validation of coagulometers in the diagnostic laboratory: new guidelines

1. Chris Gardiner Department of Haematology University College London Hospitals Validation of coagulometers in the diagnostic laboratory:New guidelines

3. Don’t believe everything that you read

4. Why validate an analyser? It improves the chance of good performance in NEQAS It is a CPA requirement

5. CPA definition Validation - confirmation, through the provision of objective evidence, that the requirements for a specific intended use or application have been fulfilled

6. CPA Standards Examination procedures, including those for sampling, shall meet the needs and requirements of users. Examination procedures shall be validated for their intended use prior to introduction, and the methods used and results obtained, recorded. The laboratory shall determine the uncertainty of results, where relevant and possible.

10. Existing guidelines/protocols Chemistry analysers CLSI (NCCLS) guidelines (EP-5, EP-12, EP-15) Well defined stable analytes Variety of matrices Haematology analysers ICSH 1984 (Clin Lab Haematol 6 (1):69-84) Measure blood cell parameters

11. Haematology

12. Clinical Chemistry

13. Haemostasis

14. First guidelines for coagulometer evaluation

18. Problems Modern coagulometers are complex Tailored reagent-instrument systems Reagent specific algorithms Unstable analytes Some tests poorly standardised with no ‘gold standard’ e.g., APTT

19. New Guidelines Recommendations for the evaluation of coagulometers Gardiner, Kitchen, Dauer, Kottke-Marchant & Adcock. (2006) Lab Hematol. 12(1):32-8 Protocol for the Evaluation, Validation, and Implementation of Coagulometers. Clinical and Laboratory Standards Institute (2008) Approved guideline H57-A

20. Levels of evaluation Type of evaluation National (eg FDA, MHRA etc) Beta site Local (Purchasing laboratory) Availability of independent evaluation data Type of work performed Range of samples available

21. Planning Time required Staff time Analyser availability Reagent quantities Contingency plan Reagents held in reserve

22. Preliminary information Price: Instrument, reagents, consumables, leasing… Requirements: Space, power, operating environment… Full technical specification: Throughput, sample volume, tests per sample, reagent storage… Closed tube sampling? Effect on dead volume Interface: Who will perform interfacing with LIS/HIS

23. Preliminary assessment Safety Mechanical Electrical – IEC 61010-1 Microbiological – aerosol, splashing, waste disposal Installation and training Training course Operator’s manual Preliminary assessment of imprecision

26. Performance assessment Comparability (accuracy) Reproducibility (precision) Ease of use Fit for purpose?

29. Precision testing Lyophilised plasma or frozen pooled plasmas Within run and total imprecision 3 replicates of 2/3 levels on 3 or 5 separate days (CLSI EP-15 H-57) Compare %CV with manufacturer’s if available Useful for establishing uncertainty of results for CPA

30. Precision testing

31. Precision testing On-board stability of reagents Analyse freshly prepared QC over the stated stability period of the reagents

32. Comparability Comparability rather than accuracy Accuracy implies a ‘true’ value Not applicable to many tests (eg APTT, PT, APCR) Differences between antigenic and activity assays

33. Comparability The reference method will normally be that currently in use, BUT…… big differences can arise when: Mechanical and optical clot detection methods are compared There are significant differences between reagents recombinant human v rabbit brain thromboplastin large differences in thromboplastin ISI natural v synthetic phospholipids in APTT reagents clotting v chromogenic factor/thrombophilia assays

34. Comparability PT/INR >50 samples from patients receiving warfarin ?20 normal samples Verify ISI assignment (use a calibration set) APTT ? 20 samples from patient receiving unfractionated heparin Lupus anticoagulant samples Factor deficient samples (FVIII, FIX, FXI etc)

35. Good comparability

38. Heparin APTT therapeutic range

39. Factor assays Calibration curve reproducibility Linearity

40. Reference ranges Where ever possible local reference ranges should be established for any new method Fresh or frozen plasmas from at least 20 healthy individuals should be tested Sex specific ranges may be required (e.g. protein S assays)

41. Reagent carryover Use when a single probe is used for all reagents and samples Clauss fibrinogen or antithrombin assays are performed on 5 aliquots of normal plasma followed by aPTT The sequence is repeated 10 times Carryover is indicated by a progressive shortening of clotting times

42. Interfering substances Local evaluation: manufacturer’s specifications Full evaluation: Analyse samples with a broad range of interfering substances Icterus: Bilirubin up to 1000 ?mol Lipaemia: Triglycerides up to 20 mmol Haemolysis: Haemoglobin up to 1.0 g/dL

43. Throughput Manufacturers’ throughput data generated using normal samples often using 2 or 3 tests eg 160 PT/APTT per hour Real situation Sick patients Wide range of tests performed simultaneously Reflex tests Can several test methodologies run at once?

44. What can go wrong…… During 2001, a hospital laboratory using a benchtop coagulometer in Pennsylvania reported 2146 tests with correct PT results but with incorrectly calculated INR results over a 7 week period This led to several major haemorrhages and two fatalities This was the result of changing INR method without validation

45. Adjusted relative odds of intracranial haemorrhage by INR