1-Intro to Chemical Pathology-1.pptx

Fgh

1. INTRODUCTION TO CHEMICAL PATHOLOGY DR. S. A. SAKYI Dept. of Molecular Medicine SMS, KNUST

2. OUTLINE Introduction to chemical pathology Specimen collection & handling Side room testing/point of care testing/ near patient testing

3. OBJECTIVES Describe the biochemical and pathophysiological mechanisms of diseases and the biochemical principles underlying their treatment. Select appropriate laboratory test and interpret the results to confirm or refute a provisional clinical diagnosis and to monitor progress during treatment. To understand the potentials and limitations of various laboratory tests. Collect the right type of specimens for laboratory investigations under the right conditions.

4. CHEMICAL PATHOLOGY Clinical Biochemistry Clinical Chemistry M edical Biochemistry Pure Blood Chemistry Physiological Chemistry

5. Chemical Pathology The systematic study of biochemical processes associated with health & disease & the measurement of constituents in body fluids or tissues to facilitate diagnosis of disease.

6. CHEMICAL PATHOLOGY Chemical Pathology is the study of the biochemical basis of disease, and the application of biochemical and molecular techniques in diagnosis Constant changes in the chemical constitution & biochemical mechanisms of the body as a result of disease. Chemical pathology is a sub-specialty within pathology which extends across most medical specialties and involves the chemical analysis of bodily fluids

7. SCOPE OF CLINICAL CHEMISTRY Biochemistry Instrumentation Computers Pharmacology Endocrinology Analytical Chemistry Toxicology Immunology CLINICAL CHEMISTRY

8. Add anticoagulants (heparin, potassium oxalate)

9. Separation of Components Plasma = Less Dense RBCs More Dense Platelets / WBCs

10. Plasma vs. serum Plasma is the liquid, cell-free part of blood, that has been treated with anti-coagulants . Anticoagulated Serum is the liquid part of blood AFTER coagulation , therfore devoid of clotting factors as fibrinogen. serum= plasma - fibrinogen Clotted

11. Components of Plasma Blood plasma Consists of: Water 90% Plasma Proteins 6-8 % Electrolytes ( Na + & Cl - ) 1% Other components: Nutrients (e.g. Glucose and amino acids) Hormones ( e.g. Cortisol, thyroxine) Wastes (e.g. Urea) Blood gases (e.g. CO 2 , O 2 )

12. CHEMICAL PATHOLOGY Primarily involves the chemical analysis of the following bodily fluids: whole blood serum or plasma urine cerebrospinal fluid Faecal material effusions seminal fluid sweat and amniotic fluid to assist in the diagnosis of various disease

13. ROLE OF CHEM. PATHOLOGY IN HEALTHCARE Diagnosis : used to help differentiate between several possibilities based on the initial history and examination Monitoring : to check disease progression or response to therapy e.g. monitoring DM patients

14. ROLE OF CHEM PATH IN HEALTHCARE Screening : to screen for the presence of disease in an apparently healthy population or detection of disease before it is clinically evident Prognosis : providing information on disease susceptibility e.g . cholesterol can predict the risk of coronary artery disease. Serves as tools to assist clinicians in the diagnosis of various disorders, as well as management and follow-up of patients

15. WHY CHEM PATH? Providing a consultation service to clinicians to advise on the most appropriate testing within specific clinical situations Interpretation of a wide variety of clinical laboratory tests Advice regarding the limitations of laboratory tests in specific circumstances Advice on the influence of "pre-analytical" factors, medications and other factors on laboratory tests which may influence clinical decision making

16. WHY CHEM PATH Ensuring the quality of laboratory testing through internal and external quality assurance Introduction of new tests, since medicine and clinical testing is a rapidly evolving science and new tests are developed continuously Training of medical students in the appropriate selection of clinical laboratory testing and the most cost effective and appropriate use of the clinical laboratory

17. Selecting the right test, at the right time, for the right patient. After making the decision that an investigation is necessary, and selecting the most appropriate test, Consideration to factors present that may affect the interpretation of results, or even the decision to proceed with the test at that time

18. Analyses performed within the Chemical Pathology laboratory include: Water and electrolyte balance and kidney function Acid base balance Liver function Minerals such as calcium, magnesium and phosphate Proteins and enzymes Lipids such as cholesterol and triglycerides and risk factors for heart disease Diagnosis of diabetes mellitus and it's complications Cardiac markers for detection of cardiac damage Iron and porphyrins Endocrinology, including: Pituitary function Sex hormones Thyroid function Adrenal hormones

19. Analyses performed within the Chemical Pathology laboratory include : Tumour markers such as Prostate specific antigen (PSA) and various other markers used in the detection and management of cancer patients Inherited metabolic diseases Therapeutic Drug Monitoring (TDM) Drugs of abuse testing Allergy testing Specialised testing including: Occupational Health testing (monitoring of exposed workers) Insurance testing Environmental testing (water analysis, etc.

20. VACUTAINER/EVACUATED TUBES These are tubes for blood collection which are color-coded based on the anticoagulant present . They come in various sizes; 2, 5, 7, and 10 ml. Blood is drawn in this order: Blood culture tubes , red top , blue top , green top , lavender top and gray top

21. COLOR ADDITIVE ACTION USE Lavender Ethylenediaminetetraacetic chelates calcium hematologic assays Acid (EDTA) lead assay, CEA * Versene (disodium salt) Determination and * Sequestrene (dipotassium salt) cell counts Red None Allows blood to clot Most chemistry, immunologic and blood bank tests Red Gray or None but contains Allows blood to clot Most chemistry tests Red Black separator material and serves as a barrier between cells and serum Yellow Citrate dextrose preserves RBCs Blood culture Green Heparin (Na+, Li+, inhibits thrombin ammonia CO- Hb or, NH 4 + ) activation and methemoglobin

22. COLOR ADDITIVE ACTION USE Orange Thrombin accelarated clot STAT serum tests Blue Buffered citrate binds calcium Coagulation assays like PT & APTT Black Buffered sodium binds calcium Westergren ESR citrate Gray NaF /K 2 C 2 O 4 Inhibits glycolytic Glucose enzyme enolase and determination act as anticoagulant Iodoacetate inhibits glycolytic Glucose enzyme glyceraldehyde determination 3-phosphate dehydrogenase

23. Anticoagulant Interference Dilution errors especially oxalates which are highly osmotic Inhibition of plasma enzyme activities activities especially with fluoride which is an enzyme poison, EDTA which chelates metallic enzymes activators. Oxalate inhibits AMS, LD and ACP, and citrate which inhibits AMS Oxalates, citrate and EDTA lower plasma calcium levels False increase in electrolyte analyses due to the anticoagulants in the salt form

24.  If multiple tubes are needed, the proper order of draw to avoid cross contamination and erroneous results is as follows:  1st -   Blood culture vials or bottles, sterile ( yellow or yellow - black top) 2nd - Coagulation tube ( light blue top) NOTE: If just a routine coagulation assay is the only test ordered, then a single light blue top tube may be drawn. If there is a concern regarding contamination by tissue fluids or thromboplastins, then one may draw a non-additive tube first, and then the light blue top tube. 3rd - Non-additive tube or Serum tube Last draw - additive tubes in this order: 1. SST ( red - gray or gold top). Contains a gel separator and clot activator. 2. Sodium heparin ( dark green top) 3. PST ( light green top). Contains lithium heparin anticoagulant and a gel separator. 4. EDTA ( lavender top) 5. ACDA or ACDB ( pale yellow top). Contains acid citrate dextrose. 6. Oxalate/fluoride ( light gray top)

25. Specimen Handling and Processing Serum 20-30 minutes is the ideal clotting time Generally more preferred than plasma Interfering substances are co-precipitated during clotting such as LPL(lipoprotein lipase) Optically clearer Free from anticoagulant interference Must ideally reach the laboratory within 45 minutes Agitation must be avoided during transport Use amber containers for photolabile substances Transport in ice (4 deg C) those specimens for BGA, renin, enzymes and catecholamines

26. Specimen Interference Lysis of cells or Laking ( Hemolyzed serum) Results in leakage of intracellular substances Lysis of RBCs is called laking or hemolysis which may occur in vivo or in vitro In vitro hemolysis is more common which may be due to: Use of vacuum tubes Vigorous mixing Use of too narrow or too wide needle bores Effect of alcohol Centrifugation and separation steps Hemolysis is visible only not until a 200 mg/L of hemoglobin level in present

27. Icteresia (Icteric serum) Intensely yellow serum sample due to elevated bilirubin value Jaundice in a patient is caused by a bilirubin level of greater than 430 µM (25 mg/L) Bilirubin interferes with tests using dyes and turbidity tests Interference due to bilirubin may be minimized by sample blanking or dual wavelength method known as the Allen correction method

28. Lactescence ( Lipemic serum) Obtained normally after a meal due to elevated chylomicrons Characterized by milky or highly turbid serum Lactescence appears when the TAG level reaches 4.6 mM (4g/L) Errors due to lipemia may be corrected by ultracentrifugation of the serum sample

29. Grounds for rejecting a specimen Inadequate sample identification Insufficient volume of specimen collection Inappropriate collection tube Hemolysis Improper transportation Interferences

30. Analytical process

31. QUALITY MANAGEMENT Quality Assurance (QA) includes maneuvers encountered in the analytic , pre-analytic and post-analytic phases of laboratory testing Pre-analytical phase includes: test ordering specimen collection transport of the specimen in the laboratory

32. Analytical phase includes specimen analysis (manual or automated) use of commercial controls record keeping Post-analytical phase includes reporting out results of analysis (manual or computerized) physician contact

33. Specimens or samples are analyzed while substances in them are measured or quantitated.

34. Substances measured in serum fall generally into the following categories: Substances normally present with a function in the circulation electrolytes TAG, cholesterol hormones vitamins glucose TP albumin individual proteins 2. Metabolites- nonfunctioning waste products in the process of being cleared urea creatinine uric acid ammonia bilirubin

35. 3. Substances released from cells as a result of cell damage & abnormal permeability or abnormal cellular proliferation enzymes such as LD, ALT, AST, CK, AMS, GGT, ALP & ACP ferritin 4. Drugs & toxic substances antibodies substances of abuse therapeutic drugs poisons

36. FACTORS TO CONSIDER PRIOR TO SPECIMEN COLLECTION Patient’s Diet : Ca , OGTT . Patient’s c urrent medication : Oral Contraceptives, Cough Mixtures Time of day: Iron & Corticosteroids

37. FACTORS TO CONSIDER AT THE TIME OF COLLECTING THE LAB SPECIMEN . Patient posture :proteins and protein-bound constituents change with posture e.g. Albumin, calcium, cholesterol, cortisol and protein bound iodine. Venostasis : raise the conc. of plasma proteins, haemoglobin , hormones, calcium & lipids . Remove the tourniquet soon after puncturing the vein Site of venipuncture : eg . site of infusion-fluid is likely not have mixed with the entire blood vol .

38. FACTORS TO CONSIDER AT THE TIME OF COLLECTING THE LAB SPECIMEN. Haemolysis : release of erthrocytic content eg . K+, Lactate dehydrogenase, acid Phosphatase. The plunger of the syringe should not be drawn too fast T here should be an easy flow of blood.

39. FACTORS TO CONSIDER AT THE TIME OF COLLECTING THE LAB SPECIMEN . Identification of specimen - Patient’s name Location/ward Identifying number Date T ime of specimen collection Suspected pathology HIS LIS PID PTS

40. FACTORS TO CONSIDER AT THE TIME OF COLLECTING THE LAB SPECIMEN Specimen container: Tubes into which the blood is expelled must be clean . FBS- Flouride oxalate tubes Anticoagulant in the tube should be mixed with the blood by gentle rotation

41. PRESERVATION OF SPECIMEN IN TRANSIT Blood Gas Analysis :Pco 2 , Po 2 sample must be kept at 4 o C from the time sample is drawn till serum or plasma is separated from cells. Transfer of specimen to the lab must be done by placing the specimen in a container of ice.

42. PRESERVATION OF SPECIMEN IN TRANSIT Specimen for hormonal assays e.g. gastrin , rennin and parathyroid hormone must be separated from the cells in a refrigerated centrifuge. Specimen for bilirubin and carotene must be protected from both sunlight and fluorescent light to avoid photo degradation.

43. CHANGES THAT OCCUR IN THE BLOOD AFTER COLLECTION D iffusion of K + and Lactate Dehydrogenase (LDH) & Aspartate Transaminase (AST) through the red cell membrane into the serum/plasma. Decrease in conc. of glucose by erythrocytic glycolysis .- prevented by the use of fluoride oxalate tubes. Loss of activity of liable enzymes such as Prostatic acid phosphatase- protease inhibitors . Photo degradation of bilirubin & B-carotene by light. -prevented by keeping the sample in aluminium foil .

44. FACTORS THAT INFLUENCE LABORATORY TESTING Increased Decreased Exercise Immediate effects: Alanine and lactate FFA Long-term effects CK, LD, AST, platelets, testosterone, androstenedione and LH Fasting Most analytes ( 8-12 hrs for common bilirubin, FFA, TAG Glucose metabolites and 12-14 hrs for lipid profile ) EFFECT ON LABORATORY RESULTS

45. Increased Decreased After eating K, TAG (chylomicrons), ALP intestinal isoenzyme, turbidity of serum and plasma Diet High meat NPNs (esp. urea, ammonia, and urate except creatinine) High ratio of unsaturated Serum to saturated fat cholesterol Purine-rich Urates High caffeine FFA, catecholamine release Bananas, pineapple, Serotonin in blood tomatoes and 5’HIAA in urine avocados Alcohol ingestion Immediate Lactate, urate, ketone bodies, and FFA Chronic abuse HDL-cholesterol, GGT, and MCV

46. Increased Decreased Tobacco smoking Acute effects Catecholamines , cortisol Eosinophil neutrophils, monocytes and FFA Chronic effects CO- Hb , MCV and leukocyte count Prolonged tourniquet application Serum enzymes, protein, protein-bound substances, cholesterol, TAG, Ca, blood cell concentration ( hemoconcentration )** Postural changes same as ** due to hydrostatic efflux of water Stress lactate, FFA and alteration i n electrolyte levels

47. Summary of Pre-analytical variables age, gender, race, and pregnancy diet, starvation, and physical activity caffeine, cigarettes, and alcohol timing of sampling diagnostic and therapeutic measures posture and tourniquet site of sampling anticoagulants transportation of samples storage, processing, centrifugation, and distribution effects of lipemia, hemolysis, and hyperbilirubinemia

48. Point-of-care Testing (POCT) Also known as near-patient testing , alternate-site testing or patient-focused testing Used in emergency dept., operating suites, clinics, health maintenance organization (HMO), physicians, offices & nursing homes Addresses acute patient needs Instrumentation includes portable chemistry analyzers, glucometers, BG Analyzers, hemoglobin meters & coagulation testing

49. SIDE ROOM TESTING/POINT OF CARE TESTING/ NEAR PATIENT TESTING Refers tests performed outside the hospital laboratory by untrained healthcare professionals or non-laboratory personnel. Bedside Clinic By patients themselves at home

50. CATEGORIZATION Simple side-room tests: qualitative or semi- quantitative : mostly performed on urine or. eg . urine dipsticks tests Simple side-room test, semi-quantitative or quantitative: mostly performed on blood specimens . e.g. Glucometers Quantitative tests performed with equipment that have microprocessor-controlled operations. can be quickly and reliably operated after little instructions.

51. ADVANTAGES OF SRT/NPT Turn Around Time- Relatively short analysis time. Early treatment and shorten the patient wait . Ease of use– can be perform by less trained personnel or by the patients themselves Prompter stabilization of life-threatening crises ( eg drug overdose)

52. ADVANTAGES OF SRT/NPT Closer therapeutic management (e.g. diabetes) Better patient compliance with therapy (diabetes, hyperlipidaemia ) Reduce : – Repeat clinic/ patient visits – Length of stay in hospital – Use of blood products (implantable biosensors)

53. DISADVANTAGES OF SRT/NPT Analytical performance can be inferior to lab ( eg . s ome glucometers) Risk of poor operator competence Risk of poor equipment maintenance Cost per test relatively more expensive.

54. SETTINGS FOR NPT DEVICES ACCIDENT & EMERGENCY Quick turnaround time for results e .g. Diagnosis of acute MI- whole blood troponin NPT device. Drug overdoses- p lasma p’mol , cocaine.

55. SETTINGS FOR NPT DEVICES DRUG ADDICTION CLINICS measure misused drugs and alcohol( Alcohol breath test) screen workers for substance abuse. Roche Diagnostics– for qualitative testing for ethanol in either saliva or urine.

56. SETTINGS FOR NPT DEVICES NEONATAL CARE AND ADULT INTENSIVE CARE N eonatal units: determination of blood bilirubin using NPT bilirubinometers . PATIENT SELF-TESTING eg . Pregnancy self- testing using over -the-counter pregnancy test kits

57. NEAR PATIENT TESTING & DIABETES MELLITUS NPT self monitoring is often used in the management of diabetes mellitus . glucose determinations in urine ketones in urine or plasma blood glucose measurements urinary microalbumin tests

58. Sakyi AS, Laing EF, Ephraim RK, Asibey OF , Sadique OK. Evaluation of analytical errors in a clinical chemistry laboratory : A 3 year experience. Ann Med Health Sci Res 2015;5:8-12.

59. The depth of your struggle determines the height of your success