1. 1 Module AFB MICROSCOPY

2. 2 Content Overview Collection of sputum specimens Quality of sputum specimens Processing of sputum specimens Registration of sputum specimens Sputum smearing and staining Smear examination Recording and reporting

3. 3 Collection of Sputum Specimens Collect 1-3 sputum specimens for diagnostic purposes according to NTP policy Follow NTP policy for declaring or excluding a smear-positive case Follow NTP policy for follow-up smears

4. 4 Instructions to Patients Give clear instructions to patients on how to collect the sputum: Importance of sputum examination for TB diagnosis Need for real sputum, not saliva How to produce good sputum How to open and close the container How to avoid contamination of outside of the container

5. 5 Characteristics of Good Sputum Specimen 3-5 ml Usually thick and mucous, but may be fluid with pieces of purulent material Color varies from opaque white to green, reddish to brown when blood is present Clear saliva is not suitable; but examine saliva if a better specimen can not be produced, especially for follow-up examinations A specimen mainly containing blood should not be examined; patient should see a doctor for immediate management

6. 6 Processing Sputum Specimens Process specimens as soon as possible for the benefit of the patients However, for microscopy time between collection and staining matters little a refrigerator is not needed to keep samples After preparation of a fixed smear, further processing can wait

7. 7 Collection and Transportation of Sputum Options if the health facility attended by the patient does not perform smear microscopy Refer patients to nearest microscopy facility Have the specimens taken by assigned person or sent by public transport to microscopy facility Have the specimens collected, smears prepared and fixed and taken to microscopy facility by assigned person

8. 8 Registration of Sputum Specimens Register all specimens for AFB microscopy in the TB Laboratory Register One line for the diagnostic smears of each patient One line for each follow-up One or more results may be registrered on one line Label the sputum containers on the side, not on the lid, with laboratory serial number and the column number or code for diagnostic smears Label the slides with the laboratory serial number and the column code

9. 9 Sputum Smearing (1) Select new, clean, grease-free, unscratched slides, free of fingerprints Use a pencil in case of a frosted slide and a diamond pencil in case of unfrosted slides to write the serial number Ensure that the number on the slide is the same as on the sputum container

10. 10 Sputum Smearing (2) Select and pick up yellowish (or whitish) particles with an applicator stick, e.g. bamboo, or a wire loop Prepare the slide in oval shape in the center of the slide, 2 (length) by 1 (width) cm or 3 by 2 cm Crush thick particles with the stick/wire loop, spread for a sufficiently long time to obtain even smears

11. 11 Sputum Smearing (3) Place the used stick in a discard container; use a new stick for each specimen Dip the wire loop in a sand-alcohol bottle; move the loop up and down to remove excess sputum; heat in flame until red-hot Air-dry the smear at room temperature If the smear is completely dry, pass the slide 2-3 times, 2-3 seconds over a flame, holding the slide upwards

12. 12 Smear Staining with ZIehl-Neelsen Stain (1) A well stained ZN smear shows strong red AFB, against a weak blue background Background should not show much remaining red and no red artifacts Proposed technique is to use 1% carbol fuchsin (strong red) and 0.1% methylene blue (weak blue) as a counter stain Destaining with 3% hydrochloric acid in alcohol or 20-25% sulphuric acid in water must be complete can be repeated to ensure complete absence of red color from the background

13. 13 ZN Stained Smear

14. 14 STEPS: Arrange slides in a serial order on a staining bridge, smear side up, avoid slides touching each other Cover slides completely with carbol fuchsin solution (through filter paper) Gently heat to steam Leave for 10-15 minutes Rinse with water and drain Cover with decolorizing solution, 3-5 min, rinse with water, drain. Repeat if needed, rinse and drain Apply methylene blue solution for 1, at most 2 minutes Rinse with water and drain Air dry on a slide rack Smear Staining with Ziehl-Neelsen Stain (2)

15. 15 Smear Staining with Auramine (1) STEPS Arrange slides in a serial order on a staining bridge, slide side up, avoid slides touch each other Apply filtered auramine solution, leave for at least 15, max. 30 minutes Rinse with water and drain Apply decolorizing solution, 1-3 minutes, rinse with water and drain, repeat if needed, rinse with water and drain Apply counter stain for max. 1 minute Rinse with water and drain Air dry on a slide rack

16. 16 AFB show yellow or green fluorescence against a more or less dark background, depending on the lamp and filters used Destaining solution must have alcohol; acid usually 0.5% HCl Usual counter stain is 0.5% potassium permanganate Keep stained slides in the dark, if examination cannot be done immediately after staining Smear Staining with Auramine (2)

17. 17 Auramine Stained Smear (1)

18. 18 Auramine Stained Smear (2)

19. 19 Examination of ZN Smears Examine 1 length of the smear (2 cm) or 100 fields with the bright field microscope, 1000x magnification Declare the smear as negative if no AFB are found If <10 AFB are found, count the number of AFB If >= 10 AFB are found, grade the smear 1+, 2+, or 3+ For high positives examination of 20-30 fields may suffice

20. 20 Examination of Auramine Stained Smears Examine 1 length of the smear (2 cm) with FM best use a 20x objective: 1 length is 30 fields with this objective, but 3 times the area seen through a 100X objective if a 40x objective is used, one length counts 40-50 fields or 2 times the area seen through a 100X objective If necessary, confirm AFB, typical curved rod shape, turning to a higher power objective (40X if screening with 20X)

21. 21 Grading of Auramine Stained Smears Examine one length if no or few AFB are seen declare negative if none at all are seen if less than 30 AFB are found using 20x objective, or less than 20 AFB using 40x objective, the result is scanty. Report the exact number of AFB per length if AFB per length count 30 to 299 using 20X objective, or 20 to 199 using 40X objective, grade the smear as 1+ Examine about 10 fields if many AFB are seen if on average there are more than 100 AFB per field with 20X objective, or more than 50 with 40X, grade the smear as 3+ if per field between 10 and 100 AFB are seen with 20X objective, or between 5 and 50 with 40X objective, grade as 2+

22. 22 Recording and Reporting Write the smear results in the TB Laboratory Register Write the smear results on the sputum examination request forms Forward the filled sputum request form to the person requesting the sputum examinations

23. 23 Grading of ZN Smears IUATLD/WHO scaleZN 1000x, 1 length=100 HPF Negative0 AFB / 1 length Scanty1-9 AFB / 1 length 1+10-99 AFB / 1 length 2+1-10 AFB / 1 HPF on average 3+>10 AFB / 1 HPF on average

24. 24 Grading of Smears in FM Using 20X Objective IUATLD/WHO scaleFM 20x, 1 length = 30 fields 1 length = 300 HPF Negative0 AFB / 1 length Scanty1-29 AFB / 1 length 1+30-299 AFB / 1 length 2+10-100 AFB / 1 field on average 3+>100 AFB / 1 field on average

25. 25 Grading of Smears in FM Using 40X Objective IUATLD/WHO scaleFM 400x, 1 length = 40-50 fields 1 length = 200 HPF Negative0 AFB /1 length Scanty1-19 AFB / 1 length 1+20-199 AFB / 1 length 2+5-50 AFB / 1 field on average 3+>50 AFB / 1 field on average

26. 26 Laboratory Register for Sputum Smear Microscopy

27. 27 Request and Reporting Form for Sputum Smear Examination

28. 28 Key Messages Good quality specimens, smearing, staining and reading are all essential for correct diagnosis of tuberculosis Ziehl-Neelsen and fluorochrome methods are both suitable, but fluorescence is better in case of high workload and/or few AFB Grading scales for quantification of AFB in smears vary with the magnification system and thus objective used The specific request form and the laboratory register must always be used to record and report smear examination results