Clinical laboratory basic


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Clinical laboratory basic

  1. 1. Clinic Lab : Basic OverviewTraining Design: Dorra Hung
  2. 2. Laboratory Roles & ResponsibilitiesThe 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 professionalsAnalyst testing is used to help diagnose, monitor or treat diseasePage 2 Mar-07 Dorra Hung
  3. 3. Laboratory WorkflowThere are six main steps in how a sample flows through the lab from order creation to final test result.1. Test is ordered.2. Sample is collected3. 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. 4. Laboratory SpecimensMost common Laboratory Specimen Types: Blood UrineAdditional Laboratory Specimens: Body fluids Sputum Stool Tissue samples Culture swabsPage 4 Mar-07 Dorra Hung
  5. 5. Why do we analyze our blood? Coronary Disease CK, LDH Kidney Disease Liver Disease BUN, Creatinine BilirubinPage 5 Mar-07 Dorra Hung
  6. 6. Medical Examination Overview Anamnesis Medical Examination Pre-diagnose Enquiry Examination RequestAnalytical 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 CheckPage 6 Mar-07 Dorra Hung
  7. 7. Sampling Types of samples : Serum,plasma, blood, urine, CSF...Page 7 Mar-07 Dorra Hung
  8. 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 samplingPage 8 Mar-07 Dorra Hung
  9. 9. Pre-AnalyticalCommon material for examination 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. 10. What is Blood?Blood Composition 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 transportPage 10 Mar-07 Dorra Hung
  11. 11. What is Blood?Blood Composition 0.1% Leucocytes Plasma White blood cells Cells Protection against bacteria viruses 0.9% Thrombocytes Platelets Coagulation at injuriesPage 11 Mar-07 Dorra Hung
  12. 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. 13. What samples do we analyze? Phlebotomist draws samplePage 13 Mar-07 Dorra Hung
  14. 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. 15. Pre-AnalyticalPossible 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 medicinesPage 15 Mar-07 Dorra Hung
  16. 16. Pre-AnalyticalDisturbing 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. 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. 18. Pre-AnalyticalSeparation of samples Centrifugation, Deproteinization Chromatography Electrophoresis, ....
  19. 19. Pre-AnalyticalAfter the centrifugation if the sample was without anticoagulant the supernatantfluid is SERUM otherwise is plasma .As anticoagulants they use EDTA K3 , EDTA K2 , Heparin, Citric acid 9:1, CitricAcid 4:1 NaF and others.If we use plasma we must know the type of the anti-coagulant due to differentinterferences f.e.Ca , Na , Fe , ALP ...
  20. 20. Pre-analyticsPre-AnalyticalPage 20 Mar-07 Dorra Hung
  21. 21. Pre-Analytical Some photometric 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. 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 codingWhole blood (without Serum Clinical Chemistry, Serology, Redagent) ImmunochemistryHeparin Plasma Clinical Chemistry GreenEDTA Plasma Haematology, Special Lilac Chemistry, ImmunochemistryCitrate Plasma Coagulation tests BleuNa-Fluoride / K-Oxalate Plasma Glucose, Lactate GreyPage 22 Mar-07 Dorra Hung
  23. 23. Pre-Analytical SerumPlasma 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 onlyPage 7 Jan-07 Velemirov / Twisk EU Sales TrainingPage 23 Mar-07 Dorra Hung
  24. 24. Pre-AnalyticalSample transport and storage 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. 25. Pre-AnalyticalExample: Potasium Plasma is 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 hoursPage 25 Mar-07 Dorra Hung
  26. 26. AnalyticalPage 26 Mar-07 Dorra Hung
  27. 27. AnalyticalAdequate test methodology Standard Operating Procedure Understandable TraceableRoutine test must be Easy to be executed Reliable Low risk on failurePage 27 Mar-07 Dorra Hung
  28. 28. Statistical Quality ControlSamples with known concentration Low Medium HighAs part of the daily routine Begin of the run Middle in the run End of the day RandomPage 28 Mar-07 Dorra Hung
  29. 29. Quality controlPage 29 Mar-07 Dorra Hung
  30. 30. Cumulative controlPage 30 Mar-07 Dorra Hung
  31. 31. Patient Result Test ReportDemographic information Patient name, Patient ID, Lab number Sample matrix, Visual distortions Date, Time Sample collection, Arrival in the lab, Time of analysesAnalytical results Test name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……)Page 31 Mar-07 Dorra Hung
  32. 32. Analytical Results Expected ValuesReference Range Normal values Based on a large pool of healthy personsDifferences between Children vs. adults Male vs. female Serum vs. plasma Population Biorhythm Page 32 Mar-07 Dorra Hung
  33. 33. DiagnoseAfter checking the reliability 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 DIAGNOSEPage 33 Mar-07 Dorra Hung
  34. 34. Methods of Clinical Chemistry Photometry Chemiluminence Potentiometry (ISE) Electrophoresis Nephelometry γ- COUNTER Mass absorption Osmometry HPLC TLC Coagulation ...
  35. 35. Optical Density A = - log 10 TPhotometry A= ε x L x CPage 35 Mar-07 Dorra Hung
  36. 36. PhotometryPage 36 Mar-07 Dorra Hung
  37. 37. End Point there is final <<stable>> colorPage 37 Mar-07 Dorra Hung
  38. 38. END POINT –linear calibration curvePage 38 Mar-07 Dorra Hung
  39. 39. CALCULATIONSPage 39 Mar-07 Dorra Hung
  40. 40. Rate Method Page 40 Mar-07 Dorra Hung
  41. 41. RATE or ZERO ORDER kineticsPage 41 Mar-07 Dorra Hung
  42. 42. RATE , ZERO ORDERDecrease :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. 43. RATE or ZERO ORDER kineticsIncrease :340 nm : LDH L->P, CK , CKMB , HBDH , ELASTASE , LAP405 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. 44. Turbidimetric assaysPage 44 Mar-07 Dorra Hung
  45. 45. Page 45 Mar-07 Dorra Hung
  46. 46. Page 46 Mar-07 Dorra Hung
  47. 47. Direct potentiometry : This is the simplest method of making ion-selective electrode measurements. The electrodes are immersed ina test solution and the electrode potential is measured directly witha millivolt meter. The concentration is then related directly to thismeasurement by reading the answer from a calibration graph ofconcentration versus millivolts.Indirect potentiometry : Dilution of the sample (less volume, lessproblems, less interventionsPage 47 Mar-07 Dorra Hung
  48. 48. Page 48 Mar-07 Dorra Hung
  49. 49. Pre-analytical factors that affect 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 storagePage 49 Mar-07 Dorra Hung
  50. 50. Patient Result Test ReportDemographic information Patient name, Patient ID, Lab number Sample matrix, Visual distortions Date, Time Sample collection, Arrival in the lab, Time of analysesAnalytical results Test name, Unit, Reference values, Comments (high/low, diluted, duplicates, ……)Page 50 Mar-07 Dorra Hung
  51. 51. Auto-validation Automatic Limits defined by the lab Delta checks Quality control with Westgard rules Messages from the systemsPage 51 Mar-07 Dorra Hung
  52. 52. RESULTS ClinicPage 52 Mar-07 Dorra Hung