2. Module 11 Compliance with established quality assurance requirements 1

3. Learning objectives At the end of this module you will be able to:  explain the three main components of the QA system and their importance;  adhere to the QC and EQA procedures. 2

4. Content outline Quality assurance (QA) in mycobacteriology: –internal quality assurance (IQA) or quality control QC –external quality assessment (EQA) –quality improvement (QI) 3

5. Definitions Quality assurance (QA) System designed to continuously improve the reliability and efficiency of laboratory services. Includes internal quality control, external quality assessment, and quality improvement. Quality control (QC) Includes all means by which the TB laboratory controls operations (instrument checks, checking new lots of staining solutions, reagents, media). 4

6. Definitions External quality assessment (EQA): –participating laboratories assess their capabilities through panel testing; –includes on-site evaluation of the laboratory. 5

7. Definitions Quality improvement (QI): –to permanently remove obstacles to success; –involves continued monitoring, identifying defects; –followed by remedial action (e.g. retraining); –often relies on effective on-site evaluation visits. 6

8. System designed to improve: –reliability –efficiency –use of laboratory services in order to achieve the required technical quality in laboratory diagnosis. Quality assurance in mycobacteriology 7

9. The process of quality assurance should be continuous and monitored Central laboratories Intermediate laboratories Peripheral laboratories Organized according to existing laboratory network levels: 8

10. Quality assurance Process of effective and systematic monitoring of all laboratory activities –establishes limits of acceptable standard of test performance; –ensures laboratory data accuracy, reliability, reproducibility, comparability; –not for punitive purposes! QA is the responsibility of all laboratory technicians and supervisors. 9

11. Quality assurance should be: Practical Workable Comprehensive 10

12. QA should be applied to: Laboratory arrangement Human resources (training, health control) Laboratory equipment Collection and transport of specimens Handling of specimens Reagents and media Culture procedures Reporting of results 11

13. QA – laboratory arrangement BSL2/BSL3 Doors in the laboratory to be kept closed. Work areas, equipment and supplies arranged for logical and efficient work- flow (clean to dirty). Benches swabbed at least once a day with an appropriate disinfectant. 12

14. QA – laboratory administration Use Standard Operating Procedures (SOPs). Keep written procedures in the laboratory for easy reference. Any changes must be dated and initialled by the laboratory supervisor. Retain all records for 2 years. 13

15. QA – human resources Training (retraining) – before starting – refreshment – in case of failure Adequate number 14

16. Training form (example for NTP) 15

17. QA – laboratory equipment Equipment should meet the manufacturer’s claims and specifications. Equipment should be calibrated. Dated service records must be kept for all equipment. Equipment must be monitored regularly to ensure constant accuracy and necessary precision. 16

18. QC – biological safety cabinet Daily checks: Minimal airflow: 0.38–0.51 metre/second (75– 100 feet/minute) depending on (sub)- classification. Check the magnehelic gauge in the exhaust duct for any pressure drop across the filters; replace the filters when the gauge indicates that the airflow across the front opening has dropped below optimal levels. 17

19. QC – other equipment Refrigerator 2–8 ºC / Freezer – 20°C –record temperature daily; –clean monthly; –defrost or check refrigerator and freezer compartment every 3 months. Glassware: –discard chipped or etched glassware; –ensure that glassware is free of detergents; –do not store sterile glassware for more than 3 weeks before it is used. 18

20. QC model for monitoring temperature of equipment TEMPERATURE REQUIRED : 36°C +/- 1°C (variation acceptable) Trimester :Year : MonthTemp.VisaMonthTemp.VisaMonthTemp.Visa °C(Initials)°C(Initials)°C(Initials) 111 222 333 444 555 666 777 888 999 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 19

21. QA – collection and transport of specimens Process specimens only in response to written requests – do not consider oral requests. Insist on specimen request forms being kept separate from the specimens. Insist on adequately completed request forms and proper labelling of specimens. Differentiate specimens for diagnosis from those for control of treatment. Lack of this information rules out the use of monitoring of bacteriological results as a method of IQA. 20

22. QC – handling of specimens Process sputum specimens in batches according to centrifuge capacity. CAUTION : If delivery of specimens is delayed, contamination rate will increase 21

23. QC – reagents and media Discard media that are discoloured or have bubbles following inspissation. Check all batches of media for sterility by incubation at 35–37 ºC for 48 hours. Keep all media in the dark in the refrigerator and discard unused drug-containing media after 4 weeks. For culture and DST media: check with M. tuberculosis H37Rv. IQC for identification test with M. terrae and M. tuberculosis H37Rv 22

24. QC – preparation of egg-based media Sensitivity The numbers of colonies obtained by inoculating 5 tubes from a previous batch of media and 5 tubes from a newly prepared/purchased batch should be comparable. Sterility No bacterial growth should be present 48 hours after the incubation at 37 ºC of each new batch. Storage Avoid the accumulation of expired media. Estimate the number of tubes to be prepared or purchased to cover culturing needs for about 2 months of work. Store media in a cool and humid place, preferably in a refrigerator at 4 ºC. 23

25. Media register Batch no. Volume (ml) Date of:Sterility control Sensitivity control Preparation or purchase delivery Start of use End of use Contamination detected Yes/No Strain/ specimen inoculated Growth detected at 20 days (no. of colonies) Growth detected at 60 days (no. of colonies) Control batch Present batch Control batch Present batch 24

26. QC – media for DST H37Rv MIC expected INH 0.06 µg/ml RIF 4.0 µg/ml EMB 0.5 µg/ml SM 2.0 µg/ml Signature MIC Observed Date of batch production: __________ Date:___________ 25 Additional concentrations tested (µg/ml) INH 0.0250.050.10 RIF 2.55.010.0 EMB 0.1250.250.5 SM 0.51.02.0

27. QC – culture procedures: decontamination Use aliquoted reagent solutions. Do not keep all tubes open at the same time. Do not reuse the reagent solution aliquots opened during the work day. The total time of contact of the sample with the decontaminant must be strictly controlled. Too short a time results in high contamination rates, too long a time causes loss of viability of the bacilli. Be suspicious of several successive positive specimens or of cultures with few colonies following a heavily positive culture. 26

28. QC – reporting of results Always check for agreement between the laboratory register number and the number written on tubes/slants. Use WHO forms for reporting results. A copy of the completed final report should be retained in the laboratory for 2 years. 27

29. Reporting – turn-around time Smear from specimen (fluorochrome or ZN) – report positive or negative and the staining method used, preferably with no more than 24 hours delay from the moment of receipt of specimen in the laboratory. Culture contaminated: report immediately and a repeat specimen requested. Culture-positive and identification of M. tuberculosis: report immediately. Upon completion of the drug susceptibility test – susceptible or resistant to each test drug. 28

30. Reporting – turn-around time Culture-negative – upon completion of the incubation protocol (6 weeks for liquid, 8 weeks for solid). For solid media: - at 4 weeks, send an interim report on all negative stating that another report will be issued in the event of the specimen becoming positive later on - at 8 weeks, issue a final report 29

31. Daily monitoring – alarm signals Smear-positive/culture-negative specimens What are the possible explanations? What are the corrective measures? Contaminated specimens What are the possible explanations? What are the corrective measures? 30

32. Alarms and remedial actions Smear-positive/culture-negative specimens Follow-up? If for diagnosis: –Does this type of result recur? –Check the quality of the media being used. –Concentration of decontaminating solution? Time of contact with specimens? Temperature of incubator? Contaminated specimens Time between specimen collection and processing too long? –Corrective measures needed in specimen transportation system. Contamination of specimens from the same patient? –Use a harsher decontamination procedure for testing further specimens from the patient. 31

33. Daily monitoring – Alarm signals Recurrent contamination What are the possible explanations? What are the corrective measures? Clustering of culture-positive specimens What are the possible explanations? What are the corrective measures? 32

34. Alarms and remedial actions Recurrent contamination Check sterility of decontamination reagent solutions. Check the whole decontamination process. If the problem is traced to the technician performing the procedure, he or she should be immediately retrained. Clustering of culture-positive specimens Check contamination of decontamination solution Check that: –aliquoted reagent solutions are discarded after single use; –processing sequence of specimens is maintained, i.e. smear-positive specimens processed last; –tubes are not uncapped immediately after removal from centrifuge; –supernatants are discarded carefully. 33

35. Periodic monitoring EQA for whole culture process NOT feasible. Necessary to evaluate indicators monthly. DO NOT inoculate H37Rv in specimens to evaluate the culture process. 34

36. Periodic monitoring – indicators Classification of specimens from adult pulmonary TB patients investigated for diagnosis (not for follow-up): a.Smear-positive and culture-positive b.Smear-positive and culture not done c.Smear-negative and culture-positive d.Smear-positive and culture-negative e.Smear-positive and culture contaminated f.Smear not done and culture-positive 35

37. Periodic monitoring – indicators c + f Contribution of culture to diagnosis = ---------------------------- x 100 a + b + c + d + e + f Expected contribution: 20% Diagnosis of adult pulmonary cases – for a specific setting 36

38. Periodic monitoring – indicators c Contribution of culture to diagnosis = ------------------------- x 100 over microscopy a + c + d + e Expected contribution: >20% (higher with liquid culture) Diagnosis of adult pulmonary cases – for a specific setting 37

39. Periodic monitoring – indicators d Percentage of smear-positive = --------------------- x 100 and culture-negative cases a + c + d + e Expected: 2–3% 38

40. Periodic monitoring – indicators contaminated tubes Percentage contamination = ----------------------------- x 100 all inoculated tubes Expected: < 5% for solid <10% for liquid 39

41. Alarms: what to investigate Indicators of culture performance Normal value (%) Much higher: investigate Much lower: investigate Contribution of culture to bacteriological diagnosis of tuberculosis 20AB and C Percentage of smear-positive/ culture-negative specimens 2–32–3C and DNot a problem Percentage of contaminated tubes< 5% (solid) < 10% (liquid) EF 40

42. DST QC and indicators – IQC: Before use, new batches of media should be tested with a reference strain – Alarms: –change in patterns of resistance; –unusual resistance rate to a drug e.g. monoresistance to RMP higher than monoresistance to INH 41

43. EQA for culture : EQA for culture procedure: Indirect external supervision based on alarm signals, see above (slide 40) for QC EQA of culture media If low sensitivity in two consecutive controls: The laboratory is requested not to use or distribute culture media until the deficiencies are overcome. Culture media are offered so that the laboratory can continue its activity Retraining in culture media preparation procedures should also be offered, if needed. 42

44. International EQA for DST EQA of NRLs. Blinded test of a panel of 20 strains from SRLs. Parameters evaluated: sensitivity, specificity, reproducibility for each drug. Minimum agreement of 90% for at least isoniazid and rifampicin. If not reached, re-evaluate crucial points: drugs, media, procedures, supervision visit. 43

45. National EQA for DST EQA of national laboratories performing culture. Blinded test of the SRL panel of 20 strains from the NRL. Parameters evaluated: sensitivity, specificity, reproducibility for each drug. Minimum agreement of 90% for at least isoniazid and rifampicin. If not reached, re-evaluate crucial points: drugs, media, procedures, supervision visit. 44

46. Quality improvement It is not sufficient in IQC simply to identify errors or weaknesses in laboratory services; remedial action must be taken to permanently eliminate them. This implies continuous monitoring of performance, an IQC programme, as well as a direct supervision programme. 45

47. Keys to successful quality control Adequately trained, interested and committed staff. Common-sense use of practical procedures. Willingness to admit and rectify mistakes. Effective communication. 46

48. True and false take-home messages 1.Quality assurance is a system designed to discover and punish people who don’t work well. 2.It is useful periodically to calculate indicators to evaluate the performance of the laboratory. 47

49. Module review: take-home messages  A quality assurance (QA) programme is essential for improving the reliability, efficiency and use of laboratory services in order to achieve the required technical quality in laboratory diagnosis.  The process of quality assurance should be continuous and monitored  Quality assurance is the responsibility of all laboratory technicians and supervisors.  It is useful periodically to calculate indicators to evaluate the performance of the laboratory.  It is not sufficient in QC simply to identify errors or weaknesses in laboratory services; remedial action must be taken to permanently remove them. 48

50. Explain the three main components of the QA system. Which components of the laboratory workflow should be checked for QC? Self-assessment 49