)
5. 17α-hydroxyprogesterone (CAH)
6. Dexamethasone suppression test: low doses can inhibit ovarian production, but
high doses can cause suppression in case of adrenal hyperplasia but not in adrenal
tumors.
The DD should include 5 conditions differentiated on basis of 5 parameters:
17αOH-
progesterone
FSH LH DHEA Testosterone
Hirsutism Normal Normal Normal Normal/high Normal/high
PCOS Normal Normal/low High Normal High
Ovarian tumor Normal Normal/low Normal/low Normal High
Adrenocortical
tumor
Normal Normal/low Normal/low High Normal/high
CAH High Normal/low Normal/low High High
3. Infertility and sterility Sterility is defined when pregnancy cannot occur during 2 years of attempts; whereas
infertility refers to the ability to get pregnant but pregnancy ends spontaneously (spontaneous
abortion).
Causes for these conditions include psychological causes, as well as anything in the
hypothalamus-vagina axis: hypothalamus, pituitary, ovary, uterus, Fallopian tubes, vagina,
androgen predominance, endocrine disorders and alcoholism. Immunological factors can also
lead to these conditions (antisperm antibodies). As some causes are reversible (hormonal
causes, some anatomical changes and immunological causes) some are irreversible
(chromosomal abnormalities, severe congenital abnormalities).
Diagnosis should include:
Avi Sayag Clinical Biochemistry
1. History taking
2. Gynecological examination
3. Sperm examination
4. Post-coital test: mucus with an adequate estrogen stimulation is clear and thin. Before
mucus dries, more than 20 motile sperm should be seen in one HPF (high power
field). This test is considered unreliable nowadays.
5. Examination of the cervical mucus: the penetration of the sperm is investigated after
the mucus and the sperm are mixed in vitro.
6. Hormone examination:
a. Progesterone measurement: its levels peak 5-9 days after ovulation
b. Basal body temperature: a rapid increase of 0.5ºC upon ovulation
c. LH surge: 24-36 hours before ovulation
d. Estrogen, FSH, PRL, androgens and other dynamic tests
Oral Contraceptives These inhibit ovulation. When the administration is withdrawn, vaginal bleeding resumes.
The pills can include estrogen and progesterone (either equally or with different
concentrations); it is taken for 21 days with a 7-day break. The first time a woman takes a pill,
it is recommended to take it on the first day of bleeding, for 21 days, and then stop for a week
during which she bleeds. If she bleeds for more than a week, the pill should be replaced with a
more suitable one for her. The other pill contains only progesterone and is taken continuously.
Side effects include post-pill amenorrhea, irregular cycles, thromboembolism, VLDL
elevation, and cortisol and thyroxine elevation.
Absolute contraindications include cardiovascular diseases, liver and renal disorders, and
history of thromboembolism; whereas relative contraindications include hormonal
disturbances, migraines, DM, smoking, preoperative state and myomas.
Avi Sayag Clinical Biochemistry
Topic 50 Lab procedures in the diagnosis of bone and skeletal disorders Some general information about bone tests and markers:
Markers of bone formation (osteoblast formation) 1. Osteocalcin: a non-collagen protein of 49 amino acids produced by osteoblasts. Active
vitamin D stimulates its production. It promotes bone mineralization by binding calcium
after undergoing post-translational carboxylation (a vitamin K-dependent process). It also
has a chemotactic and nitrogen effect on osteoclasts (a negative regulator of bone
formation, but the mechanism is not fully understood). Serum osteocalcin concentration is
a good indicator of osteoblast activity. Its half life in the serum is 5 minutes, and the
kidney eliminates it. Its fast proteolysis requires special sampling processing. False
elevated results can be obtained by renal insufficiency (less clearance of osteocalcin),
immobilization and therapy with active vitamin D (as it stimulates its production).
2. BSAP (bone specific alkaline phosphatase): a tetrameric glycoprotein found on the
surface of osteoblast cells. The gene for alkaline phosphatase expresses isoenzymes
which are non-tissue specific, intestinal, placental and germ cells. 3 isoforms are derived
from the non-specific isoenzymes: liver, bone and kidney, which undergo post-
translational modification (glycosylation) in the specific tissue. The liver isoform travels
furthest in electrophoresis, while the intestinal one travels the shortest distance. BSAP has
advantages over osteocalcin: its half life is longer (1-2 days), it has no diurnal variation, it
is stable in the serum (no proteolysis), it does not require special sampling, transport and
storage, and the kidney's function has no effect on its serum concentration.
3. Pro-collagen type I- terminal peptides: collagen type I is the sole collagen type found in
bones and tendons. Carboxyterminal propeptide, derived and cleaved from procollagen
type I (PICP) during collagen synthesis, and aminotermianl (PINP), are delivered into the
blood, where they can be measured. According to current knowledge, PICP correlates
with bone collagen synthesis and bone formation rate. However, these peptides are neither
specific nor sensitive for bone formation, because collagen type I is formed in other
tissues as well.
Markers of bone resorption (osteoclast function) 1. Clearance of calcium, hydroxyproline, hydroxylysine and galactosyl-hydroxylysine (the
last 3 are part of collagen type 1, and their clearance indicates the degradation of
collagen).
2. Pyridine/deoxypyridine crosslaps (measured in the urine)
3. C- and N- telopeptide crosslaps (CTX, NTX): the assay is specific for an octapeptide in
the C-terminus of the α1 chain of type 1 collagen and accurately reflects osteoclast-
mediated bone resorption.
4. Tartrate resistant acid phosphatase (TRAP): in osteoclasts, TRAP is localized within the
ruffled border area, within lysosomes, and in Golgi cisternae and vesicles. Its elevation
indicates osteoclastic function. There are 4 diseases in the skeleton that are worth mentioning:
1. Paget's disease A chronic disease of the adult skeleton, in which localized areas of bone become hyperactive
and are replaced by a softened and enlarged osseous structures. About 3% of persons over the
age of 40 suffer from this disease (more males than females).
Diagnosis:
1. X-ray: bones with increased bone density, abnormal architecture, cortical thickening,
bowing and overgrowth. Mainly localized in the pelvic bones, spine and skull.
2. Lab: se BSAP (bone-specific alkaline phosphatase) is increased, se Ca+2
and
phosphate are normal and collagen crosslaps are elevated.
2. Rickets and osteomalacia A metabolic bone disease resulting from vitamin D deficiency (in children it's called Rickets
and in adults – osteomalacia).
Avi Sayag Clinical Biochemistry
Causes include:
a. Vitamin D deficiency (low UV light exposure, inadequate intake,
malabsorption of vitamin D, hepatic 25-hydroxylase deficiency)
b. Vitamin D-dependent rickets type 1: 1α-hydroxylase deficiency
c. Vitamin D-dependent rickets type 2: receptor defect (resistance to the actions
of calcitriol (vitamin D)).
d. Vitamin D-resistant rickets: X-linked; defective bone mineralization due to
an inadequate supply of phosphate. The cause is usually a renal tubular
phosphate leak.
e. Calcium deficiency: excess loss, malabsorption and inadequate intake
f. Drugs: heparin, methotrexate and anticonvulsive drugs.
Diagnosis:
1. X-ray: bowing legs, deficient mineralization of bone matrix; in children, there is an
inability to mineralize cartilage and remodel new bone at the epiphyseal growth
plates.
2. Lab: se BSAP is elevated, calcium and phosphate are low.
3. Renal osteodystrophy Pathogenesis:
Diagnosis: serum phosphate, BSAP and creatinine are all elevated; serum calcium and
vitamin D are low.
4. Osteoporosis Osteoporosis is characterized by reduced bone mass and abnormalities of bone micro-
architecture, which render it more fragile and susceptible to fracture. It is defined as a bone
mineral density >2.5 standard deviations below the mean for young people or by the
occurrence of a typical fracture. The lifetime risk of fracture due to osteoporosis is about 40%
in women and 15% in men; they are particularly likely to occur in the proximal femur (both
sexes) and in the vertebral bodies (to a much greater extent in women). Fractures of the distal
radius (Colles' fracture) are also more common in women. Risk factors are associated with
female sex, age (menopausal women), immobility, hypogonadism, alcoholism, chronic renal
disease, RA and cortisol therapy.
Diagnosis is based on bone densitometry and increased CTX (collagen crosslaps).
Secondary osteoporosis is related to hyperthyroidism, hypogonadism, multiple myeloma and
chronic liver disease.
Hyperthyroidism: test TSH, T3 and T4
Hypogonadism: test serum testosterone (estrogen)
MM: test ESR, serum protein electrophoresis and serum calcium
Chronic liver disease: test GOT, GPT, GGT and ALP.
Avi Sayag Clinical Biochemistry
Topic 51 Lab procedures in the diagnosis of muscle disorders There are 3 main muscle disorders to address in this topic: periodic paralysis, malignant
hyperthermia and muscular dystrophies.
1. Periodic paralysis: Periodic paralysis is a group of rare genetic diseases that lead to weakness or paralysis (rarely
death) from common triggers such as cold, heat, high carbohydrate meals, not eating, stress or
excitement and physical activity of any kind. The underlying mechanism of these diseases are
malfunctions in the ion channels in skeletal muscle cell membranes that allow electrically
charged ions to leak in or out of the muscle cell, causing the cell to depolarize and become
unable to move (a channelopathy). The attacks can be hypo- or hyperkalemic, depending on
the inducing trigger. In the hypokalemic form, K+ flows into the myocytes from the
bloodstream, while in the hyperkalemic form K+ leaks out of the myocytes into the blood.
Hypokalemic Hyperkalemic
Induced by Glucose (insulin causes reduction of K+) KCl or exercise
Duration Hours 30-40 minutes
Serum K+ <3 mmol/l Increased
Urine K+ Decreased Normal
inheritance AD (chromosome 1) AD (chromosome 17)
2. Malignant hyperthermia:
Malignant hyperthermia is a rare life-threatening AD condition that is triggered by exposure
to certain drugs used for general anesthesia and the neuromuscular blocking agent
succinylcholine. The defect lies in the ryanodine receptor gene (Ca+2
channel). In susceptible
individuals, these drugs can induce a drastic and uncontrolled increase in skeletal muscle
oxidative metabolism, which overwhelms the body's capacity to supply oxygen, remove CO2
(leading to respiratory acidosis), and regulate body temperature (heat in the muscle is
increased as well as the body temperature), eventually leading to circulatory collapse (shock)
and death if not treated quickly.
Therefore, Lab tests may reveal:
i. Acidosis (pH<7.2)
ii. Increased lactic acid
iii. CK, AST, LD greatly increased
iv. Myoglubinuria
v. Tubular necrosis
vi. Acute renal failure
Screening can be done by measuring a 100-fold increase in CK levels.
The diagnosis should rest on muscle biopsy and molecular biology (Ryanodin receptor defect)
Muscular dystrophies: A large group of inherited progressive muscle weakness diseases that can be inherited as AD,
AR or X-linked. The lab diagnosis is made with a combination of DNA, RNA and protein
analysis to identify the molecular defect and to distinguish between the many forms of MDs
that have an overlapping phenotype.
MD involve problems in the dystrophin-glycoprotein complex. In a nutshell, dystrophin, actin
and syntrophin (α and β1) are located intracellularly; sarcoglycans (α,β,γ,δ) and β-
dystroglcans are transmembrane structures; α-dystroglycan and α2-laminin (aka merosin –
connects dystroglycans to integrins in the basal lamina) are extracellularly located.
The dystrophin gene (Xp21) is a very large gene (100 times larger than average). Its product
(dystrophin) is produced by alternative splicing yielding at least 7 tissue specific isoforms
(muscle, brain, retina, kidney, etc.). The muscular type is 175 nm long, with 4 domains: N
terminal binds actin, the 2nd
domain is proline rich and is similar to α-actinin, the 3rd
domain is
cystein-rich and binds β dystroglycan, and the C terminal binds syntrophins. It functions in
assembling and maintaining the complex, and it connects contractile element within the cell to
merosin located extracellularly.
Avi Sayag Clinical Biochemistry
Dystrophin-related disorders: Duchenne and Becker MD
DMD (Duchenne) BMD (Becker)
Wheelchair by age 12 Still ambulant at age 16
Reduction in IQ No mental deficiency
Sarcolemma destroyed Slower progression
Approx. 28:100,000 males Approx. 5:100,000 males
50-100-fold increase in CK 50-100-fold increase in CK
Immunostaining of muscle biopsy- dystrophin
absent
Immunostaining of muscle biopsy-
interrupted staining of dystrophin
Immunoblotting- no protein detected Immunoblotting- altered dystrophin (larger
or truncated)
Deletion/duplication/point mutations of genes Deletion/duplication/point mutations of genes
Reading frame alteration (frame shift) No reading frame alteration
X- linked X- linked
Diagnosis: all those in bold are used for diagnosis. Deletion screening is done using
multiplex-PCR in which more than one pair of primers used to amplify and detect dystrophin
deletions (deletions account for 60% of cases, point mutation for 35% and duplications for
5%). MLPA (multiplex ligation dependent probe amplification) can also be used. It is one of
the best quantitative methods to detect small differences. The method uses a pair of probes
that are amplified only when both are attached AND ligated to each other. The results are
given in a graph, and the peak intensities are compared and analyzed.
Prenatal testing is performed at the 11th week when 30 mg of villi are taken to determine the
sex of the fetus (by cytogenetics). If it is a male, then further examinations are carried on the
same biopsy. In case of a male fetus, multiplex PCR/MLPA are carried out, and the results are
obtained in 1-2 weeks.
Other MD:
Limb Girdle MD- LGMD: inherited as AD/AR, 10-120-fold increase in CK, mainly
with sarcoglycans (α type is deficient mostly)
Merosin deficiency: AR, no merosin, no mental problems.
Oculopharyngeal MD: AR/AD, repeat of GCG creating stabile fibril, which
accumulates in the nuclei causing cell death. It is prevalent in French-canadian and
Bukharik Jews in Israel.
If SG are normal,
merosin should be tested
(merosin def. MD) and
calpain (calpainopathy).
However, if both are
normal, then Emerin
immunostain should be
performed. A deficient
Emerin indicates
Emerin-Dreiffus
Avi Sayag Clinical Biochemistry
Topic 52 Clinical biochemistry at the extremes of age Lab values are greatly affected by some factors such as:
1. Age
2. Sex
3. Race
4. Circadian changes
5. Posture
6. Pregnancy
7. Altitude
The reference ranges of normal adults would not apply to the values received from newborns,
children during puberty and the elderly.
Infants and neonates Testing neonates, there are some problems to confront. Neonates have a limited amount of
blood (up to 300 ml) and immature ones have even less (100-200 ml). The reference intervals
are different for them, and differ even further for whole blood samples and for serum ones.
Lastly, it is problematic to get a neonate to fast.
Changes in lab parameters which are age related-
a. Glucose: from 1.6-3.3 mmol/l in neonates to 3.9-5.6 mmol/l in healthy adults (caused
by small amount of glycogen stores and adrenal immaturity)
b. Bilirubin: in the cord <34 µmol/l and rises up to <273 µmol/l on days 3-5 and then
falls again. Adult values: <17 µmol/l.
c. Urea: 3.5-6.6 mmol/l in the cord and drops to 0.7-2 mmol/l in the neonate and starts
to rise again (due to increased protein synthesis). Adult values: 2.9-8.2 mmol/L.
d. Leukocyte count: high at birth (18.1 G/L), and drops with the years to 7.8 G/L (at 16
years of age).
e. Other changes can be seen also in MCV, Hb, PLT, neutrophils, lymphocytes,
monocytes and reticulocytes.
Elderly Testing the elderly, there are also some problems to confron. Similar to neonates, the
reference range is different. There are prevalent diseases to take into consideration, and some
of them present as atypical form of the disease. Lastly, most elderly patients are taking
medications that might interfere with the lab results.
1. Endocrinological changes-
a. Estrogen/testosterone decrease
b. FSH/LH increase
c. Hypothyroidism (T3 decreases up to 40% over age 40)
d. Peripheral blood glucose uptake is decreased and the Tm for glucose is
reduced as well.
e. PTH decreases
f. Aldosterone decreases up to 50%
2. Renal concentrating capacity decreases.
3. Comparison (also see Topic 34 for further details of the normal ranges):
Cholesterol (mmol/l) Normal: <5.2 Triglycerides (mmol/l). Normal: 1.35
Newborn 1.37-3.5 0.36-1.12
Adolescent 3.11-5.44 0.45-1.81
Elderly 3.63-8.03 0.62-2.79
For both the elderly and neonates, CSF values are also different (protein level in CSF can
reach up to 0.9g/L, while in adults it is 0.15-0.45g/L).
Avi Sayag Clinical Biochemistry
Topic 53 Clinical biochemistry and lab diagnostics of porphyrias Heme synthesis begins when glycine and succinylCoA combine to form AminoLevulinic
Acid (catalyzed by ALA synthase). Then, 2 ALAs condense to form porphobilinogen (PBG,
catalyzed by PBG synthase), and 4 PBGs condense to form the first porphirins. Porphyrias
are a group of inherited diseases in which a partial deficiency of one of the enzymes of
porphyrin synthesis leads to decreased formation of heme and thus, by releasing ALA
synthase from inhibition, results in the formation of excessive quantities of porphyrin
precursors (ALA and PBG) or porphyrins (heme, the end product, normally inhibits ALA
synthase). When precursors are produced in excess, the clinical manifestations are primarily
neurological (the precursors are neurotoxins). When porphyrins themselves are the major
product, the predominant feature is photosensitivity: the porphyrins absorb light and become
excited, inducing the formation of toxic free radicals.
The porphyrias are classified as acute or chronic, according to their clinical presentation, and
hepatic or erythropoietic, depending on the major site of abnormal metabolism. The 3 acute
forms are acute intermittent porphyria, hereditary coproporphyria and variegate porphyria.
All acute forms are hepatic. The chronic forms are porphyria cutanea tarda (hepatic), and
congenital erythropoietic porphyria and erythropoietic protoporphyria (both are
erythropoietic). All forms are AD except congenital erythropoietic porphyria and ALA
dehydratase deficiency porphyria (an hepatic form). All hepatic acute forms are characterized
by neurological symptoms, and all the chronic forms are characterized by photosensitivity.
Acute intermittent porphyria:
The defect lies in HMB synthase (aka PBG deaminase). The inheritance is AD with over 60
mutations described. Women are affected more than men but around 90% of carriers are
asymptomatic. The acute attacks, which begin over several hours, may last between days to
weeks, and are followed by complete remissions. Some factors may precipitate the attacks
(e.g. anesthesia, drugs, alcohol, stress, pregnancy and infection). The heme precursors are
neurotoxic and cause vascular damage. Symptoms usually begin around puberty and include
GI symptoms (vomiting, constipation), peripheral neuropathy (muscle weakness and
numbness), seizures, depression, hysteria and psychosis, sinus tachycardia and systemic
hypertension. Recall that there is no photosensitivity.
Diagnosis includes:
i. Urinary PBG during an attack
ii. Quantitative analysis of other precursors in the urine (e.g ALA)
iii. The results might be normal if the patient is not in the midst of an attack
Treatment includes control of the fluid and electrolytes balance, IV glucose, pain relief if
needed, IV hamatin (which decreases ALA synthase action) and avoiding the precipitating
factors.
Porphyria cutanea tarda:
The most common cutaneous porphyria caused by a decreased activity of uroporphyrinogen
decarboxylase enzyme in the liver. 15-20% are inherited (type 2), while the sporadic cases
(type 1) occur among alcoholics or people who consume drugs/other heptotoxic substances.
Photosensitivity dominates the clinical picture (due to accumulation of porphyrins) and it
manifests in photodermatitis, blisters, necrosis of the skin, itching, dark urine after an
exposure to light (when the porphyrins are excreted) and red teeth in case they accumulate
there. In contrast to AIP, there are no neuro-visceral symptoms and the porphyrins can be
found in the urine AS WELL AS in the stool.
Congenital erythropoietic porphyria:
The defective enzyme is the uroporphyrinogen synthase in RBC. Photosensitivity is very high
in comparison to the other types mentioned. The porphyrins bind to the dental Ca+ leading to
their red color in fluorescent light (erythrodontia). In contrast to AIP, there are no neuro-
visceral symptoms, the porphyrins are excreted in BOTH the urine and stool, and accumulate
within the RBC themselves.
Avi Sayag Clinical Biochemistry
Topic 54 Lab diagnostics of CNS diseases; lab investigation of the CSF While the diagnosis of some metabolic neurological disorders requires complex investigations
that will usually be performed only in specialist laboratories, simple investigations that should
be in the repertoire of most laboratories are frequently required in the initial investigation of
patients presenting with neurological signs and symptoms. While most of these investigations
are performed on plasma or serum, assays of CSF are also valuable in certain conditions:
CSF sampling is indicated in cases of meningeal infection, subarachnoid hemorrhage (SAH),
CNS malignancy and a demyelinating disease. The results should be correlated with the
plasma values, with the clinical symptoms and with the radiological findings. The CSF can be
sampled from the lumbar region (anywhere below L1, preferably L3-L4). Alternative
methods of CSF collection are rarely used, but may be necessary if the person has a back
deformity or an infection. Cisternal puncture uses a needle placed below the occipital bone. It
is always done with fluoroscopy. Ventricular puncture is even more rare, but may be
recommended in people with possible brain herniation. This test is usually done in the
operating room. A hole is drilled in the skull, and a needle is inserted directly into one of
brain's ventricles. CSF may also be collected from a tube that is already placed in the fluid,
such as a shunt or a venitricular drain. These sorts of tubes are usually placed in the intensive
care unit. The opening pressure in adults reaches 180mmH2O, while that of infants reaches
only 10-100 mmH2O. Blood should be drawn 2-4 hours before sampling the CSF. Once the
CSF needle is inserted, 5 tubes are collected: tube 1- cell count; tube 2 – Stat gram stain and
culture; tube 3 –glucose and protein; tube 4 – cell count (for comparison to tube 1); tube 5 –
virology, mycology, cytology, etc. (an optional tube). Those tubes destined to cell count
procedure need not be centrifuged, while those sent to immunochemical investigations are
centrifuged.
The examination of the CSF starts with a gross examination: healthy patients have a colorless
CSF sample, and its viscosity is similar to water. A viscous sample may indicate metastatic
mucin-secreting adenocarcinoma or cryptococcal meningitis. Also, a clot can sometimes be
seen, which implies a traumatic tap, suppurative and tuberculous meningitis or spinal block.
Note that a clot does not suggest SAH. Lastly, following hemorrhage into the CSF, red cells
undergo lysis and phagocytosis, and the oxyhemoglobin that is released is slowly converted
into bilirubin (and sometimes methemoglobin). In some cases, the bilirubin imparts a yellow
colour (xanthochromia) to the CSF that is visible to the naked eye. Scanning of apparently
colourless CSF for xanthochromia may help to distinguish SAH from a traumatic tap.
The next step is a microscopic examination:
Cells
Fuchs-Rosenthal chamber is used for cell counting. In adults, there are 0-5 cells/µl, while
infants have 0-30 cells/µl.
The differential for leukocytes is also obtained. As known, the differential of neutrophils in
the serum is around 60%. In the CSF, however, it is 2% in adults and 3% in newborns.
Increased neutrophils indicate meningitis (mainly bacterial), CNS hemorrhage or infarction,
foreign materials in the SAS (such as contrast media), and metastatic tumor in contact with
the CSF.
Lymphocytes in the CSF are 60% (+/-30) in adults and 20% (+/-18) in newborns. In the
serum they are 20-40%. Increased lymphocytes in the CSF suggests meningitis (mainly non-
bacterial), degenerative disorders (e.g. MS, Guillain-Barre) and other conditions such as
sarcoidosis and polyneuritis.
Proteins
More than 80% of proteins in the CSF are derived from the plasma, and most are albumin.
The normal reference range in the CSF is 0.15-0.45 g/L (while in the plasma it is 60-80g/L).
Note that neonates and the elderly usually have higher values (up to 0.9g/L). CSF protein
level can be increased in cases of BBB malfunction, decreased resorption, mechanical
obstruction and elevated intrathecal Ig synthesis. Decreased levels can be found in youns
Avi Sayag Clinical Biochemistry
children (not neonates) 0.5-2 years old, when the turnover of CSF is increased and in
hyperthyroidism.
Albumin: albumin is 230 times more abundant in the serum than in the CSF (the ratio
between albumin in the CSF and in the serum is 1:230). As long as this ratio is below 9, the
barrier is intact. A slight impairment is suggested by a ratio of 9-14, and it becomes a
moderate one when it rises to 14-30. The barrier impairment is said to be severe when the
ratio is 30-100.
The ratio between CSF Ig and serum Ig is also an indicator. Normally, it is kept between 3-8.
It significantly increases in MS where there is an increased intrathecal Ig synthesis.
S-100B: this is a calcium-binding protein found in Schwann cells, astroglia, melanocytes and
others. It is found both in the serum (<0.15µg/L) and in the CSF (<5µg/L). When hypoxia
occurs, the glia cells are damaged, protein S-100B leaks to the intercellular space and the
CSF, and as the BBB is damaged, it reaches the serum. Cranial trauma results in increased
levels by the same mechanism. The levels of S-100B are elevated in viral encephalitis, less so
in bacterial meningitis and even less in viral meningitis.
CRP: elevated CRP in the CSF is highly suggestive of bacterial meningitis (the predictive
negative value of a low result ruling out bacterial meningitis is 97%).
Enzymes Neuron specific enolase: this is a prognostic marker after cerebral infarct and intracerebral
hemorrhages. In the serum it is found in less than 12.5µg/L, and in the CSF it is 2.5 times
more (<32µg/L).
LDH: should be less than 40 U/L
CK: normally less than 5 U/L and usually the CK-BB isoforms is present. Its levels increase
in demyelinating diseases, CVA, brain malignancies, meningitis and head injuries.
Glucose The normal value is 60% of that in the plasma. Lower levels suggest meningitis (bacterial,
TB, fungal and sometimes viral), and other conditions involving the meninges (SAH, tumors,
sarcoidosis). During recovery from meningitis, the glucose levels normalize before these of
proteins.
Ammonia: the levels should be 30-50% of that in the plasma. Its elevation is proportional to
the degree of hepatic encephalopathy. α-ketoglutarate protects the CNS by binding NH3 to
form glutamine.
There are 5 conditions to address:
1. CSF leakage: diagnosed by clinical symtoms (otorrhea, rhinorrhea), lab tests for
glucose, S-100B and protein electrophoresis with immunofixation for transferrin.
Radiological examinations will complete the picture.
2. CVA: lactate, CK-BB, LDH, S-100B and NSE are all elevated.
3. Tumors: CRP is elevated, neutrophils are elevated, tumor cells are present in a
microscopic slide, electrophoresis shows a monoclonal band, and tumor markers are
detected (AFP, CEA, hCG and NSE). Flow cytometry is used to characterize the
cells.
4. SAH: xanthochromia is detected, and D-dimers, LDH, CK-BB and S-100B are all
elevated.
5. Meningitis: total protein, lactate, LDH, Ig, CRP and S-100B are elevated (more in
bacterial than in viral meningitis) and glucose is reduced (more in bacterial than in
viral meningitis).