External Quality Assessment EQA for AFB Microscopy

1. External Quality Assessment (EQA)for AFB Microscopy

2. Area 2 a microscope table, (if there is no electricity, this table should be placed directly in front of a window) Area 2 a microscope table, (if there is no electricity, this table should be placed directly in front of a window)

3. Labeling of Specimens   It is critical to label a specimen properly to avoid confusion and delay.   A request form must accompany each specimen. The information on the form must exactly match the information on the container. Always label the container on the side, never on the lid.   Using an indelible marking pen, write the name of the TB suspect and the date of collection. Labeling of Specimens   It is critical to label a specimen properly to avoid confusion and delay.   A request form must accompany each specimen. The information on the form must exactly match the information on the container. Always label the container on the side, never on the lid.   Using an indelible marking pen, write the name of the TB suspect and the date of collection.

4. Washing Hands   Always wash hands with soap and water after handling specimens and containers. Washing Hands   Always wash hands with soap and water after handling specimens and containers.

5. The Laboratory Register   The WHO or IUATLD laboratory register is strongly recommended as a guideline. The format of the register should never be altered.  The Laboratory Register   The WHO or IUATLD laboratory register is strongly recommended as a guideline. The format of the register should never be altered.  

6. If the slide is not frosted, engrave the laboratory serial number using a diamond pointed stylus.   Never use a grease pencil, since markings could wash off during the staining process. If the slide is not frosted, engrave the laboratory serial number using a diamond pointed stylus.   Never use a grease pencil, since markings could wash off during the staining process.

7. Using a continuous rotation, cover an oval area approximately 2 cm in length on the slide. Put only one smear on each slide. Using a continuous rotation, cover an oval area approximately 2 cm in length on the slide. Put only one smear on each slide.

8. Gently wash the stain from each slide with a stream of cold water until all free stain has washed away. Always rinse gently so the smear is not washed off of the slide. Individually tilt each slide to drain off any excess rinse water. This prevents puddles remaining on the slide which can dilute the next reagent. Gently wash the stain from each slide with a stream of cold water until all free stain has washed away. Always rinse gently so the smear is not washed off of the slide. Individually tilt each slide to drain off any excess rinse water. This prevents puddles remaining on the slide which can dilute the next reagent.

9. Arrange the following supplies next to the microscope: a bottle of immersion oil, organic solvent, lens paper, laboratory register and a slide box for storage.   Use the 40X objective to focus and determine a suitable reading area of the slide.   Place a drop of immersion oil on the stained smear. Let the drop fall freely onto the slide.   Never touch the slide with the oil applicator. This could lead to carry over of AFB in the immersion oil to the next slide.   Turn the nosepiece to bring the 100X objective into place. Now, gently lower the 100X objective.   It should barely touch the oil. Never allow the lens to touch the slide. This can damage the lens and possibly break the slide. While looking through the eye-piece, adjust the immersion lens slowly and focus until the image on the smear appears. To fine focus, turn the fine adjustment knob carefully. Arrange the following supplies next to the microscope: a bottle of immersion oil, organic solvent, lens paper, laboratory register and a slide box for storage.   Use the 40X objective to focus and determine a suitable reading area of the slide.   Place a drop of immersion oil on the stained smear. Let the drop fall freely onto the slide.   Never touch the slide with the oil applicator. This could lead to carry over of AFB in the immersion oil to the next slide.   Turn the nosepiece to bring the 100X objective into place. Now, gently lower the 100X objective.   It should barely touch the oil. Never allow the lens to touch the slide. This can damage the lens and possibly break the slide. While looking through the eye-piece, adjust the immersion lens slowly and focus until the image on the smear appears. To fine focus, turn the fine adjustment knob carefully.

11. External Quality Assessment Early warning-system for problems Measure of laboratory quality Valuable benchmarking tool (standardization and traceability) Indicator of where to direct improvement efforts Monitor of changes in technology and testing practices (evaluation component)

13. Take Home Message The primary responsibility for National TB Reference Laboratories is supporting AFB microscopy networks through implementation of EQA and training

14. International GuidelinesEQA – AFB Microscopy External Quality Assessment (EQA) Components onsite evaluation rechecking proficiency testing

15. Key Features of EQA guidelines Resource analyses and phased approach for EQA Identify laboratories with performance problems---not intended to identify individual errors or validate patient diagnosis Emphasizes the need for onsite evaluation of laboratories using standard indicators Recognizes panel/proficiency testing as a method of EQA

16. Key Features of EQA Guidelines Rechecking Emphasis on blinded random sample Emphasis on the smallest possible sample size that provides some information on test performance (sensitivity) Provides information to measure performance---based on the number and types of errors

17. EQA -- AFB MicroscopyOn-site Evaluation -- Background Many countries lack the resources for annual visits of peripheral laboratory by central laboratory staff Laboratories in most countries are visited by a non-laboratory District supervisor Optimum evaluation is performed by trained laboratory staff in supervisory role

18. EQA – AFB Microscopy Guidelines Key FeaturesOn-site Evaluation Develop a standard checklist of questions and indicators Include minimal evaluation that can be performed by non-laboratory trained personnel (e.g., inventory supplies, reagents, equipment) Include detailed evaluation that can be performed by supervisory laboratory staff Train laboratory and non laboratory staff to assure consistent application

19. On-site Evaluation Results - Uganda

20. On-site Evaluation Performed by Non-laboratory Staff Examples: Are all staining reagents available and within expiration dates? How are wire loops cleaned? Is the laboratory register present and all columns completed properly? How is maintenance on the microscope performed?

21. Quality Control in the TB Laboratory   Quality Control (QC) should be routinely performed in the laboratory to ensure reliability as well as reproducibility of results.   Perform QC on each new batch of stain. Observe these results before reading patient slides. This important step will verify correct staining procedure and microscope function.   When controls are not demonstrating proper staining, determine the cause and correct it.   If the negative control appears red after the staining procedure, this is the result of incomplete decolorization. Quality Control in the TB Laboratory   Quality Control (QC) should be routinely performed in the laboratory to ensure reliability as well as reproducibility of results.   Perform QC on each new batch of stain. Observe these results before reading patient slides. This important step will verify correct staining procedure and microscope function.   When controls are not demonstrating proper staining, determine the cause and correct it.   If the negative control appears red after the staining procedure, this is the result of incomplete decolorization.

22. On-site Evaluation Performed by Laboratory Staff Examples: Does the technician verify that the container is properly labeled? How are slides labeled? How often is the carbol fuchsin filtered? How many fields are examined to report a negative smear?

23. EQA - AFB MicroscopyProficiency Testing - Background Uncommon in resource-limited countries Prepared smears (South Africa) or patient slides (Senegal) sent from central laboratory Consistent challenge of laboratory test performance PT test performance may be different from testing routine patient specimens

24. EQA – AFB Microscopy Guidelines Key FeaturesProficiency Testing Laboratory may re-use patient slides but a procedure is provided to produce test slides for consistent slide sets Recommended slide set is 10 slides: 5 stained and 5 unstained Simple forms for slide production and collection of test results

25. PT Implementation in Mexico Inspected 587 of 637 laboratories 604 microscopists given a 2 hour, 10 slide test 54% had score >80 33% had score 60-79 13% had score <60 536/604 (88.7%) finished all 10 slides 216 persons with score <80 received training followed by second PT: average scores improved from 65 to 90 (P-value < 0.0001).

26. EQA - AFB MicroscopyRechecking - Background Recommended by IUATLD and WHO Usually 100% of positive and 10% of negative smears Usually un-blinded — adds bias* Reviews patient testing; including smear preparation, staining, and interpretation *Lan N.T.N. et al, 1999 Int J Tuberc Lung Dis 3(1): 55-61

27. EQA – AFB Microscopy Guidelines Key FeaturesRechecking Emphasizes “blinding” and random sample using the laboratory register Sample size is based on Lot Quality Assurance Sampling (LQAS) with parameters selected for test volume and desired sensitivity Positives and negatives sampled Minor errors (FP or FN with 1-9 AFB/ 100 f) are included as a measure of performance

28. Comparison of random blinded re-checking to non-blinded rechecking of AFB Smears in Mexico

29. Comparison of random blinded re-checking to non-blinded rechecking of AFB Smears in Mexico

30. Sample Size Comparison of the old method* and new EQA guidelines

31. EQA - AFB Microscopy Guidelines Key FeaturesResource Analysis Inventory available resources (actual/projected) Manpower, supplies, communication, administrative, financial Examine effectiveness of current EQA activities Gather laboratory service information Planning—options for the evolution of EQA Pilot test and document changes Expansion based on availability of resources

32. Resources to implement on-site evaluation The NTP will need to consider: Capacity of the reference laboratory staff to provide on-site evaluation of all intermediate laboratories at least annually. Capacity of intermediate laboratories to provide on-site inspection of the peripheral laboratories at least annually, and more frequently to correct problems identified. Availability of properly trained non-laboratory personnel to make supervisory visits at least quarterly (as required for DOTS). Capacity to implement necessary QI measures.

33. Resources to implement panel testingThe NTP will need to consider: Available financial support. Proficiency of reference laboratory staff to perform ZN AFB smear microscopy. Capacity of the reference laboratory staff to prepare panel testing slide sets for the laboratories to be evaluated Available mechanisms to deliver slides to the peripheral sites, including mail and couriers. Capacity of the reference laboratory staff to review and evaluate results from peripheral laboratories, and provide recommendations and follow-up for corrective action.

34. Resources to implement blinded rechecking The NTP will need to consider: Available financial support. Capacity of peripheral laboratories to store smears for rechecking. Availability of properly trained personnel to collect appropriate samples of slides from peripheral sites. Capacity of the reference laboratory staff at central and intermediate level laboratories to reread smears from peripheral sites, including second rereading to resolve discrepancies as needed. Capacity of reference laboratories to provide results of rechecking as well as feedback to implement effective corrective action.

35. EQA – AFB Microscopy GuidelinesResource AnalysisPhased Approach Assure the five elements of DOTS Develop a central reference and intermediate laboratories to carry out EQA Determine the existing capacity for EQA Train district health officials to evaluate the minimal functions of microscopy laboratories

36. EQA – AFB Microscopy GuidelinesResource AnalysisPhased Approach (cont) Proficiency testing to evaluate performance Pilot rechecking program Determine resources: additional PT or phased implementation of rechecking

38. The Keys to Successful Quality Assurance Include: Adequately trained, interested, and committed staff Common sense use of practical procedures A willingness to admit and rectify mistakes Effective communication Ref: Laboratory Services in Tuberculosis Control, WHO