clinical biochemistry assays

8/13/2019 Clinical Biochemistry Assays

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PRACTICAL I

CLINICAL BIOCHEMISTRY ASSAYS

GLUCOSE

PROFILE

LIVER

FUNCTION

TEST(LFT)

KIDNEY

FUNCTION

TEST(KFT)

LIPID

PROFILE

CARDIAC

PROFILE

IRON

STUDIES

BONE

PROFIL

FBS/RBS

(FPG)

AST BUN TOTAL

CHOLES

AST TOTAL

IRON

CREAT

GTT ALT CREAT TRIG LDH TIBC ALB

2HR PP ALP Na+

HDL-C CK-MB Ferritin Ca2+

HbA1c GGT K+

LDL-C MYOGLO PO42-

URINE

sugar/ketones

TOTAL

BILIRUBIN

Cl-

VLDL TROPONIN ALP

Direct BUE Mg2+

Indirect URIC ACID

TOTAL

PROTEIN

Albumin

Globulins

CREAT

CLEAR.

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HORMONAL ASSAYS

THYROID FN

TEST

FERTILITY

HORMONES

TUMOUR

MARKERS

OTHER

HORMONES

OTHER

CHEMISTRIES

T4 FSH Total PSA INSULIN AMYLASE

T3 LH B-HCG CORTISOL

TSH PRL CEA

PRG AFP

E2 CA 125

TESTOSTERONE CA19-9 /CA15-3

SPECTROPHOTOMETRY

Measurement of light intensity of multiple wavelengths

LAMBERT-BEERS LAW: The [substance] is directly proportional to the amount of light

absorbed or inversely proportional to the logarithm of the transmitted light.

CA log 100

%T

A=abC

Where A= absorptivity, b=length of light path, C= [ ]

a and b are constants.

C1=A1, C2=A2,

C1 = A1

C2 A

2

[Ctest= CstdA test]

Astd

Beers law is the basis of all the spectrophotometric measurements.

Advantages of spectrophotometric measurements:

1. High sensitivity

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2. Ease of measurement3. High degree of specificity4. Accuracy

Urinalysis is an indispensable part of chemical pathology. It is used to uncover dx anywhere in the

urinary tract.

TESTS ON URINE COMMONLY INCLUDES

A) URINE COLOUR AND APPEARANCE (MACROSCOPY):Its colour is determined by its concentration, the presence of drugs, exogenous and endogenous

compounds and its pH.

Colourless urine---normal or 20to diuretic use, high fluid intake,DIorDM.

Cloudy/Hazy------phosphates, pyuria,or bacteruria

Yellow to orange----- presence of bile, riboflavin , bilirubin, urobilinogen

Red----------------presence of haemoglobin,

Amber

B)CHEMICAL COMPONENTS

TEST REFERENCE

Specific gravity 1.003-1.029

pH 4.5-7.8

Protein Negative

Glucose Negative

Ketones Negative

Bilirubin Negative

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Occult blood Negative

Leukocyte Negative

Nitrite Negative

Urobilinogen 0.1-1.0 EU/dL

C )MICROSCOPY COMPONENTS:

Microscopy components consists of epithelial cells, pus cells, RBCs,WBCs,casts. These are

examined microscopically.

PRACTICAL II

An essential metabolic fuel and its concentration in the blood are normally tightly controlledby the action of insulin and the counter regulatory hormones like Glucagon, cortisol, catecholamine,

and growth hormone.

[Glu] rises after meals and stabilizes at lower levels on fasting.Syno: blood sugar, FBS, FBG sugar.

Specimen: plasma: Note: whole blood taking in fluoride oxalate vacutainer.

Patient care: Pt should be fasting for 8hours-12hours prior to the test.

Source of error: blood not in fluoride oxalate tube. Glycolysis by red cells will consume glu and may

lower concentrate in vitro in absence of fluoride.

BLOOD & PLASMA GLUCOSE

Plasma [glu] is 10-15% higher than whole blood [glu] since red cells contain less water per unit

volume than plasma. The discrepancy can be greater them this if [glu] is changing rapidly because

glu will not have reached equilibration across the red cell membrane. Therefore plasma yields more

reliable results.

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Types of glucose test:

RBS, 1hr pp, 2hr pp.

2hr Postprandial: glucose levels 2hrs after meal or after measured glucose level. It is used

extensively to establish the diagnosis of DM

Ref Ranges: FBS : 3.6-6.4 mmol/L

RBS :3.3-7.0mmol/L (Dependant on time & meal content)

PRACTICAL III

GTT/OGTT

The GTT is a provocation test to examine the efficiency of the body to metabolise glucose and is

used to establish the presence of glu intolerance. It is used in pts with borderline fasting and PP glu

to support or rule out the diagnosis of DM. It is used to work up glycosuria without hyperglycemia

and also used to predict prenatal mortality in pregnancy, to diagnose gestational diabetes.

PRECAUTION BEFORE TEST

1. Pt. should fast 12 hours prior to test.

2. Pt. should not smoke due to glu stimulation by nicotine.

3. Pt should not take any drug prior to test.

4. Pt should not be stressed.

5. Pt should not eat nor drink nor smoke during test.

6. Pt should not indulge in unaccustomed amounts of exercise liking jogging.

7. Pt should be sitting up or lying over on the right side so as to facilitate rapid emptying of the

stomach.

Normal Glucose load: 75G in 300ml H2OChildren: 1.75 x weight ( up to 75g)

Pregnant women: 100g in 300ml

Take samples of Times 0. FBS 1. 1 hr pp 2. hr pp 3. 3hr pp. with urine samples H2O

GLUCOSE LOAD: g in 300 ml H2O

RESULTS OF OGTT Vs TIME

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TIME (Min)BLOOD GLUCOSE

CONC (mmol/L)URINE SUGAR

0

FASTING BLOOD

SUGAR

60

120

180

METHODOLOGY: Glucose oxidase method or Hexokinase method.

Principle of method: glucose oxidase (GOD) catalyzes the oxidation of glucose to

give hydrogen peroxide (H2O2) and gluconic acid. In the presence of enzyme

peroxidase (POD), the hydrogen peroxide is broken down and the oxygen

released reacts with 4-aminophenazone

(4-aminoantipyrine) and phenol to give a pink colour. The absorbance of the

colour produced is measured in a spectrophotometer at 520nm.

Glu+O2 + H2O Glucose + phenol + 4-G Oxidase

Aminophenazone

PROCEDURE

STD STD TEST BLK

Glucose oxidase

Rgt

1 ml 1ml 1ml 1ml

STD 10L 10L - -

TEST - - 10L -

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PRACTICAL IV

TOTAL SERUM PROTEIN

1. There are over a hundred individual plasma proteins: many are present in very small

concentrations.

2. Approximately half of the total protein is albumin; much of the rest is immunoglobulins

(principally IgG)

i.e. Total Protein Albumin = Total Immunoglobulin

Ref. Range: 60 80g/LSPECIMEN: Serum ( Total plasma protein is slightly higher than total serum protein because of

coagulation factor present).

SOURCES OF ERROR:

1. Venous stasis during venipuncture can lead to increased values.

2. Haemolysis can falsely elevate (total protein)

METHODOLOGY: BIURET REACTION

PRINCIPLE: Cu2+

ions react with peptide bonds in alkaline medium to form a purple complex

which absorbs maximally at 540nm.The [ Complex] = [ protein ]

STD STD BLANK T1 T2

Biuret Rgt. 2.5mL 2.5mL 2.5mL 2.5mL 2.5mL

Std 0.05mL 0.05mL - - -

Sample - - - 0.05mL 0.05mL

Mix well and incubate at RT for 10mins and read absorbance at 540nm against rgt. blank.

Use Beers Law to calculate the [test].

ABNORMAL LEVELS OF PROTEINS CAN BE DUE TO:

HIGH LOW

1. Sarcoidiosis 1. Poor nutrition

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2. Chronic inflammation 2. Over hydration

3. Myeloma 3. Hepatic insufficiency

4. Hypergammaglobulinemia 4. Protein losing enteropathy

5. Agammaglobulinemia

ALBUMIN

The major plasma protein and is involved non-specifically in the transport of numeroussubstances including unconjugated bil, Ca

2+, free fatty acids, vitamins A, K and other

drugs.

It is the most important determinant of oncotic pressure (osmotic pressure due to ptn) andhence the distribution of fluid between the vascular and interstitial spaces.

USES:

Evaluation of nutritional status. Diagnosis of renal diseases with proteinuria. Diagnosis of chronic diseases.

SPECIMEN: Serum

Ref. Range: 35 50g/L

SOURCES OF ERROR:

1. Stasis during venipuncture can cause an apparent increase.

2. Taking blood sample close to site of intravenous infusion.

METHODOLOGY

Bromocresol Green (BCG) is widely used.

PRINCIPLE: BCG reacts with albumin in acidic medium of PH 4.2 to form a green complex

which is measured at 600nm. The reading of the complex has to be done at 30s to avoid thetendency of other proteins reacting with the BCG.

STD STD BLANK TEST TEST

BCG 2.0mL 2.0mL 2.0mL 2.0mL 2.0mL

STD 10L 10L - - -

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BLANK - - - - -

TEST - - - 10L 10L

Mix well and read absorbance at 600nm at exactly 30s. Use Beers law to calculate [test].

ABNORMAL VALUES OF ALBUMIN CAN BE DUE TO:

INCREASED CONC DECREASED CONC

1.Dehydration 1. Renal disease2. Liver insufficiency with decreased

albumin synthesis.3. Severe malnutrition4. Acute inflammation5. Chronic inflammation6. Pregnancy7. Burns

PRACTICAL V

An orange-yellow pigment, the end product of haem degradation normallymetabolized in the liver and excreted into bile.

Accumulation causes jaundice which usually become apparent when [plasma] exceedtwice normal.

Frequently elevated in Hepatobiliary dx of any type when bilirubinuria is usually alsopresent.

Differential diagnosis of liver dx requires total and direct bil values as well as othertests.

Total bil=Direct (conjugated) bil+Indirect (unconjugated) bil

Direct bil is the water soluble fraction and indirect is water insoluble.

SPECIMEN: SERUM

N.B. Protect samples from since bil is photosensitive.

Ref. Range: DBil: (< 3.4mol/l)

Total Bil: 2-21mol/l

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Neonates values vary with age in days, prematurity and maturity.

[Total bil] are measured in neonates in mol/l

AGE Premature Full term

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Bilirubin conc. [mg/dL] = A540x 13.0 or

[mol/L] = A540x 17.1x 13

ABNORMAL VALUES (CAUSES)

TOTAL DIRECT

1. Liver insufficiency 1. Neonatal jaundice due to atresia of bile

ducts.

2. Extra-hepatic obstruction 2. Obstructive jaundice

3. Haemolysis

4. Neonate from a variety of caused including neonatal

physiological hyperbilirubinemia.

5. Gilbert syndrome

6. Erythroblastosis fetalis

7. Dubin-Johnson syndrome

PRACTICAL VI

LIVER ENZYMES

ALKALINE PHOSPHATASE (ALP)

An enzyme occurring particularly in osteoblasts (bone-forming cells) and the liver. Plasma activities occur in various bone and Hepatobiliary disease. Measured as part of bone and liver profiles.

SPECIMEN: Serum

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Ref. Range: 53 128 U/L (in adults).

Levels up to 3x this may be normal in children.

SOURCE OF ERROR: None.

CAUSES OF ABNORMAL VALUES

Increased levels are characteristics of bone disease associated with increased osteoblastic activity

and Hepatobiliary disease with partial or complete biliary obstruction.

Increased ALP due to bone disease Increased ALP due to Hepatobiliary disease

1. Paget disease of bone 1. Cirrhosis

2. Hyperparathyroid bone dx 2. Hepatic tumours (1oand 2

o)

3. Ricketts 3. Extrahepatic biliary obstruction( eg. carcinoma of

pacreas)

4. Renal osteodystrophy

5. Healing fractures

6. Osteomalacia

7. Bone tumours (1oand 2

o)

Increased ALP can occur in late pregnancy due to secretions of the enzyme in the placenta.

Decreased ALP

1. Hypophosphotasemia

2. Hypothyroidism

3. Scurvy

PROCEDURE

Working Rgt 20mL of buffer + substrate

TEST 1 TEST 2

Sample 10L 10L

Working Rgt. 500L 500L

Mix well and read absorbance and start time simultaneously. Read again after 1,2 and 3 min [Conc.

In U/L] = 2760 x A405

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GAMMA-GLUTAMYL TRANSFERASE (GGT)

GGT is an enzyme that is mainly in the liver, often measured as part of a panel of LFT.

Increased plasma activities of GGT can occur in all types of liver disease but it is a particularly

sensitive (though not specific) indicator of excessive consumption of alcohol.

Ref. Range (12-64) U/L

SPECIMEN: Serum

CAUSES OF ABNORMAL RESULTS

Increased GGT

1. Hepatitis (alcoholic)

2. Excessive alcohol consumption

3. Cholestatic liver disease of any cause.

4. Treatment with certain anticonvulsant and other drugs.

5. Fatty liver (eg. in obesity, poorly controlled diabetes).

PRACTICAL VII

ALANINE TRANSAMINASE (ALT, GPT)

A soluble cytoplasmic enzyme widely distributed in body tissues but present in particularlyhigh amounts in the liver.

Released into the plasma when there is tissue damage (eg. Hepatitis) ALT activity is most often measured as one of LFT as a measure of hepatocellular damage

since it is more specific indicator than AST.

Parallel measurement of ALT and AST is therefore applied to distinguish liver from heart orapplied to distinguish liver from heart or skeletal muscle damages, the AST/ALT is used for

differential diagnosis in liver dx.

1 severe or chronic dx.

SPECIMEN: Serum

Ref. Range : 0 50 U/L

SOURCE OF ERROR: None

METHODOLOGY

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Enzymatic kinetic method + Enzymatic end point method.


HIGH LEVELS LOW LEVELS

1. Hepatitis 1. Renal hemodialysis or renal insufficiency

2. Cirrhosis 2. End stage of liver disease

3. Cholestatic Jaundice

4. Congestive cardiac failure (due to hepatic

congestion)

ASPARTATE TRANSAMINASE (AST/GOT)

A soluble cytoplasmic and mitochondrial enzyme widely distributed in body tissues,particularly in the liver, skeletal and cardiac muscles.

Tends to released more than ALT in chronic hepatocellular dx. e.g. cirrhosis.

SPECIMEN: Serum

SOURCES OF ERROR: Haemolysis

METHODOLOGY

Enzymatic end point reaction/ enzymatic kinetic reactions

CAUSES OF ABNORMAL TESTS:

1. Liver dx

a. Hepatitis

b. Preceded jaundice

c. Less elevation in cholestatic dx.

d. Excessive alcohol, obesity, drug toxicity, DM

2. Myocardial infarction

3. Skeletal muscle damage dx.

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PRACTICAL VIII

CREATININE

Creatinine is used to diagnose impaired renal function.

Creatinine is a catabolic product of creatinine phosphate, which is used in skeletal musclecontraction.

It is excreted entirely by the kidney s and is directly proportional to renal excretory function. Age-Related Concerns: The elderly and young children normally have lower creatinine levels

as a result of reduced muscle mass. This may potentially muscle renal dx in patients of these

age groups.

Creatinine is the most common clinical RFT providing a rough approximation of glomerularfiltration.

GFR must fall to approximately half normal before serum creatinine becomes reliablyincreased.

SPECIMEN: Serum

Ref. Ranges: Adult males: < 106mol/L

Adult females: < 97mol/L

PRINCIPLE OF THE METHOD

Creatinine reacts with picric acid in an alkaline medium. The absorbance of the yellow-red colour

produced is measured at 490nm wavelength. A number of other compounds similarly react with

picric acid giving artificially high results for creatinine if one simply measures the total yellow-red

colour produced. A second reading is therefore made after making the solution acid. The colour

produced by creatinine is quickly destroyed by acid whereas that given by non-creatinine

chromogens is destroyed more slowly. By subtracting the second reading which is due to non-

creatinine substances from the first reading (due to creatinine and non-creatinine substances) the

colour produced by the true creatinine can be obtained.

PROTOCOL

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WORKING REAGENT: EQUAL VOLUMES OF REAGENT 1 & 2

ABSORBANCE:490nm on spectrophotometer.

STD BLK TEST

WORKING RGT 1 mL 1 mL 1 mL

STD/ SAMPLE 100L - 100L

Mix well and incubate for 20seconds and read A1 at 490nm against rgt blank and incubate at room

temperature for exactly 80 seconds and read A2.

CALCULATION: [CREAT] in mol/L = 176.8 mol/L X ASAMPLE

ASTD

Increased levels

Dx affecting renal function, such as glomerulonephritis, pyleronephritis, acute tubular necrosis, U.T

obstruction, reduced renal blood flow (eg. shock, dehydration, congestive heart failure, and

atherosclerosis), diabetic nephropathy, and nephritis. Renal function impaired and creatinine

increases.

Acromegaly

Gigantism: Associated with increased muscle mass.

Decreased levels

1. Debilitation2. Decreased muscle mass (e.g. muscular dystrophy)

CREATININE CLEARANCE [CrCl]

CrCl is a measure of the GFR.Urine and serum creatinine levels are assessed and the clearance rate is calculated as :

CrCl = UV

P

U = [urine creatinine] over 24 hrs (mol/L)

V = volume of urine in mL/min= vol of urine

24 x 60

P = [serum creatinine] in mol/L.

PATIENT PREPARATION

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24 hr urine collection. Discard the initial specimen and start 24hr timing at that point.

Collect all the urine passed during the test.

Keep urine sample refrigerated.

Indicate the starting time.

Patient should drink fluids during the test.

Avoid vigorous exercise during the test.

Collect last specimen as close as possible to the end of the 24hr collection.

UREA:

urea is the major end product of nitrogen (protein) metabolism and is synthesized in the liverand excreted by the kidneys.

Urea increases in renal failure but can be increased in dehydration and in individuals on ahigh protein in take in the absence of renal impairment.

Often increased as part of renal profile i.e BUE but creatinine provides a better index of renalfunction for most purposes.

SPECIMEN: serum/plasma

REF. RANGE: 2.5-6.7mmol/l

SOURCES OF ERROR: noneMETHODOLOGY: Berthelot Reaction

Causes of abnormal results

HIGH LEVELS LOW LEVELS

1. Dehydration 1. Starvation

2. Renal failure

3. Gastrointestinal bleeding (due to catabolism of

retained blood)

4. Recent protein intake

PRACTICAL IX

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ELECTROLYTES

Sodium (Na+), Potassium (K

+) and Chloride (Cl

-)

SPECIMEN: Serum/Plasma

SOURCES OF ERROR:

Haemolysis Delay in separation of serum Dont allow patient to clench and unclench Minimal venestasis Dont force blood through needle or syringe

METHODOLOGY: Iron selective Electrode and Flame Photometry

Potassium (K+)

K+is the principal intracellular cation but its plasma concentration has a major effect on the

excitability of nerve and muscle membranes.

REF. RANGE: 3.6-5.0 mmol/l

CAUSES OF HYPOKALAEMIA

Gastroinstinal loss (diarrhoea Renal loss

Hyperkalaemia

Renal failure Accumulation of potassium supplements

Sodium (Na+)

Principal extracellular cation and determinant of extracellular fluid osmolality and volume. Abnormalities of potassium concentration can be related to abnormal water or sodium

heamostasis.

REF. RANGE: 135-145mmol/

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HYPONATREMIA HYPERNATREMIA

1. constant diuretic treatment

2. cancer

3. diarrhea and vomiting

4. congestive cardiac failure

5.chronic liver dysfunction

6.chronic renal failure

1. Dehydration

Chloride (Cl-)

The principal negatively charged ion in the ECF Provides additional diagnostic information on plasma [ Na+]Ref. range: 95-105 mmol/l

Methods used

1. Flame photometry2. Ion selective electrode (ISE)

URIC ACID

Urate is the end product of nucleic acid metabolism Increased urate can predispose of gout, due to the deposition of monosodium urate crystals in

joints.

Hyperuricaemia does not always cause gout, but gout is usually related to hyperuricaemiaSpecimen: serum (ideally fasting)

Ref. Range :( 208-428) mol/L

ABNORMAL RESULTS

Increased [urate] Decreased [urate]

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1. Gout2. Renal failure3. Diuretic therapy4. Lesch-Nyhan syndrome5. Polycystic kidney6. Leukaemia,lymphoma,polycythaemia7. Diet(high protein intake)8. Drugs(uricostatic drugs e.g. diuretic)9. Lactate excess e.g. ethanol ingestion10.Chronic lead nephropathy

1. Administration of uricosuric drugs(high doses of

salicylates,cortisone,allopurinol)

2. Renal tubular defects(Fanconissyndrome, Wilson disease)

PRACTICAL X

LIPID PROFILE

Acts as energy stores (triglyceride) and structural component.

Insoluble in water are transparent in pleasure as perimeter complexes the proteins thelipoprotein

It includes total cholesterol,triglycerides,HDL choles, LDL choles andVLDL choles

PATIENT CARE PREPARETION:

Patients should be on stable diet ideally for 2-3 weeks prior to collection of blood. Patients should fast for at least 12hours before collection of blood. Patients must abstain from alcohol at least 72hours. Posture may be a significant factor: cholesterol value may be 10% to 15% hour after 20 min

in a recumbent position. From standing to a sitting position values are about 6% hours after

20mins.

Increases of 2-5% in cholesterol may be seen if tourniquet is applied for 2mins duringsampling

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Cholesterol is the most prominent member of steroid family and is an important componentof many cell membranes

Procure of 3 major classes of steroids: steroid hormones and bile acid High serum (choles) are an important risk factor for atherosclerosis (esp. affecting the

coronary )

Serum

Sources of error: Avoid recent food intake

Ideal conc. TOTAL

4.5mmol/L (>400mg/dL).

Calculation of LDL formula

LDL= TC- (HDL+0.46trigly) mmol/l

=T C- (HDL+0.20trigly) mg/dl

Acquired [Choles]

1. Hyperthyroidism2. the cholestatic liver dx3. renal failure4. nephrotic syndrome

Inherited [Choles]

1. hypercholesterolemia2. common or polygenic hypercholesterolemia3. Hyperlipidaemia

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HDL AND LDL

The 2 principal lipoproteins which contain cholesterol are HDL and LDL. The risk of coronary

disease is associated with increased levels of LDL, while [HDL]s are inversely correlated with

risk,ie are beneficial. LDL is normally present in [high] and thus has the greater effect on total serum

cholesterol.

LDL is a major atherogenic particle and its [plasma] can therefore function as a key for

1. Identifying those with high risk of CHD2. Clinical decision making with respect to cholesterol- lowering therapy.

METHODOLOGY

The methodology for total cholesterol and HDL cholesterol.

1. TOTAL CHOLESTEROL

STD BLK TEST1 TEST 2

SAMPLE/STD 10L - 10L 10L

REAGENT/BLK 1mL 1mL 1mL 1mL

Mix well and incubate for 10mins at RT and read absorbance at 500nm.

Use Beer Lamberts Law to calculate the [test].

HDL CHOLESTEROL

The chylomicrons, VLDL and LDL are precipitated by addition of phosphotungstic acid and

MgCl2. After centrifugation, the supernatant contains HDL fraction.

Test 1 Test 2

Sample/serum 200L 200L

Precipitant 500L 500L

Mix well and incubate for 10mins at RT and centrifuge at High speed for 5mins.

Std. Blk Test 1 Test 2

Supernatant 100L - 100L 100L

Cholesterol rgt. 1mL 1mL 1mL 1mL

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Mix well and incubate at RT for 10mins and read absorbance at 500nm against rgt. blk. Use

Beers law to calculate.

LOW [HDL] can be due to

1. Obesity

2. NIDDM

3. Physical inactivity

4. Oestrogen deficiency

TRIGLYCERIDES

Triglyceride is the principal form in which energy is stored in the body. They are the majorconstituent of adipose tissue

Triglycerides circulate in the plasma as component of various lipoproteins, particularlyVLDL and chylomicron(CM)

VLDLs are made in the liver and are always present in the plasma. CM transport dietary trigfrom the gut and are not normally present in the plasma in fasting state.

SPECIMEN: Serum

Reference range:


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High Triglycerides Low Triglycerides

1. Inherited

a. Familial combined hyperlipidaemia (common)

b. Familial hypertriglyceridemia (uncommon)

2. Acquired

a. Drugs

b. Corticosteroids

c. DM

d. Obesity

e. Excess alcohol ingestion

PRACTICAL XI

FERTILITY HORMONES

It consists of LH, FSH, PRL, PRG, TESTO, ESTROGEN. LH and FSH are anterior-pituitary hormones which regulates male and female gonadal

function.

Measurement of FSH and LH in serum or plasma can be of value in suspected disordersof gonadal function.

SPECIMEN: Serum: For females, the LMP should be taken.

Ref. range: for females depends on the menstrual cycle thus luteal, follicular midcycle,

premenopausal and post menopausal.

SOURCE OF ERROR:

1. In pregnancy

2. In tumor secreting hCG

CAUSES OF ABNORMAL VALUES

High FSH low or normal LH = primary gonadal failure

High FSH = azoospermia, ovulatory failure.

LH > FSH = PCOS( polycystic ovary syndrome).

PROLACTIN

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A hormone secreted by the anterior pituitary which stimulates lactation and inhibitsthe action of gonadotropins.

SPECIMEN: Serum

Ref. range: Female: (1.35 24.59)ng/ml

Male: (1.5 18.75)ng/ml

SOURCES OF ERROR

1. Stress by venipuncture

2. Fasting sample

3. Draw sample between 8.00am to 10.00am.

INCREASED LEVELS

1. Hyperthyroidism

2. PCOS

3. Chronic renal failure (CRF)

4. Adenomas (micro and macro adenomas)

5. Drug treatment eg. methyldopa, oestrogen

PROGESTRONE

PRG is made by the corpus luteum. Its major source in pregnancy is the placenta.

SPECIMEN: Serum

Ref. Range: Depends upon the menstrual cycle.

OESTRADIOL

E2 is a principal female gonadal steroid and its measurements with other gonadal hormones.

It is useful in menstrual disorders.

Ref. Range: Depends on the stage of menstrual cycle.

TESTOSTERONE

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It is the major androgen responsible for sexual differentiation and male secondarysexual characteristics. It is carried in the blood by the sex hormone binding globulin

(SHBG).

SPECIMEN: SerumRef. Range:

PRACTICAL XII

TUMOUR MARKERS

Tumour markers are substances secreted into the body fluids or expressed on cellsurface which are characteristic of the presence of a tumour.

They are of potential value in screening for cancer, for diagnosis, in prognosis, inassessing the response to treatment and in long term follow up.

The most frequently used TM are substances secreted by tumours, and measuredin either in the blood or urine.

PSA

It is a glycoprotein secreted by normal prostate and [Serum] increases withincreasing age and are increased in BPH (Benign Prostatic Hypertrophy)

SPECIMEN: Serum

Ref. Range: 0 4.0ng/mL (male). Female: < 0.5

SOURCES OF ERROR

1. Constipation

2. Rectal examination 48hrs before test.

ASSAY Significance REFERENCE

-HCG

Molar pregnancy,

Choriocarcinoma,

gestational trophoblastic

(0 - 5) mlU/ml

CA 125

Cancer antigen 125Ovarian cancer (0 - 35) U/ml

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CA 15-3

Carbohydrate antigenBreast carcinoma

(0 - 37) U/ml

CA 19-9

Carbohydrate antigen

Hepatocellular

carcinoma(25 30) U/ml

CEA

Carcinoembryonic antigen

Colorectal neoplasm,

rectal(0 2.5) ng/ml

AFP

Hepatoma, teratoma,

neural tube defect(spina

bifide)

(2 16) ng/ml

PSA Prostate cancer (0 4) ng/ml

THYROID FUNCTION TEST (T3, T4, TSH)

Thyroid hormones (thyroxine {T4} and tri-iodothyronine {T3} are extensively protein bound in the

plasma and measurements of total hormone concentrations can give misleading information if the

concentration of binding proteins are abnormal. Measurements of the free ( and physiologically

active) hormones (fT4, fT3, but particularly the former) are now replacing total hormone

measurements.

Measurement of thyroid stimulating hormone (TSH) is valuable since in primary thyroid disease (

which is far commoner than thyroid disease secondary to a pituitary cause), its concentration

inversely reflects thyroid activity.

Reference ranges

TSH 0.3-5.0 mIU/L

Thyroxine (total) 60-150 nmol/LTri-iodothyronine (total) 1.2-2.9 nmol/L

Thyroxine (free) 9.0-26 pmol/L

Tri-iodothyronine (free) 3.0-8.8 pmol/L

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T3 T4 TSH

Hyperthyroidism

T3Thyrotoxicosis

Thyrotoxicosis

Hypothyroidism

PRACTICAL XIII

RADIONIMMUNOASSAY (RIA) AND ELISA

RIA and ELISA are widely used because these tests are extremely sensitive. RIA and ELISA are methods used for the determination of the concentration of thehormones, glycoproteins, peptide hormones and other hormones.

PRINCIPLE

For both the RIA and ELISA tests, the human antigen is put into a plastic test tube or on a plastic

microtitre plate. Some of the antigen will adsorb to the surface of the tube or plate and the excess is

then washed away. The serum to be tested is added to the tube or plate and incubated for a short

time. If antibodies are present, they will bind the Ag.

For the RIA, the excess Ab is washed away and a radioactively labeled Ag that can react with the Ab

is added. The excess radioactive Ag is washed away and the tubes are then counted using a gamma

counter.

The ELISA is done in a similar manner except instead of using a radioactive Ag, the Ag is coupled

to an enzyme such as peroxidase. The Ag binds to the test Ab, excess ligand is washed away, and

chromogen is added. Chromogen is a colorless substrate that produces a colour end point when acted

upon by an enzyme such as peroxidase. The colour change can then be observed and measured using

a microplate reader.

clinical biochemistry assays

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