Prof. Dr. Serdar ÖZTEZCAN

1. Prof. Dr. Serdar ÖZTEZCAN Introduction to Clinical Biochemistry

2. Contents • What is the Clinical Biochemistry ? • Historical Background • Field of Clinical Biochemistry • Types of diagnostic questions addressed in laboratory • Workflow in laboratory • Preanalyitic Phase • Analytic Phase • Postanalytic Phase

3. Clinical BiochemistryClinical Biochemist • Clinical Biochemistry refers to the discipline involved in the selection, provision, and interpretation of diagnostic testing that uses primary samples from patient • Clinical Biochemist is usually physicians who use the tools of contemporary science to diagnose disease and to assist and guide the other physicians in the diagnosis and management of disease

4. Terminology • Clinical Biochemistry • Clinical Chemistry • Clinical Pathology • Laboratory Medicine • Tıbbi Biyokimya

5. Historical Backround • The clinical biochemistry is both an old and a new science • Diagnosis of diabetes mellitus dates back to the Middle Ages 19th century • Friedrich Wöhler synthesized a compound urea, which had been thought to be produced only by living beings • The compositon of starch and fats were known • A number of proteins from blood had been isolated and characterized • Cholesterol was shown to be present in gallstone • The chemical make-up of urine was elucidated • The measurement of urine sugar in diabetics was carried out • The first clinical chemistry text was written in 1836.

6. Historical Background • 20th century, • pH meter • colormeter • As the demends on the laboratory grew, the human capability for meeting those demands became less and less adequate • Dr. Leonard Skeggs, clinical biochemist, began to develop a device that would automatically pipet a sample, add reagent to it, mix and incubate the resulting solution, and measure the color change with a colormeter

7. Importance of laboratory • The laboratory plays a central role in healthcare. It involves all branches of medicine • By one estimate*, 70% of all medical decisions are based on laboratory results (*Silverstain 2003) • 94% of the all objective data in a typical medical records comes from laboratory • Test request is increasing 5-8 % every year • There are more than 12.000 tests in scope today • The laboratory offers high clinical value at relatively low cost which is only 3.5 % of total healthcare expenses

8. The field of the Clinical Biochemistry • Diagnostic medicine • Administration of Laboratory • Instrumentation • Information technology • Quality assurance (quality control, proficiency testing, audit, benchmarking) • Organization, management and communication with other clinics • Research • Develop, evaluate and improve a diagnostic procedure • Research on clinical and experimental biochemistry projects • Teaching • Technologists, Medical students, Residents, Fellows, Attendings • Papers, lectures,journal clubs, clinical conferences

9. The Scope of Clinical Biochemistry • Analitical Chemistry • Hematology • Coagulation, Hemostasis and Thrombosis • Endocrinology, Organ - function testing • Tumor markers • Drug Monitoring • Toxicology • Biochemical and Moleculer Genetics • Immunology, Allergy • Forensic Medicine • Blood banking (transfusion medicine)

10. Diagnostic medicine • Types of diagnostic questions addressed in laboratory • What is the diagnosis? • Can another diagnosis be rule out? • What is this patient’s prognosis? • How is the patient doing?

11. Diagnostic medicine • Confirming a clinical suspicion • Excluding a diagnosis • Providing a prognosis • Assisting in the selection, optimization, and monitoring of treatment • Screening for disease in the absence of clinical sign or symptoms

12. Confirming a clinical suspicion(Rule in) • Question • A- Is the breathless patient suffering from heart failure? • B- Has this patient had a myocardial infarction? • Request / Result • A- BNP 450 ng/L • B- cTrI 7,2 ug/L • Possible Action • A- Confirm with cardiac ultrasound, decide to admit and treat • B- Decide to admit, intensity of care required, and treat • Potential outcome • A- Reduced symptoms, decresead morbidity and mortality • B- Decresead morbidity and mortality

13. Excluding a diagnosis (Rule out) • Question • Is the breathless patient suffering from heart failure? • Has this patient had a myocardial infarction? • Request / Result • BNP 56 ng/L( N ) • cTrI <0,1 ug/L ( N ) • Possible Action • Seek alternative diagnosis • Consider other possible diagnosis and early discharge • Potential outcome • Avoid incorrect diagnosis and treatment with its potential for harm • Less worry for patient, reduce unneccesary admissions to cardiac care unit

14. Providing a prognosis • Question • Is the patient’s heart failure deteriorating? • What is these patient’s risk of a futher cardiac event? • Request / Result • BNP Increase from 450 to 650 ng/L in last year • cTrI 0,9 ug/L • Possible Action • Adjust therapy, perhaps advise on palliative care • Consider intervention • Potential outcome • Poor prognosis • Increased risk without intervention

15. Assisting in the selection, optimization, and monitoring of treatment • Question • Is the patient taking the correct dosage of b -blocker? • Is the patient complying with treatment protocol? • Request / Result • BNP fallen from 216 ng/L to 160 ng/L • HbA1c 10,6 % (no change in a year) • Possible Action • No change to dosage, encourge patient • Consider changing treatment, closer monitoring of compliance, clinic visits and consultations with diabetes nurse • Potential outcome • Reduced symptoms and reduced risk of cardiac event • Persistently high HbA1c carries increased risk of complications; intervention necessary to decrease risk

16. Screening for diasease in the absence of clinical sign or symtoms • Question • Does new born have hypothyroidism? • Does this pregnant women has risk for baby for Down Syndrome ? • Request / Result • TSH 22,2 mU/L • 11-14 weeks test + • Possible Action • Treat with throxine • After confirming with amniocentesis, medical abortus ? • Potential outcome • Decresead morbidity and mortality • To decrease burden of family and society

17. Workflow in Clinical Biochemistry Laboratory • The pre-analytic phaseis defined as all procedures from the time the test is ordered until the specimen is analyzed • The analytic phaseis defined as analysis of the specimen by automated, semi automated, or manual methods • The post-analytic phaseis defined as all procedures from the time a result is produced in the laboratory until the providers interpret the result

18. Steps in obtaining a laboratory test • Written order is placed • Specimen is collected and properly labeled • Specimen and order are transported to the lab • The specimen is accessioned in the lab • The specimen is processed • The specimen is delivered • The specimen is analyzed • The results are reviewed and verified by a biochemist • The results are released to the patient’s record

19. Specimen collection • The completed request (order) should include: • patient's name, sex and date of birth • hospital or other identification number • ward/clinic/address • name of requestingdoctor (telephone/pager number for urgent requests) • clinical diagnosis/problem • test(s) requested • type of specimen • date and time of sampling • relevant treatment (e.g. drugs)

20. Specimen in clinical biochemistry? (preanalytic phase) • Blood • phlebotomy / venipuncture • syringe or evacuated tube

21. Specimen in clinical biochemistry ? (preanalytic phase) • 18g • 20g • 21g • 22g

22. Specimen in clinical biochemistry ? (preanalytic phase) • Blood • Whole blood • Serum • Plasma

23. Whole blood (preanalytic phase) • Both the liquid portion of the blood called plasma and the cellular components • Arterial (Blood gas, pH ) • Venous (CBC, ESR, HbA1c etc.) • Capillary • Tubes / hct • Filter paper / new born screening • Smear / evaluate cell distribution and morphology

24. Serum (preanalytic phase) • The fluid portion of the blood obtained after removal of the fibrin clot and blood cells

25. Plasma (preanalytic phase) • The fluid portion of the blood in which the cells are suspended • This requires blood collection into a tube containing an anticoagulant • Plasma consists of 93% water and 7% solids (mostly proteins, the greatest proportion of which is albumin) • Plasma contains fibrinogen and related compound that are removed from serum when blood clots

26. Tubes and Anticoagulants (preanalytic phase) • Red-top tubes • contain no anticoagulants or preservatives • are used for collecting ...................... • Gold (and “tiger”) top tubes • noanticoagulantsbut contain a gel that forms a physical barrier between the serum and cells after centrifugation • Green-top tubes • contain either the sodium or lithium salt of heparin • Heparin inhibits thrombin, so blood does not ............... • Lavender-top tubes • contain EDTA, which chelates calcium and inhibits coagulation • Used for hematology, and some chemistries

27. Tubes and Anticoagulants (preanalytic phase) • Blue-top tubes • contain sodium citrate, which chelates calcium and inhibits coagulation • Used for coagulation tests • Gray-top tubes contain either: • Sodium fluoride and potassium oxalate, orSodium iodoacetate • Both perservatives stabilize glucose in plasma • NaF/oxalate inhibits enolase • Iodoacetate inhibits glucose-3-phosphate dehydrogenase • Brownand Royal Blue top tubes • specially cleaned (acid washed) for trace metal studies (Brown for Pb, Royal blue for others)

28. Order of tubes

29. Others (preanalytic phase) • Urine • Feces • Saliva • Stones • Sweat • Cerebrospinal fluid • Other body fluids • Synovial • Pleural • Pericardial • Ascitic • Amniotic

30. Errors in laboratory (preanalytic phase) • 32 – 75% of errors occurred in pre-analytical phase, most common in all studies are; • Improperly patient preparation • Misidentification of patient • Mislabeling of specimen • Short draws / wrong anticoagulant to blood ratio • Wrong tubes / wrong anticoagulant • Hemolysis / lipemia • Hemoconcentration • Exposure to light / extreme temperatures • Improperly timed specimens / delayed delivery to laboratory • Improperly transport of specimen • Processing errors; incomplete centrifugation, incorrect log-in, improper storage

31. Hemolysis (preanalytic phase) • The destruction or dissolution of red blood cells, with subsequent release of hemoglobin • K, LDH, AST etc.

32. Controllable variables Posture Exercises Circadian variation Menstural cycle Travel Diet Fasting Smoking Alcohol ingestion Drug administration Normal biological variability Noncontrollable variables Age Sex Race Altitute Ambient temperature Location of residence Seasonal influences Obesity Blindness Pregnancy Stress Shock and travma Transfusion and infusions Pre-collection variables (preanalytic phase errors)

33. Analytical techniques and instrumentation (Analytic phase) • Optical technics • Spectrophotometry and photometry • Atomic absorption spectrophotometer • Flame emission photometry • Fluorometry • Chemiluminescence • Turbidity and nepholometry • Laser applications • Electrochemistry • Ion selective electrodes • pH electrodes • Gas-sensing electrodes • Enzyme electrodes • Coulometric chloridometers • Empedans

34. Analytical tecniques and instrumentation (Analytic phase) • Electrophoresis • Isolectric focusing • Capillary electrophoresis • Immunfixation electrophoresis • Chromatography • High performance liquid chromatography • Gas chromatography • Immunochemical technics • Unlabeled immunoassays • Labeled immunoassays • Radioimmunoassays • Magnetic Detection • Nucleic acide probes • Point-of-care testing • Mass spectroscopy

35. Method selection (Analytic phase) • Before a new test or methodology is introduced into the laboratory both managerial and technical information must be complied and carefully considered • The technical information include; • Analytic sensitivity • measures low concentrations of the analyte • Analytic specificity • is not subject to interference by other substances • Linear range • Estimates of imprecision and inaccuracy

36. Imprecision and inaccuracy (Quality Control)

37. Imprecision and inaccuracy

38. Monitoring (Quality Control) • In practice, no test is ideal, but the biochemist must ensure that the results are sufficiently reliable to be clinically useful • Laboratory staff make considerable efforts to achieve this and analytical methods are subject to rigorous quality control and quality assurance procedures • Levy-Jennings graphics • Wesgard rules vb. • External control

39. Monitoring (Quality Control)

40. Interpretation of result (Postanalytic phase) • When the result of a biochemical test is obtained, the following points must be taken into consideration: • is it normal? • is it significantly different from previous results? • is it consistent with the clinical findings?

41. Is it significantly different from previous results?(Postanalytic phase) • If the result of a previous test is available, the clinician will be able to compare the results and decide whether any difference between them is significant • This will depend upon • the precision of the assay itself(a measure of its reproducibility) • Analytical variation: typical standard deviations for repeated measurements made using a multichannel analyser on a single quality control serum with concentrations in the normal range • the natural biological variation • Biological variation: means of standard deviations for repeated measurements made at weekly intervals in a group of healthy subjects over a period of ten weeks, corrected for analytical variation

43. Case • A GP measured the serum creatinine concentration of a 41-year-old man newly diagnosed as having diabetes mellitus and hypertension. The result was 105 μmol/L. • Six months later, both conditions were well controlled and the test was repeated. Serum creatinine 118 μmol/L • The patient was alarmed at the apparent increase, but the GP was uncertain as to whether this was a significant change. • Comment • The analytical variation for creatinine is 5.0 μmol/L, the biological variation 4.1 μmol/L • The critical difference is: 18 μmol/L. • Thus the apparent increase in creatinine is not significantat a level of p = 0.05

44. Is it consistent with the clinical findings?(Postanalytic phase) • If the result is consistent with clinical findings, it is evidence in favour of the clinical diagnosis • If it is not consistent, the explanation must be sought There may have been a mistake in the collection, labelling or analysis of the sample, or in the reporting of the result • In practice, it may be simplest to request a further sample and to repeat the test • If the result is confirmed, the utility of the test in the clinical context should be considered and the clinical diagnosis itself may have to be reviewed

45. Is it normal? Reference intervals • To interpret a test, one must first compare the result to a reference (‘normal’) interval • A reference interval is usully defined as the range of values that represent the central 95% tendency of measurements from a population of nondiseased or ‘normal’ individuals • Gender, age ect. • A referance interval is the laboratory’s attemp to separate ‘normal’ and abnormal’ patient population to help a clincian interpret results and make a decision

46. Reference Intervals

47. Postanalytic phase • Diagnostic Accuracy • Sensitivity • Spesifity

48. Test have four results Condition Present Absent Test TP FP Positive Negative FN TN

49. Overlap • To separate ‘normal’ and abnormal’ patient population is not always easy • False-positive and false-negative results occur when there is an overlap between these population

50. Test Diagnostic Accuracy • The ability of a test discriminate disease from no disease is described by the sensitivity and specificity of the test • Sensitivity is the probability of a positive result in a person with the disease (true positive rate) • Sn = TP / (TP + FN) • Specificity is the probability of a negative result in a person without disease (true negative rate) • Sp = TN / (TN + FP)