INTRODUCTION TO LABORATORY MEDICINE

DEFINITION Laboratory medicine a specialty in which

pathologists provide testing of patient samples (usually blood or urine) in several different areas.

Determination of the level of enzymes in blood in case of heart attack or

Level of glucose (sugar) in the blood of a patient with diabetes.

The presence of bacteria and other microorganisms.

Blood cells studies for various types of anemias

COURSE OBJECTIVES application of basic science to those clinical

disciplines practiced by the medical laboratory scientist.

the scope of Laboratory Medicine, and of its potential applications.

How to analyze various samples under certain circumstances.

COURSE CONTENTS Clinical Biochemistry

Cardiac profilesLiver and renal panelsBone metabolismLipid chemistry

Special chemistryCardiovascular markersTumour markersNutritional markersCalculi

Immunology and ImmunodiagnosticsDrug monitoringUrine and serum proteinsAutoimmune disease testingEndocrinology testsFertility testing

Point-of-Care TestingCardiac markersGlucose monitoring programBlood gases and metabolitesRoutine chemistry panelsRoutine urinalysis and pregnancy screeningCoagulationComplete Blood Counts Urine toxicology screening

Laboratory Hematology Routine and special hematology

Hemoglobinopathy studiesSpecial stains

HematopathologyBone marrow consultations and

interpretive report Flow cytometry

CD 34 (stem cell) enumerationCD4/CD8 monitoringLeukemia/lymphoma immunophenotypingPNH

Special coagulationCoagulation profiles, screening and factors

and inhibitorsPlatelet studiesThrombophilia testing

MicrobiologyBacterial culture and sensitivitiesBlood culturesMolecular typing of organismsViral detection methodologiesHIV viral load -public health lab accredited

site for viral loadChlamydia detection

Infection control Reference centre for medical microbiology and infectious

diseases Detection and typing of epidemiologically significant

organisms Serology

Clostridium difficile toxin testing Wide range of viral and non-viral serologies

Molecular Diagnostic Testing Wide range of molecular testing for viral and bacterial

agents Mycology Fungus detection Cells/tissues/organ donor testing

Blood Bank/Donor Center Concepts of immunohematology and histocompatibility Blood transfusion services and quality assurance Blood donation and storage of blood Blood grouping Compatibility testing

RECOMMENDED BOOKS

Textbook:

District laboratory practice in tropical countries by Monica Cheesbrough.

Clinical chemistry by William J Marshall.

Reference books:

Medical Laboratory technology by Ramnik Sood.

 

REASONS FOR ORDERING TESTS

Aid in diagnosis

Confirm diagnosis

Evaluate prognosis

Monitor therapy

Screen for a disease

SECTIONS OF THE LABORATORY CLINICAL PATHOLOGY

1. Clinical Chemistry  BUN  Cholesterol  FBS

2. Clinical Microscopy  Analysis of body fluids Urin analysis Fecal anaysis Semen analysis

3. Microbiology  Cultures (sputum,blood, urine)

4. Hematology Biggest section Includes CBC,coagulation,

PT, PTT 

BLOOD BANKVery critical section Bec. May have errors Blood typing Cross match AB Identification Goes hand in hand with

serology and immunology Tests done for MALARIA SYPHILIS HIV

Serology/Immunology  Cardiac and thyroid fxntest

II. ANATOMY PATHOLOGY

Histopathology Submission of tissues for

tests

LABORATORY SERVICES Laboratory investigations of the patient. Laboratory aspects of detection and

prevention of diseases. Request System of analysis System for interpretation of results and

timely advice relevant to the urgency of the clinical problem

FUNCTIONS OF A HOSPITAL LABORATORY To meet the request for laboratory

investigations by maintaining adequate diagnostic facilities.

To arrange for laboratory investigations from referral laboratories if not available in the premises.

To provide professional advice on the management of the patient.

To monitor individual patients. To provide laboratory facilities for research

projects under taken by clinicians. To collaborate in development, study and

control of new methods of treatment.

To understand applied research on pathology related problems.

To arrange for the training of the medical and paramedical staff.

NATURE OF REQUESTSTAT  Performed immedi

ately and by itself.  Run control and

standard  20-50% More

expensive  TAT is shortened  Request is needed

Today  Confusing  Performed as

soon as possible, given priority

 Based on “running time”

Routine Done with the batch Wait for TAT stated

by laboratory

VALUES

REFERENCE VALUES Better term than

“normal value” Pulled value, usually

95%of population Vary in diff. hospitals

but not that far

SIGNIFICANT VALUES Clinical decision

should be made if higher or lower than reference value

Usually when 2x to 3x

CRITICAL VALUES

Needs immediate attention “panic values” Should call physician Patient is at risk

REFERENCE VALUESNot fixed for allShould consider:

Age  Sex  Pregnancy  Diurnal Variation  Race  Blood type

ROUTINE EXAMINATIONSROUTINE ADMISSION TESTS 

CBC, Urinalysis, FecalysisROUTINE CHEMISTRIES 

BUN, Creatinine, Glucose, Uric Acid, Cholesterol

  Sometimes triglycerides

BASIC LAB EQUIPMENTS The Light Microscope. Colorimeters and photometers Water bath Laboratory centrifuge Balance Cold incubators refrigerators pH meters Mixers Ovens De-ionizers Safety cabinets. Glassware and plasticware

SAMPLING Pathologist should try to answer the question

which is imposed by the clinician. Correct specimen for requested test with

necessary information so that right test is carried out And result is delivered to the requesting clinician with the minimum of delay.

Patient identification must be correct.

SPECIMEN TYPES Venous blood serum or plasma. Arterial blood. Capillary blood Urine Feces Cerebrospinal fluid Sputum and sliva Tissue and cells Aspirates (pleural fluid, ascites, joint fluid,

intestinal (duodenal) fluid, pancreatic pseudocysts.

Calculi

BLOOD SPECIMENS Serum Plasma

Urine specimen Preservative may be added to prevent bacterial

growth or acid may be added to stabilize metabolites.

Other specimen typesDangerous specimen Labelled as “dangerous specimen” yellow sticker. Similar label should be attached on the request

form. HBV and HIV

SAMPLING ERRORS Blood sampling techniques Prolonged stasis during venepuncture Insufficient specimen Errors in timing Incorrect specimen container In appropriate sampling site Incorrect sample storage.

LIPID CHEMISTRY AND CARDIOVASCULAR PROFILE Main lipids in the blood are the triglycerides

and cholesterol.(phospholipids, FFA) These are insoluble in the water. Transport in the blood is via lipoproteins.

(protein) 4 major classes of lipoproteins.

Chylomicrons Very low density lipoproteins (VLDL) Low density lipoproteins (LDL) High density lipoproteins (HDL)

LIPOPROTEINS COMPOSITIONS

COMPOSITION OF LIPOPROTEINS

Class Diameter (nm)

 % protein

 % cholesterol

 % phospholipid

 % triacylglycerol& cholesterol ester

HDL 5–15 33 30 29 4LDL 18–28 25 50 21 8IDL 25–50 18 29 22 31VLDL 30–80 10 22 18 50Chylomicrons 100-1000 <2 8 7 84

LIPOPROTEINS Chylomicrons carry triglycerides ( dietary fat) from

the intestines to the liver, to skeletal muscle, and to adipose tissue.

Very-low-density lipoproteins (VLDL) carry (newly synthesised or endogenous) triglycerides from the liver to adipose tissue and metabolized to LDL through IDL.

Intermediate-density lipoproteins (IDL) are intermediate between VLDL and LDL. They are not usually detectable in the blood.

Low-density lipoproteins (LDL) carry cholesterol from the liver to cells of the body. LDLs are sometimes referred to as the "bad cholesterol" lipoprotein.

High-density lipoproteins (HDL) collect cholesterol from the body's tissues, and take it back to the liver. HDLs are sometimes referred to as the "good cholesterol" lipoprotein.

LIPOPROTEIN METABOLISM

60% of plasma cholesterol is present in LDL, 25% in HDL and small quantity in VLDL.

Lipoprotein metabolism is controlled by their protein component apolipoproteins.

Apo A-1 in HDL and Apo B-100 in LDL are very important ones.

Lipoprotein (a) in also present in human plasma. It is synthesized in the liver.

Smaller but denser than LDL. Cholesterol esters are major lipids and it is

an independent risk factor for IHD.

LDL and VLDL are associated with premature atherosclerosis.

HDL high levels are negative risk factors for IHD.

HYPERLIPIDEMIA Coronary heart disease Acute pancreatitis Failure to thrive and weakness Cataract

Endothelial dysfunction Lpid accumulation. Migration of inflammatory cells into the

arterial wall.

Atherosclerosis and plaque formation Plaque stability SCAD (asymptomatic)

Chest pain at rest(angina, non ST elevation MI, STEMI)

PATHOPHYSIOLOGY Atherosclerotic plaque, rupture and thrombus

formation. Obstruction of coronary circulation. Necrosis of the heart tissue. Irreversible cardiac injury if occlusion is

complete for 15-20 mins. Starts from endocardium and spreads

towards epicardium. If full thickness of myocardium is involved

then it is transmural infarct.

PRECIPITATING FACTORS Physical exertion After surgical operation Early in the morning (adrenergic activity,

fibrinogen level, platelet adhesiveness is increased).

In the winter months Emotional stress

DIAGNOSIS OF MI Detection of rise and fall of cardiac biomarker

troponinT/I with one of the following: Symptoms of ischemia ECG changes Q wave

ECG CHANGES

CARDIAC PROFILE TEST

ENZYMES Creatinine Kinase –MB(CK-MB) Lactate Dehydrogenase(LDH 1 and 2) Aspartate Aminotransferase(AST)/Serum

Glutamate Oxaloacetate Transaminase(SGOT) Alanine Aminotransferase(ALT)/ Serum Pyruvate

Transaminase(SGPT)

LIPID PROFILE CHOLESTEROL TRIGLYCERIDE HDL LDL

PROTEINS Myoglobin Troponins

CARDIAC PROFILECardiac Enzymes

Cardiac Profile assesses the function of the heart’s muscle and the increased level of enzymes following a myocardial infarction. The cardiac enzymes include the following: Aspartate aminotransferase (AST) Lactate dehydrogenase (LD) Creatine Kinase (CK)

ASPARTATE AMINOTRANSFERASE (AST) (SGOT)

found in all tissue, especially the heart, liver, and skeletal muscles it catalyzes the transfer of the amino group of aspartic acid to alpha-

ketoglutaric acid to form oxaloacetic acid and glutamic acid Reaction catalyzed:Amino group Alpha-keto group Oxaloacetate &In aspartic acid In alpha-ketoglutaric acid

glutamate   Considerations in AST assays -Serum is the best specimen-Hemolyzed samples must be avoided-Alcohol lowers AST values-Muscle trauma like intramuscular injections, exercise, or surgical

operation can significantly increase AST levels

CLINICAL SIGNIFICANCE

Myocardial infarction In myocardial infarction, AST levels are usually

4-10 times the upper limit of normal These develop within 4-6 hours after the onset

of pain Peak on the 24th – 36th hour Usually normalize on the 4th or 5th day

Muscular dystrophy Hepatocellular disorders Skeletal muscle disorders Acute pancreatitis

INCREASED LEVELS OF AST Drug hepatoxicity Pulmonary infarction Pericarditis Acute hepatitis Skeletal muscle disorders

DECREASED LEVELS OF AST Pregnant women Falsely elevated results Bilirubin Aceto-acetatae N-acetyl compounds P-aminophenol Sulfathiozole Isoniazid Methyldopa L-dopa Ascorbic acid

LACTATE DEHYDROGENASE (LDH)

Catalyzes the reversible oxidation of lactate to pyruvate

Used to indicate AMI Is a cytoplasmic enzyme found in most cells

of the body, including the heart Not specific for the diagnosis of cardiac

disease

DISTRIBUTION OF LD ISOENZYMES

LD1 and LD2 (HHHH, HHHM) Fast moving fractions and are heat-stable Found mostly in the myocardium and erythrocytes Also found in the renal cortex

LD3 (HHMM) Found in a number of tissues, predominantly in the white

blood cells and brain

LD4 and LD5 (HMMM, MMMM) Slow moving and are heat labile Found mostly in the liver and skeletal muscle

CONSIDERATIONS IN LD ASSAYS

Red cells contain 150 times more LDH than serum, therefore hemolysis must be avoided

LDH has its poorest stability at 0°C

Clinical Significance In myocardial infarction, LD increases 3-12

hours after the onset of pain Peaks at 48-60 hours and remain elevated for

10-14 days In MI, LD1 is higher than LD2, thus called

“flipped” LD pattern

FLIPPED LDH

An inversion of the ratio of LD isoenzymes LD1 and LD2; LD1 is a tetramer of 4 H–heart subunits, and is the predominant cardiac LD isoenzyme;

Normally the LD1 peak is less than that of the LD2, a ratio that is inverted–flipped in 80% of MIs within the first 48 hrs DiffDx. LD flips also occur in renal infarcts, hemolysis, hypothyroidism, and gastric CA

INCREASED LEVELS OF LD Megaloblastic anemia Pulmonary infarction Granulocyte leukemia Hodgekin’s disease Hemolytic anemia Infectious mononucleosis Progressive muscular dystrophy (PMD) 

CREATINE KINASE (CK)

Is a cytosolic enzyme involved in the transfer of energy in muscle metabolism

Catalyzes the reversible phosphorylation of creatine by ATP

-Is a dimer comprised of two subunits, resulting in three CK isoenzymes The B, or brain form The M, or muscle form

Three isoenzymes isolated after electrophoresis:

CK-BB (CK1) isoenzyme

Is of brain origin aand only found in the blood if the blood-brain barrier has been breached

CK-MM (CK3) isoenzyme Accounts for most of the CK activity in skeletal muscle

CK-MB (CK2) isoenzyme Has the most specificity for cardiac muscle It accounts for only 3-20% of total CK activity in the heart Is a valuable tool for the diagnosis of AMI because of its

relatively high specificity for cardiac injury Established as the benchmark and gold standard for

other cardiac markers

Considerations in CK assays

CK is light sensitive and anticoagulants like

oxalates and fluorides inhibit its action CK in serum is very unstable and rapidly loss

during storage Stored specimens are not used since it

contains cellular products and intermediate like adenylate kinase, ATP and G-6-Phosphate which affect the assay

Exercise and intramuscular injections causes CK elevations

Clinical Significance -In myocardial infarction, CK will rise 4-6

hours after the onset of pain -Peaks at 18-30 hours and returns to normal

on the third day -CK is the most specific indicator for

myocardial infarction (MI)

Raised levels of CK Progressive muscular dystrophy Polymyositis Acute psychosis Alcoholic myopathy Delirium tremens Hypothyroidism Malignant hyperthermia Acute cerebrovascular disease Trichinosis and dermatomyositis

Normal Value: a. Male – 25-90 IU/mL b. Female – 10-70 IU/mL

CHOLESTEROL

Normal values: range varies according to age Total Cholesterol: 150-250mg% Cholesterol esters: 60-75% of the total

cholesterol

CHOLESTEROL IS ADVISED IF YOU have been diagnosed with coronary heart disease, stroke or mini-stroke (TIA) or peripheral arterial disease (PAD) are over 40 have a family history of early cardiovascular disease have a close family member with cholesterol-related condition are overweight have high blood pressure, diabetes or a health condition that can increase cholesterol levels, such as an underactive thyroid

FACTORS LEADING TO RAISED CHOLESTEROL an unhealthy diet: some foods already contain

cholesterol (known as dietary cholesterol) but it is the amount of saturated fat in your diet which is more important

smoking: a chemical found in cigarettes called acrolein stops HDL from transporting LDL to the liver, leading to narrowing of the arteries (atherosclerosis)

having diabetes or high blood pressure(hypertension) having a family history of stroke or heart disease There is also an inherited condition known as familial

hypercholesterolaemia (FH). This can cause high cholesterol even in someone who eats healthy diet.

TRIGLYCERIDES Ester  derived from glycerol and three fatty acids. Main lipids in the blood and important energy substrate. Insoluble in water. Hypertriglyceridemia

Not an important risk facotr for coronary artery disease. It can cause pancreatitis when severe.

Both hypertriglyceridemia and hypercholesterolemia are associated with various types of cutaneous fat deposition and xanthomatas.

Hypertension Very common clinical problem. Usually essential type

meaning that have no identifiable cause. Investigations for treatable causes like endocrine is

necessary.

HYPERLIPIDEMIAS

LIVER

Anatomy of liver

LIVER HISTOLOGY

I. TESTS BASED ON EXCRETORY FUNCTIONS

LABORATORY RESULTS

II. TESTS DUE TO DETOXIFICATION

TESTS B/O SYNTHETIC FUNCTION

Liver is the main source of synthesis of Plasma proteins

Albumin Globulin

Blood clotting factors Prothrombin Factors V, VII, and X

SERUM ALBUMIN 3.5- 5.5 gm/dl

SERUM GLOBULIN 2 -3.5 gm/dl

TOTAL PROTEINS 6-8 gm/dl

Albumin/ Globulin ratio 1.2:1 – 2.5: 1

Prothrombin time

TESTS B/O METABOLIC FUNCTIONS

SERUM TRANSAMINASES SERUM ALKALINE PHOSPHATASES

REFERENCE RANGE ALT ( upto 42 U/L) AST (0-37 U/L) ALP (65-306 U/L) raised in obstructive

jaundice.

OTHER ENZYMES GGT (11-60 u/l) 5- NUCLEOTIDASE (2-17u/L) LDH (180-360 u/l)

pathogenesisDifferent cells have different enzymes inside them,

depending on the function of the cell. Liver cells happen to have lots of AST, ALT, and GGTP inside them. When cells die or are damaged, the enzymes leak out causing the blood level of these enzymes to rise; that is why the levels of these enzymes in the blood are considered good indicators of liver cell damage. ALT is more specific for liver disease than AST because AST is found in more types of cell (e.g. heart, intestine, muscle). The AST, for instance, will rise after a heart attack or bruised kidney. GGTP and AP are said to be more specific for evaluating biliary disease since they are made in bile duct cells. In liver disease caused by excess alcohol ingestion, the AST tends to exceed the ALT, while the reverse is true to for viral hepatitis. However, this particular generalization is often wrong. There are several things to remember:

GGT (OR GGTP)  Gamma Glutamyl Transpeptidase. This

enzyme level is elevated in case of liver disorders.   In contrast to the alkaline phosphatase, the GGT tends not to be elevated in diseases of bone, placenta, or intestine

PROTHROMBIN TIME

good correlation between abnormalities in prothrombin time and the degree of liver dysfunction.

Expressed in seconds and compared to a normal control patient's blood

SPECIALIZED TESTS serum iron, the percent of iron saturated in blood, the storage protein ferritin for hemochromatosis. accumulation of copper in the liver in wilson

disease.

RENAL PANEL

Creatinine Calcium Sodium Chloride Carbon dioxide Albumin Blood urea nitrogen (BUN) Protein Phosphorus Glucose Potassium

Glucose Potassium Phosphorous Sodium Albumin BUN Creatinine BUN/Creatinine Ratio Calcium Chloride Carbon Dioxide (CO2), Total

BONE METABOLISM Bone is constantly remodelling Bone resorption= bone formation Why remodelling is necessary?

To withstand changing environment To cope with workload To repair damage caused by recurrent

microtraumasBONE METABOLISMOsteoclasts and OsteoblastsOsteocytes\Encased osteoblasts which are connected to each

other by long cellular processes forming a network connected by gap junctions.