Introduction to laboratory medicine

1. Introduction to laboratory medicine

2. 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

3. 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.

4. Course contents • Clinical Biochemistry • Cardiac profiles • Liver and renal panels • Bone metabolism • Lipid chemistry • Special chemistry • Cardiovascular markers • Tumour markers • Nutritional markers • Calculi

5. Immunology and Immunodiagnostics • Drug monitoring • Urine and serum proteins • Autoimmune disease testing • Endocrinology tests • Fertility testing • Point-of-Care Testing • Cardiac markers • Glucose monitoring program • Blood gases and metabolites • Routine chemistry panels • Routine urinalysis and pregnancy screening • Coagulation • Complete Blood Counts • Urine toxicology screening

6. Laboratory Hematology • Routine and special hematology • Hemoglobinopathy studies • Special stains • Hematopathology • Bone marrow consultations and interpretive report • Flow cytometry • CD 34 (stem cell) enumeration • CD4/CD8 monitoring • Leukemia/lymphoma immunophenotyping • PNH

7. Special coagulation • Coagulation profiles, screening and factors and inhibitors • Platelet studies • Thrombophilia testing • Microbiology • Bacterial culture and sensitivities • Blood cultures • Molecular typing of organisms • Viral detection methodologies • HIV viral load -public health lab accredited site for viral load • Chlamydia detection

8. 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

9. Recommended Books Textbook: District laboratory practice in tropical countries by Monica Cheesbrough. Clinical chemistry by William J Marshall. Reference books: Medical Laboratory technology by RamnikSood.

10. Reasons for ordering tests • Aid in diagnosis • Confirm diagnosis • Evaluate prognosis • Monitor therapy • Screen for a disease

11. 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 

12. Blood bank Very 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

13. 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

14. 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.

15. To understand applied research on pathology related problems. • To arrange for the training of the medical and paramedical staff.

16. Nature of Request STAT • Performed immediately 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

17. 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

18. CRITICAL VALUES • Needs immediate attention • “panic values” • Should call physician • Patient is at risk

19. Reference Values Not fixed for all Should consider: • Age • Sex • Pregnancy • Diurnal Variation • Race • Blood type

20. Routine Examinations ROUTINE ADMISSION TESTS CBC, Urinalysis, Fecalysis ROUTINE CHEMISTRIES BUN, Creatinine, Glucose, Uric Acid, Cholesterol Sometimes triglycerides

21. 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

22. 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.

23. 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

24. Blood specimens • Serum • Plasma Urine specimen • Preservative may be added to prevent bacterial growth or acid may be added to stabilize metabolites. Other specimen types Dangerous specimen • Labelled as “dangerous specimen” yellow sticker. • Similar label should be attached on the request form. • HBV and HIV

25. Sampling errors • Blood sampling techniques • Prolonged stasis during venepuncture • Insufficient specimen • Errors in timing • Incorrect specimen container • In appropriate sampling site • Incorrect sample storage.

26. 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)

27. Lipoproteins compositions

28. Composition of lipoproteins

29. 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.

30. Lipoprotein metabolism

32. 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.

33. 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

34. 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)

35. 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.

36. 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

37. 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

38. ECG changes

39. CARDIAC PROFILE TEST • ENZYMES • CreatinineKinase –MB(CK-MB) • Lactate Dehydrogenase(LDH 1 and 2) • AspartateAminotransferase(AST)/Serum Glutamate OxaloacetateTransaminase(SGOT) • AlanineAminotransferase(ALT)/ Serum PyruvateTransaminase(SGPT) • LIPID PROFILE • CHOLESTEROL • TRIGLYCERIDE • HDL • LDL

40. Proteins • Myoglobin • Troponins

41. Cardiac profile Cardiac 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: • Aspartateaminotransferase (AST) • Lactate dehydrogenase (LD) • CreatineKinase (CK)

42. 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

43. 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

44. Increased levels of AST • Drug hepatoxicity • Pulmonary infarction • Pericarditis • Acute hepatitis • Skeletal muscle disorders

45. Decreased levels of AST • Pregnant women Falsely elevated results • Bilirubin • Aceto-acetatae • N-acetyl compounds • P-aminophenol • Sulfathiozole • Isoniazid • Methyldopa • L-dopa • Ascorbic acid

46. 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

47. 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

48. 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

49. 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

50. Increased levels of LD • Megaloblastic anemia • Pulmonary infarction • Granulocyte leukemia • Hodgekin’s disease • Hemolytic anemia • Infectious mononucleosis • Progressive muscular dystrophy (PMD)