“Quality Control” & Hematology Cell Counter

1. “Quality Control” & Hematology Cell Counter June 2011 M R Tiwari M.Sc-TQM

2. Laboratory test results (Good laboratory practice, GLP) Clinical diagnosis Patient management

3. Good Laboratory Practice Quality can be assured at • Pre-analytical stage • Analytical stage • Post-analytical stage

4. Quality management at the Pre-analytical phase

5. Requisition form QC at Pre-analytical stage

6. Requisition form Reference: Clause 5.4 (ISO 15189) Name of the organization Complete address/telephone no./fax no./e mail/website Patient’ case no. / Name:______________________________________________ Date of Birth/Age:____________________________________________________ Sex: _______________________________________________________________ Referring Doctor’ name:_______________________________________________ Address (OPD/WARD/Unit):___________________________________________________ __________________________________________________________________ • Primary Sample Type: Blood/Fluid/Sputum/Stool/Microbiological Specimen/Slides/Tissue/_________________________________________________ (Any other specify) • Date and Time of Primary Sample Collection: ___________________________________________________________________________________________________ • Date and Time of Receipt of Primary Sample by the Laboratory: ____________________________________________________________________________________ Clinical History of the patient: ________________________________________________________________ ________________________________________________________________ Treatment History of Patient: __________________________________________________________________ __________________________________________________________________ Examinations Requested Requisition number 1. 2. 3. 4. 5. Signature ______________ QC at Pre-analytical stage

7. Sample collection QC at Pre-analytical stage

8. Example 1 • Stress and exercise • Increases cell concentrations • Increases coagulation factors (VIII) & also tissue plasminogen activator (t-PA) with increased fibrinolytic activity (2-4). Reference: (2) Standardization of blood specimen collection procedure for reference values. Clin Lab Haematol 4:83- 86, 1982. (3) Van Assendelft OW, Simmons A. Specimen collection, handling, storage and variability, in Lewis SM, Koepke JA (eds): Hematology Laboratory Management and Practice. Oxford, Butterworth Heinemann, 1995, p 109-127. (4) Dacie JV, Lewis SM. Practical Haematology, 8th ed. Edinburgh, Churchill Livingstone, 1995, p 9-19. X QC at Pre-analytical stage

9. Example 2 • Prolonged use of a tourniquet • haemoconcentration; the patient’s posture (standing, sitting or lying) and even the position of the arm during venous sampling will cause fluctuations of 5-10% in the blood count(1). Reference : (1) International Committee for Standardization in Hematology. QC at Pre-analytical stage

10. Example 3 • Causes significant shrinking of the red cells with a decrease of 1-2% in the MCV. . • K2EDTA in a concentration of 1.5-2.2 mg/ml (4.55 ± 0.8 mmol/ml) as this cause less cellular change(5). The International Council for Standardization in Hematology (ICSH) has recommended K3EDTA K2EDTA Reference: (5) Bachmann F. Molecular aspects of plasminogen, plasminogen activators and plasmin, in Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD (eds): Haemostasis and Thrombosis. Edinburgh, Churchill Livingstone, 1994, p 575-613 QC at Pre-analytical stage

11. Squeezing Example 4 Improper technique results in : • Presence of microclots or platelet clumps • Low platelet count reported by cell counter QC at Pre-analytical stage

12. Example 5 • Sodium citrate (↑) • Sodium citrate (↓) • Blood (↑) • Blood (↓) Excess sodium citrate consumes Ca+2 present in reagents Micro clots Shortening of APTT Prolongation of PT & APTT QC at Pre-analytical stage

13. Example 6 • Blood (9) : Sodium citrate (1) Delayed sample leads to Factors deterioration Contact with heparin Prolongation of APTT Prolongation of PT & APTT QC at Pre-analytical stage

14. Example 7 • Withdraw the blood slowly using 22 or 21 gauze needles • Remove the needle from the syringe and deliver the blood gently into the containers • Avoid vigorous mixing as it may cause foaming and hemolysis Avoid hemolysis QC at Pre-analytical stage

15. Example 8 • While drawing blood form indwelling lines or catheters errors due to dilution and or contamination from flushing solution should be avoided. Reference : NABL 112, Issue 02, Page 18/39 QC at Pre-analytical stage

16. Example 9 • When an intravenous solution is being administered in a patient's arm, blood should be drawn from the opposite arm. • If an intravenous infusion is running in both arms, samples may be drawn after the intravenous infusion is turned off for at least two minutes before venipuncture and applying the tourniquet below the intravenous infusion site. Reference : NABL 112, Issue 02, Page 19/39 QC at Pre-analytical stage

17. Example 10 Mix well by gently inverting the tubes 8 to 10 times Avoid clotting of blood collected in anticoagulant QC at Pre-analytical stage

18. Example 11 • A serious, and potentially fatal, cause of mishap is • collection from the wrong patient • subsequent specimen mix-up • transcription error These can occur at any stage. It is essential to have a cross-check procedure. QC at Pre-analytical stage

19. Example 12 • Sample transport • trays having protective covering • providing suitable environment required preventing deterioration of the sample • person carrying the sample should be following the universal safety norms QC at Pre-analytical stage

20. Sample receiving • Laboratory checks • quantity, • quality, • labeling, • request forms, • clot presence etc. If the required criteria are not met than the samples may be rejected QC at Pre-analytical stage

21. Sample rejection form QC at Pre-analytical stage

22. Atsampleacceptancecounter… Pl.. accept honey… QC at Pre-analytical stage

23. Example # 1 Quantity not sufficient QC at Pre-analytical stage

24. Example # 2 Sample hemolysed / lipemic QC at Pre-analytical stage

25. Example # 3 Name on vaccutainer and name on the requisition form do not match X QC at Pre-analytical stage

26. Example # 4 Outside of container contaminated by specimen QC at Pre-analytical stage

27. Example # 5 Sample partially / fully clotted QC at Pre-analytical stage

28. Example # 6 Sample received without requisition form X QC at Pre-analytical stage

29. Analysis # 1 QC at Pre-analytical stage

30. Analysis # 2 32% 33% QC at Pre-analytical stage

31. Sample rejection analysis A feedback of this kind to the concerned ward of the hospital may enhance a positive attitude towards quality improvement at pre-analytical stage Quality Indicator Rejection analysis helps in ‘Continual improvement’ QC at Pre-analytical stage

32. Quality Management of the Analytic Phase

33. Terminologies = QC at Analytical stage

34. PRECISION (REPRODUCIBILITY) • Definition Precision refers to the reproducibility of a result. • Comparing QC terms to a targetFigure illustrates that the results are precise (close together) but not accurate (they are not in the bull’s-eye). • Checking precision is required while -calibration -troubleshooting QC at Analytical stage

35. ACCURACY • Definition Closeness of a result to the true (accepted) value. NOTE: Before determining accuracy, first determine precision. • Comparing QC terms to a targetFigure illustrates that the results are accurate (in the bull’s-eye) and precise (close together). NOTE • You cannot have accuracy without precision. • However, you can have precision without accuracy. QC at Analytical stage

36. NEITHER ACCURACY NOT PRECISION • This figure illustrates that the results are neither accurate nor precise. • None of the results are close together, and none of them are in the bull’s-eye. QC at Analytical stage

37. Statistics involved… • Mean • Standard deviation (SD) • ± 1SD • ± 2SD • ± 3SD • Coefficient of variation (%CV) QC at Analytical stage

38. In 1931, Dr. Walter Shewhart, a scientist at the Bell Telephone Laboratories, proposed applying statistical based control charts to interpret industrial manufacturing processes. In 1950, S. Levey& E.R. Jennings suggested the use in the clinical laboratory. Control Chart's Inventor QC at Analytical stage

39. L-J chart Interpretation Dr. James O Westgard QC at Analytical stage

40. Modification of 10x Dr. James O Westgard QC at Analytical stage

41. I prefer the ‘xyz’ analyzer because its 2SD QC charts are amazingly flat 2SD QC at Analytical stage

42. Hematology analyzers QC at Analytical stage

43. Internal quality control (IQC) Hematology analyzers

44. IQC in hematology analyzers • Commercial controls • Retained sample • Moving averages (Bull’s algorithm) QC at Analytical stage

45. Establishing Laboratory’s acceptable ranges for controls Reference : CELL-DYN Sapphire Operator’s Manual, 11-16 Establishing laboratory control mean Commercial controls • New lot of control material should be analyzed in parallel with current lot. • This may be accomplished by running the new controls twice a day for five days (we run three times for three days and one time on fourth day). • The mean of the ten runs is then used. • A control file is set up for the new mean. • The same file is used to run control for the reminder dating of period. QC at Analytical stage

46. Establishing Laboratory’s acceptable ranges for controls Reference : CELL-DYN Sapphire Operator’s Manual, 11-16 Establishing laboratory acceptable ranges (SD) Commercial controls • The expected ranges published by the manufacturers do not represent standard deviations and are generally too broad for effective quality control. • These ranges are determined by evaluating three to six months of data (data from the previous IQC) for a particular level of control. • The individual SD values may be averaged as follows: N = number of values in a group SD = Standard Deviation of the values in that group i = the last group of values The resultant long-term instrument SD and the laboratory established mean is used to monitor instrument performance QC at Analytical stage

47. Retained sample testing • A previous days (retained) sample, stored at 2-80C, with normal counts is run as 1st sample after the controls are analyzed. This is done on a daily basis. • This sample is considered as the precision sample. • This sample is then analyzed every one hour or / after 30 patient samples and also as the last sample before the analyzer is shut down. • The mean, SD and CV is monitored. QC at Analytical stage

48. DAILY IQC of Hematology analyzer QC at Analytical stage

49. When a control point is outlier… Warning rule = use other rules to inspect the control points Rejection rule = “out of control” • Stop testing • Identify and correct problem • Repeat testing on patient samples and controls • Do not report patient results until problem is solved and controls indicate proper performance QC at Analytical stage

50. Other additive IQCs QC at Analytical stage