Internal quality control (IQC) in coagulation lab
- 1. Internal Quality Control in coagulation lab Dr Ankit Raiyani Haematology Department Sahyadri Speciality Hospital
- 2. Presentation layout • Introduction to Quality Assurance and Quality Control • Definitions of common terms, Accuracy vs. Precision • Preparation/selection of reagents- • PTT reagent PT reagent MNPT • QC for manual coagulation tests • Quality control procedures- schedule • Internal Quality Control • LJ charts • Westgard rules • Other measures for IQC
- 3. Introduction In the haematology laboratory it is essential to ensure that the right test is carried out on the right specimen and that the correct results are delivered to the appropriate recipient without delay. Quality assurance (QA) is defined as the overall programme for achieving these objectives. Quality control (QC) is defined as measures that must be included during each assay run to verify that the test is working properly.
- 4. Quality Assurance vs. Quality Control Quality Assurance Quality Control An overall management plan to guarantee the integrity of data (The “system”) A series of analytical measurements used to assess the quality of the analytical data (The “tools”)
- 5. The Quality Assurance Cycle •Data and Lab Management •Safety •Customer Service Patient/Client Prep Sample Collection Sample Receipt and Accessioning Sample TransportQuality Control Record Keeping Reporting Personnel Competency Test Evaluations Testing
- 6. Hurdles in Quality Assurance 6
- 7. Hurdles in Quality Assurance 7 Pre-analytical
- 8. Hurdles in Quality Assurance 8 Analytical
- 9. Hurdles in Quality Assurance 9 Post-analytical
- 10. Terms relating to quality control in clinical laboratory practice Precision Can be controlled by replicate tests and by repeated tests on previously measured specimens Accuracy Can be checked only by the use of reference materials that have been assayed by reference methods Controls Are preparations that are used for either internal quality control or external quality assessment. They should not be used as standards Error The discrepancy between the result of a measurement and the true (or accepted true) value Random error An error which varies in an unpredictable manner, in magnitude and sign, when a large number of measurements of the same quantity are made under effectively identical conditions Systemic error (bias) An error which, in the course of a number of measurements of the same value of a given quantity, remains constant when measurements are made under the same conditions or varies according to a definite law when conditions change
- 11. Accuracy vs. Precision Accuracy Precision Meaning How well a measurement agrees with an accepted value How well a series of measurement s agree with each other Achieved with… Calibration Quality control When low… Increases systemic errors Increases random errors
- 12. Calibration Calibration is a comparison of measuring equipment against a standard instrument of higher accuracy to detect, correlate, adjust, rectify and document the accuracy of the instrument being compared.
- 14. Reagent selection for PTT LA-sensitive reagents- (low-phospholipid reagents) • Siemens Actin FSL • IL HemosIL aPTT-SP • Stago PTT-LA Intermediate responsiveness to LA • Stago STAPTT-LA • IL HemosIl SynthASil Low responsiveness to LA • Siemens Actin FS • Stago C.K. Prest
- 15. Reagent selection for PTT Selection of aPTT reagent depends on indication of aPTT-- The routine laboratory aPTT • used for evaluating the factor VIII, IX, XI deficiency and effect of heparin • sensitive to deficiencies of factors VIII, IX and XI at concentrations of 0.35–0.4 iu/ml. • responsive to unfractionated heparin over the therapeutic range of approximately 0.3–0.7 iu/ml. • low responsiveness to LA LA screening • High responsiveness to LA (silica+ low PL content) • Two phospholipid based clotting assays, one of which may be aPTT based, are required1 Intermediate responsiveness to both factor levels and LA • Used in labs with mixed sample load, when only one reagent is feasible/desired 1PENGO, V., TRIPODI, A., REBER, G., RAND, J. H., ORTEL, T. L., GALLI, M. and DE GROOT, P. G. (2009), Update of the ISTH guidelines for lupus anticoagulant detection. Journal of Thrombosis and Haemostasis, 7: 1737–1740.
- 16. Reagent selection for PT (Thromboplastin) Contains tissue factor and coagulant phospholipids Should have ISI value as close to 1 as possible. Source- human, bovine, rabbit brain/lung, human placenta, recombinant. Animal/human source- potential hazard of viral, prion and other infections from handling Recombinant thromboplastins- • Manufactured using recombinant human tissue factor produced in Escherichia coli and synthetic phospholipids, • Do not contain any other clotting factors such as prothrombin, factor VII and factor X. • Highly sensitive to factor deficiencies and oral anticoagulant-treated patient plasma samples
- 17. What is ISI? International Sensitivity Index It is the quantitative measure, (in term of first international reference preparation of thromboplastin, human, combined, coded 67/40,) of the responsiveness of PT system to the defect induced by oral anticoagulants.
- 18. Calculation of ISI Calibration of a test thromboplastin must be against a reference thromboplastin of the same species e.g. human against human, rabbit against rabbit Tests are performed By using reference thromboplastin and test thromboplastinon 20 normal donors not on anticoagulants and 60 patients who have been on oral anticoagulant treatment for at least 6 weeks and has stable INR. Prothrombin Times are performed in duplicate for each sample and the mean for each pair of tests derived. If there is more than a 10% difference in the clotting times between duplicate samples, the tests on that plasma sample should be repeated Mean of each pair of PT results are plotted on double-log paper with the reference sample on the Y axis and the test plasma on the X-axis.
- 19. • A best fit line is drawn with points above the highest recorded PT and the lowest PT (see figure above). The slope of the line is calculated • Slope= AC/BC • ISItest = ISIStandard X Slope • INR= (PT/MNPT)ISI
- 20. Preparation of pooled normal plasma and derivation of MNPT Citrated blood samples from at least 20 healthy volunteers (with PT, aPTT in normal range) should be collected. Equal numbers of male and female (not pregnant, not on OC pills) volunteers. Platelet poor plasma should be prepared. After pooling it should be immediately frozen at -80⁰ C in aliquots. The mean of PT of individual samples is calculated and taken as mean normal prothrombin time (MNPT). SD is calculated and reference range is taken as -2 SD to +2 SD. This pooled plasma is used as control with each batch of samples.
- 21. MNPT calculation- 18/6/15 No PT PTT 1 11.5 28.6 2 12.4 27.6 3 10.5 29.2 4 10.6 26.2 5 10.6 32.1 6 11.7 29.8 7 11.1 30.7 8 12.2 31.6 9 11.9 30.9 10 11.3 31.5 11 10.6 29.1 12 12.3 29.7 13 10 26.3 14 11.9 28.7 15 11.6 33.3 16 11.2 31.4 17 10.5 28.2 18 12.5 28.7 19 10.6 27.8 20 11.1 27.7 21 10.9 29.5 22 11.3 31.9 23 11.3 29.9 24 12.5 32 total 272.1 712.4 Arithmetic Mean 11.3 29.6 SD 0.724 1.899 Range 9.8 to 12.7 25.8 to 33.4
- 22. QC for manual coagulation tests All tests and controls should be performed in duplicates. Operator skill Required instruments should be properly calibrated. Centrifuge • Serviced and Calibrated every 6 months or after break down • Put on sturdy platform to reduce vibrations. • Sample load should be well balanced. Waterbaths • Temperature- 37⁰ C (checked in all four corners) • Transparent walls for transillumination • Filled with distilled water till indicated level. Pipette • Calibrated. • Disposable tips.
- 23. Calibration of centrifuge Tachometer is used for calibration of RPM. Actual RPM is measured with tachometer at uniform intervals (minimum 5) for 5 times. Difference between actual and set values is calculated. If difference is more than +/- 5%, then repeat the test. If repeat reading again shows significant difference corrective measures are required. Timer and Temperature, when applicable, should also be calibrated.
- 24. Calibration of pipettes Analytical balance is used to weigh pipetted volume in controlled environment. Distilled water is used as substrate. The pipette is checked 5 times with the maximum volume (nominal volume), the minimum volume or 10% of the maximum volume, whichever is higher. With multichannel pipettes, both volumes are tested with the two edge channels Conversion of mass to volume V = (w + e) x Z • V = Volume (µl), w = Weight (mg), e = Evaporation loss (mg), • Z = Conversion factor for mg/µl conversion (temperature, pressure) Acceptable accuracy and precision range varies with pipette capacity.
- 26. Quality control procedures The procedures that should be included in a quality assurance programme vary with the tests undertaken, the instruments used and (especially if these include a fully automatic system) the size of the laboratory and the numbers of specimens handled. Some control procedures should be performed daily and other performance checks should be done at appropriate intervals. The latter is particularly important when there is a change in staff and after maintenance service or repair has been carried out on equipment.
- 27. Schedule for quality control procedures 1. Calibration with reference standards Instruments, pipettes 6-month intervals or more frequently if control chart or EQA indicates bias or fluctuation in results and after any repair/service Others: thermometers, scales, etc Annually 2. Control chart with control material Daily or more frequently with each batch of specimens Duplicate tests on two or three patients’ samples: if control chart or delta check shows discrepancies 3. Analysis of patients’ results Cumulative results: following previous tests and if changes in clinical state 4. EQAS performance Assessment monthly
- 28. Components of Quality control Internal quality control (IQC) • Monitoring the haematology test procedures to ensure continual evaluation of the reliability of the daily work of the laboratory with validation of tests before reports are released External quality assessment (EQA) • Evaluation by an outside agency of the between-laboratory and between-method comparability. It can be organized nationally, regionally or internationally
- 29. Internal Quality Control IQC is based on monitoring the haematology test procedures that are performed in the laboratory and includes measurements on specially prepared materials and repeated measurements on routine specimens, together with daily statistical analysis of the data. Primarily a demonstration of Precision. It ensures continual checks that the established reliability of the laboratory’s work does not fluctuate and that reports are validated before they are released
- 30. IQC includes Control charts with tests on control materials Duplicate tests on a proportion of the specimens Consistency of mean values of patient data Correlation check
- 31. Control charts These were first applied in clinical chemistry by Levey and Jennings. They are now widely used in haematology for both automated and manual procedures. Samples of the control specimen are included in every batch of patients’ specimens and the results are checked on a control chart. To check precision, it is not necessary to know the exact value of the control specimen.
- 32. Controls Level 1 controls- normal controls Level 2 controls- low abnormal controls Level 3 controls- high abnormal controls (not used routinely) Minimum of one level QC at least once a day. Patient samples >25 per day Employ 2 levels of QC at least once a day. Patient samples >75 per day Employ 2 levels of QC at least twice a day at appropriate intervals Controls are intended to simulate random sampling, they must be treated exactly like the patients’ specimens.
- 33. Preparation of LJ charts Mean value and standard deviation (SD) of the control specimen should first be established in the laboratory where the tests are performed. Using arithmetic graph paper, a horizontal line is drawn to represent the mean (as a base), and on an appropriate scale of quantity and unit, lines representing +2SD and −2SD are drawn above and below the mean. The results of successive control sample measurements are plotted. If the test is satisfactory, sequential results oscillate about the mean value and <5% of the results fall outside 2SD.
- 34. LJ chart PT / low abnormal May 2015
- 35. LJ chart PT / low abnormal June 2015
- 36. LJ chart PT / low abnormal June 2015 Reason ??
- 37. LJ chart aPTT / NCPNP
- 38. LJ chart F VIIIc / NCPNP
- 39. Westgard rules “Multirule Quality Control” developed by Dr. James O. Westgard based on statistical concepts Uses a combination of decision criteria or control rules Allows determination of whether an analytical run is “in-control” or “out-of-control”
- 40. Westgard multirule system 12S rule 13S rule 22S rule R4S rule 41S rule 10X rule
- 41. Westgard 12S rule “warning rule” One of two control results falls outside ±2SD Alerts tech to possible problems Not cause for rejecting a run Must then evaluate the 13S rule
- 42. 12S Rule = A warning to trigger careful inspection of the control data 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 12S rule violation
- 43. Westgard – 13S Rule If either of the two control results falls outside of ±3SD, rule is violated Run must be rejected If 13S not violated, check 22S
- 44. 13S Rule = Reject the run when a single control measurement exceeds the +3SD or -3SD control limit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 13S rule violation
- 45. Westgard – 22S Rule 2 consecutive control values for the same level fall outside of ±2SD in the same direction, or Both controls in the same run exceed ±2SD Patient results cannot be reported Requires corrective action
- 46. 22S Rule = Reject the run when 2 consecutive control measurements exceed the same +2SD or -2SD control limit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 22S rule violation
- 47. Westgard – R4S Rule One control exceeds the mean by –2SD, and the other control exceeds the mean by +2SD The range between the two results will therefore exceed 4 SD Random error has occurred, test run must be rejected
- 48. R4S Rule = Reject the run when 1 control measurement exceed the +2SD and the other exceeds the -2SD control limit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD R4S rule violation
- 49. Westgard – 41S Rule Requires control data from previous runs Four consecutive QC results for one level of control are outside ±1SD, or Both levels of control have consecutive results that are outside ±1SD
- 50. Westgard – 10X Rule Requires control data from previous runs Ten consecutive QC results for one level of control are on one side of the mean, or Both levels of control have five consecutive results that are on the same side of the mean
- 51. 10x Rule = Reject the run when 10 consecutive control measurements fall on one side of the mean 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mean Day +1SD +2SD +3SD -1SD -2SD -3SD 10x rule violation
- 52. Alternate ways of checking the precision of routine work Duplicate tests on patients’ samples • Test of precision can be performed by repeat analysis on samples from two or three patients Correlation Check • any unexpected result of a test must be checked to see whether it can be explained on clinical grounds or whether it correlates with other tests “Delta” check • Blood count from the same patient checked repeatedly over 2-3 weeks • Values should not change significantly providing the patients condition has not altered significantly Patient data • Need enough through put (i.e. >100 samples /day) • MCV, MCHC, MCH should not vary, providing the patient population is stable and samples from different sets of patients are not batched together. • Mean of 20 successive patients can be plotted and any drift in the calibration of the analyzer will be recognized
- 53. THANK YOU!!
Editor's Notes
- #12: Target, bull’s eye
- #16: Silica, celite, ellagic acid, kaolin Hemosil synthasil- silica
- #17: Screening using a single aPTT assay is inadequate to rule out the presence of LA
- #18: RecombiPlasTin 2g
- #23: When to calculate MNPT- 1. fresh pooled plasma, 2. change in reagent lot, 3. post instrument calibration.
- #25: If 7 sample tubes then use one tube with water to balance
- #26: Using view port in centrifuge. Source- SOP, John Hopkins