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Clinical laboratory basic
- 1. Clinic Lab :Basic Overview Training Design: Dorra Hung
- 2. Laboratory Roles &Responsibilities The clinical lab provides diagnostic test data to aid in the detection, diagnosis and treatment of disease. Data is used by physicians, nurses, pharmacists and other healthcare professionals. The responsibilities of the clinical lab include: Correct identification, collection and processing of patient specimens Accurate performance of testing Timely reporting of results Communication with physicians and other healthcare professionals Analyst testing is used to help diagnose, monitor or treat disease Page 2 Mar-07 Dorra Hung
- 3. Laboratory Workflow There aresix main steps in how a sample flows through the lab from order creation to final test result. 1. Test is ordered. 2. Sample is collected 3. Sample is delivered to the lab. 4. Sample is processed. 5. Sample is analyzed. 6. Results are reported. Page 3 Mar-07 Dorra Hung
- 4. Laboratory Specimens Most commonLaboratory Specimen Types: Blood Urine Additional Laboratory Specimens: Body fluids Sputum Stool Tissue samples Culture swabs Page 4 Mar-07 Dorra Hung
- 5. Why do weanalyze our blood? Coronary Disease CK, LDH Kidney Disease Liver Disease BUN, Creatinine Bilirubin Page 5 Mar-07 Dorra Hung
- 6. Medical Examination Overview Anamnesis Medical Examination Pre-diagnose Enquiry Examination Request Analytical Pre-Analytical -Analytical Sensitivity -External Influences -Method Specificity -Patient Sampling -Statistical Quality Control -Sample Transport -Sample Preparation Analytical Result (Value and Unit) Reference Interval (Normal Values) Patient Result Post-Analytical Phase -Plausibility Check Diagnostic Sensitivity -Alarm Values and Specificity -Trend Check Diagnose -Constellation Check Page 6 Mar-07 Dorra Hung
- 7. Sampling Types of samples : Serum,plasma, blood, urine, CSF... Page 7 Mar-07 Dorra Hung
- 8. Medical Examination Objective is to get an answer about the health status of a patient The physician determines on the basis of the anamneses, his clinical examination and on the basis of additional known information an Enquiry Examination Request This is followed by the necessary preparation of the patient and blood sampling Page 8 Mar-07 Dorra Hung
- 9. Pre-Analytical Common material forexamination Venous Blood (Serum or Plasma) Capillary blood Urine (Single shot or 24 hour collection) Cerebrospinal fluid (CSF) Puncture Fluids Others, such as Faeces, Saliva, Gastric acid, Hair….. Page 9 Mar-07 Dorra Hung
- 10. What is Blood? BloodComposition 55% Plasma Plasma Yellow, sticky liquid Cells Transport of Nutrients (proteins, fats, carbon hydrates) Hormones 44% Erythrocytes Red blood cells Contain Haemoglobin O2 and CO2 transport Page 10 Mar-07 Dorra Hung
- 11. What is Blood? BloodComposition 0.1% Leucocytes Plasma White blood cells Cells Protection against bacteria viruses 0.9% Thrombocytes Platelets Coagulation at injuries Page 11 Mar-07 Dorra Hung
- 12. Sample containers -What do we use? Tube size (mm) Draw Volume ID x Height (mL) 13 x 75 3.5 Red top Vacutainer® collection 13 x 100 4.0, 5.0, tubes are used for 16 x 100 5.0, 6.0, 7.0, 8.0 serum determinations 16 x 100 8.5 in chemistry. Stopper Coagulant Use They contain NO coagulant. color Lavender EDTA Hematology The grey and red speckled SST™ Light Blue Sodium citrate Coagulation tube at left (“tiger top”) contains a Green Lithium Plasma polymer gel for serum separation heparate chemistry and has a Hemoguard™ tube Light Green Lithium Serum heparate + gel chemistry closure. Grey Sodium citrate Glucose testing The Vacutainer® at right has a conventional tube stopper, Tubes are now made of plastic to help protect personnel from injury and bloodborne pathogens. Vacutainer® and Hemogard™ are trademarks of Becton, Dickinson & Company. Page 12 Mar-07 Dorra Hung
- 13. What samples dowe analyze? Phlebotomist draws sample Page 13 Mar-07 Dorra Hung
- 14. Plasma versus Serum Blood to which an anticoagulant has been Blood to which no anticoagulant has added will not clot. Blood cells will settle to the been added will clot. Blood cells get bottom of the tube leaving plasma at the top of caught in the clot leaving serum the tube. behind. Page 14 Mar-07 Dorra Hung
- 15. Pre-Analytical Possible Influences Age, gender Genetic influences Nutritional influences Pregnancy Biorhythm (diurnal rhythm causing analytical fluctuations) Muscular mass, body weight Physical activity or inactivity Psychological stress (fear for blood collection, surgery) Use of medicines Page 15 Mar-07 Dorra Hung
- 16. Pre-Analytical Disturbing Influences Sample collection (body position, venous congestion, ….) Sample condition (haemolytic, lipemic, icteric) Normal serum obtained from an individual in good health is usually clear, pale yellow in color. However, the color of the patient’s serum may appear different for various reasons such as disease or improper handling of the blood specimen. Lipemia (Lipe) results from increased levels of lipoproteins associated with triglycerides, and it can cause the serum to appear white. Hemolysis (Heme) is caused usually by the release of hemoglobin from ruptured red blood cells during sample collection and/or sample handling. This interference can cause the serum to appear red. Page 16 Mar-07 Dorra Hung
- 17. Pre-Analytical Icterus (Icte) is the result of increased levels of bilirubin, and it can cause the serum to appear yellow. Page 17 Mar-07 Dorra Hung
- 18. Pre-Analytical Separation of samples Centrifugation, Deproteinization Chromatography Electrophoresis, ....
- 19. Pre-Analytical After the centrifugationif the sample was without anticoagulant the supernatant fluid is SERUM otherwise is plasma . As anticoagulants they use EDTA K3 , EDTA K2 , Heparin, Citric acid 9:1, Citric Acid 4:1 NaF and others. If we use plasma we must know the type of the anti-coagulant due to different interferences f.e. Ca , Na , Fe , ALP ...
- 20. Pre-analytics Pre-Analytical Page 20 Mar-07 Dorra Hung
- 21. Pre-Analytical Somephotometric assays may be influenced by the presence of these abnormal serum colors and the reliability of the test results may be decreased. haemolysis can cause analytical interferences such as high K+ caused by release from erythrocytes, or can interfere with the measuring technique (photometry) Inadequate sample transport Wrong centrifugation Inadequate sample storage (Bilirubin) Page 21 Mar-07 Dorra Hung
- 22. Pre-Analytical Different type of sample collection in commercially available blood collection systems (Beckton Dickinson Vacutainer, Sarstedt Monovetten, …..) Tube with additional Extraction of Application Colour- Anti-Coagulation agent coding Whole blood (without Serum Clinical Chemistry, Serology, Red agent) Immunochemistry Heparin Plasma Clinical Chemistry Green EDTA Plasma Haematology, Special Lilac Chemistry, Immunochemistry Citrate Plasma Coagulation tests Bleu Na-Fluoride / K-Oxalate Plasma Glucose, Lactate Grey Page 22 Mar-07 Dorra Hung
- 23. Pre-Analytical Serum Plasma versus Serum 30-45 minutes clothing (preferably in the dark) 10-15 minutes centrifugation @ 1000-1500 g Plasma Blood to which an anticoagulant has been added will not clot. Blood cells will settle to the Blood to which no anticoagulant has been added will clot. Blood cells get Immediate 10-15 minutes bottom of the tube leaving plasma at the top of caught in the clot leaving serum the tube. behind. centrifugation @ 1000-1500 g For internal use only Page 7 Jan-07 Velemirov / Twisk EU Sales Training Page 23 Mar-07 Dorra Hung
- 24. Pre-Analytical Sample transport andstorage Properly packed Transport must be save Bio hazardous material 4 hours stable @ 15-25 oC Closed to avoid evaporation 24 hours stable @ 4-8 oC Dry ice, cool packs, refrigerator, etc. Page 24 Mar-07 Dorra Hung
- 25. Pre-Analytical Example: Potasium Plasmais recommended for rapid centrifugation Use only serum or plasma from single patients Sample preparation (heparin plasma) Centrifuge within 30-45 minutes after collection Erythrocytes produce Homocysteine, which continues after sampling Store on ice if centrifugation within 30-45 minutes is not possible Store plasma at -20 oC if sample can not be measured within 48 hours Page 25 Mar-07 Dorra Hung
- 26. Analytical Page 26 Mar-07 Dorra Hung
- 27. Analytical Adequate test methodology Standard Operating Procedure Understandable Traceable Routine test must be Easy to be executed Reliable Low risk on failure Page 27 Mar-07 Dorra Hung
- 28. Statistical Quality Control Sampleswith known concentration Low Medium High As part of the daily routine Begin of the run Middle in the run End of the day Random Page 28 Mar-07 Dorra Hung
- 29. Quality control Page 29 Mar-07 Dorra Hung
- 30. Cumulative control Page 30 Mar-07 Dorra Hung
- 31. Patient Result Test Report Demographic information Patient name, Patient ID, Lab number Sample matrix, Visual distortions Date, Time Sample collection, Arrival in the lab, Time of analyses Analytical results Test name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……) Page 31 Mar-07 Dorra Hung
- 32. Analytical Results Expected Values Reference Range Normal values Based on a large pool of healthy persons Differences between Children vs. adults Male vs. female Serum vs. plasma Population Biorhythm Page 32 Mar-07 Dorra Hung
- 33. Diagnose After checking thereliability of the analysis Analytical range Statistical Quality control Pre-analytical and analytical disturbances Plausibility of the result Compared with previous results Fit with the situation of the patient DIAGNOSE Page 33 Mar-07 Dorra Hung
- 34. Methods of ClinicalChemistry Photometry Chemiluminence Potentiometry (ISE) Electrophoresis Nephelometry γ- COUNTER Mass absorption Osmometry HPLC TLC Coagulation ...
- 35. Optical Density A= - log 10 T Photometry A= ε x L x C Page 35 Mar-07 Dorra Hung
- 36. Photometry Page 36 Mar-07 Dorra Hung
- 37. End Point thereis final <<stable>> color Page 37 Mar-07 Dorra Hung
- 38. END POINT –linearcalibration curve Page 38 Mar-07 Dorra Hung
- 39. CALCULATIONS Page 39 Mar-07 Dorra Hung
- 40. Rate Method Page40 Mar-07 Dorra Hung
- 41. RATE or ZEROORDER kinetics Page 41 Mar-07 Dorra Hung
- 42. RATE , ZEROORDER Decrease : 340 nm AST/GOT-ALT/GPT,LDH P--L,ALDOLASE FACTOR or FV = (Vtotal x 1000) / (Vsample x Light Path xMEC) Page 42 Mar-07 Dorra Hung
- 43. RATE or ZEROORDER kinetics Increase : 340 nm : LDH L->P, CK , CKMB , HBDH , ELASTASE , LAP 405 nm : ALP(AMP) , ALP(DEA) , ACP , NP ACP , AAMY , PAMY FACTOR or FV = (Vtotal x 1000) / (Vsample x Light Path xMEC) Page 43 Mar-07 Dorra Hung
- 44. Turbidimetric assays Page 44 Mar-07 Dorra Hung
- 45. Page 45 Mar-07 Dorra Hung
- 46. Page 46 Mar-07 Dorra Hung
- 47. Direct potentiometry :This is the simplest method of making ion- selective electrode measurements. The electrodes are immersed in a test solution and the electrode potential is measured directly with a millivolt meter. The concentration is then related directly to this measurement by reading the answer from a calibration graph of concentration versus millivolts. Indirect potentiometry : Dilution of the sample (less volume, less problems, less interventions Page 47 Mar-07 Dorra Hung
- 48. Page 48 Mar-07 Dorra Hung
- 49. Pre-analytical factors thataffect serum proteins concentrations 1)Time of the day 2)Position 3)Exercise 4)Fasting vs non fasting 5)Medications 6)Time of year (season) 7)Age and gender 8)Geographic location 9)Venipuncture technique 10)Sample handling and storage Page 49 Mar-07 Dorra Hung
- 50. Patient Result Test Report Demographic information Patient name, Patient ID, Lab number Sample matrix, Visual distortions Date, Time Sample collection, Arrival in the lab, Time of analyses Analytical results Test name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……) Page 50 Mar-07 Dorra Hung
- 51. Auto-validation Automatic Limits defined by the lab Delta checks Quality control with Westgard rules Messages from the systems Page 51 Mar-07 Dorra Hung
- 52. RESULTS Clinic Page 52 Mar-07 Dorra Hung