Afb microscopy and quality assurance copy

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Afb microscopy and quality assurance copy

  1. 7. <ul><li>SPUTUM CONCENTRATION </li></ul><ul><li>NEBULISED SPUTUM </li></ul><ul><li>BRONCHIAL WASH CENTRIFUGE </li></ul><ul><li>BRONCHOALVEOLAR LAVAGE 3000 RPM × 15MINS </li></ul><ul><li>GASTRIC L AVAGE NEUTRALISATION </li></ul>
  2. 8. <ul><ul><ul><li>less laborious </li></ul></ul></ul><ul><ul><ul><li>less robust </li></ul></ul></ul><ul><ul><ul><li>higher concentration fuchsin </li></ul></ul></ul><ul><ul><ul><li>longer staining time errors !! </li></ul></ul></ul><ul><ul><ul><li>NOT RECOMMENDED for low-income countries </li></ul></ul></ul>
  3. 10. <ul><li>SMEAR PREPARATION </li></ul><ul><li>With applicator stick from the thick/yellowish part make 15 mm by 20 mm oval smear </li></ul><ul><li>FIXATION </li></ul><ul><li>Once dried in air pass the slides 2-3 times over flame or by sprit for 3 mins </li></ul><ul><li>STAINING </li></ul><ul><li>Cover the slide with Carbol fuschin 0.3% after filtering through Whatman filter paper no.1 </li></ul><ul><li>Heat the slide until it steams and continue it for 5 minutes (don’t allow the stain to dry) </li></ul><ul><li>Wash it under tap water </li></ul><ul><li>Cover the slide with 3% acid alcohol (HCl and 95% alcohol or 20% Sulphuric acid (1 minute) </li></ul><ul><li>Counterstain with malachite green for 1 minute </li></ul>
  4. 11. <ul><li>Each lab staff must know that the reagents for staining are highly corrosive substances and must ensure personal protection </li></ul><ul><li>Efforts must me made to minimize and control lab operations that induce potentially infectious aerosols </li></ul><ul><li>Opening of container/preparing smears on slides/flaming </li></ul><ul><li>Use safety cabinet always!! </li></ul><ul><li>Sterilize the loop after smear </li></ul>
  5. 12. <ul><li>Note: Color of AFB may vary with filter system on microscope </li></ul>
  6. 14. Counter Stain Background
  7. 20. Accuracy : The closeness of measurements to the true value. Precision : The amount of variation in the measurements. Bias: The difference between the expectation of a test result and an accepted reference value.
  8. 21. Location Category False Negative False positive Pre-laboratory Administrative <ul><li>Specimen quality </li></ul><ul><li>Specimen labeling </li></ul><ul><li>Patient identification </li></ul><ul><li>Transport conditions </li></ul><ul><li>Specimen </li></ul><ul><li>Labeling </li></ul><ul><li>Patient identification </li></ul><ul><li>Specimen container </li></ul>Laboratory Administrative <ul><li>specimen handling </li></ul><ul><li>specimen registration </li></ul><ul><li>recording and/or reporting result </li></ul><ul><li>specimen registration </li></ul><ul><li>recording and/or reporting result </li></ul>Technical <ul><li>smear preparation </li></ul><ul><li>stain formulations </li></ul><ul><li>staining technique </li></ul><ul><li>microscope performance </li></ul><ul><li>smear examination technique </li></ul><ul><li>smear preparation </li></ul><ul><li>stain formulations </li></ul><ul><li>staining technique </li></ul><ul><li>smear examination technique </li></ul>
  9. 22. <ul><li>What is a good sample? </li></ul><ul><ul><li>What is saliva? </li></ul></ul><ul><ul><li>Good sample = yellow? mucous fluid? </li></ul></ul><ul><ul><li>Discharge from the bronchial tree </li></ul></ul><ul><ul><li>May contain solid or purulent substances </li></ul></ul><ul><ul><li>Minimal amounts of oral/ nasal material </li></ul></ul><ul><ul><li>May contain macrophages and other cells indicative of infectious disease </li></ul></ul><ul><li>Leakage– ask for new sample </li></ul><ul><li>Mucopurulent or purulent required; NOT SALIVA </li></ul><ul><li>Purulent:thick sticky greenish </li></ul><ul><li>Mucopurulent: thick brown or green </li></ul><ul><li>Mucoid:viscous and clear </li></ul><ul><li>Mucosalivary: mucoid </li></ul><ul><li>Minimum 2 ml </li></ul><ul><li>Correct labeling </li></ul>
  10. 23. <ul><li>Smearing </li></ul><ul><ul><li>delay: no problem (keep away from sun) </li></ul></ul><ul><ul><li>&quot;good particle“ homogenization may be more reliable </li></ul></ul><ul><ul><li>standard size: for ease of quantification </li></ul></ul><ul><ul><li>thickness, evenness: find a balance </li></ul></ul><ul><ul><li>Sensitivity to light, counter-stain </li></ul></ul>
  11. 24. <ul><li>Are they worth the effort? </li></ul><ul><ul><li>NaOH digestion + centrifugation : CULTURE </li></ul></ul><ul><ul><li>Hypochlorite (NaOCl) digestion & concentration </li></ul></ul><ul><ul><ul><li>homogenization, easy background </li></ul></ul></ul><ul><ul><ul><li>oxidation: staining easier </li></ul></ul></ul><ul><ul><ul><li>co-flocculation with proteins ?? </li></ul></ul></ul><ul><ul><ul><li>concentration by sedimentation, centrifugation, filtration or flotation </li></ul></ul></ul><ul><ul><li>Contradictory reports on efficiency </li></ul></ul>
  12. 26. <ul><li>Carbol fuchsin staining </li></ul><ul><ul><li>Uses higher fuschsin concentration </li></ul></ul><ul><ul><li>Dissolve well !!! </li></ul></ul><ul><ul><ul><li>IUATLD/WHO : 0.3% </li></ul></ul></ul><ul><ul><li>Heat well-apply long enough </li></ul></ul><ul><ul><li>Cold staining!! </li></ul></ul><ul><ul><li>Batch staining- great potential for cross- contamination </li></ul></ul>
  13. 27. <ul><li>Decolorization </li></ul><ul><ul><li>must be complete </li></ul></ul><ul><ul><li>not possible to de-stain too much </li></ul></ul><ul><ul><li>repeat as needed </li></ul></ul><ul><ul><li>use strong acids </li></ul></ul><ul><ul><li>alcohol not absolutely needed </li></ul></ul>
  14. 28. <ul><ul><li>Provides good contrast for observation of AFB </li></ul></ul><ul><ul><li>background for focusing, not too strong </li></ul></ul><ul><ul><li>methylene blue 0.3% ? </li></ul></ul><ul><ul><ul><li>diluted or < 1 min </li></ul></ul></ul><ul><ul><ul><li>use of malachite green? </li></ul></ul></ul>
  15. 29. <ul><ul><li>Red slender rods on blue background </li></ul></ul><ul><ul><ul><li>accept only typical shape, at least some </li></ul></ul></ul><ul><ul><li>depends condition of microscope! </li></ul></ul><ul><ul><li>light! binocular, mechanical stage, good optics,100x oil immersion objective, 10x eyepieces </li></ul></ul><ul><ul><li>Requires: patience, sincerity </li></ul></ul><ul><ul><ul><li>AFB microscopy is not difficult but tough </li></ul></ul></ul>
  16. 30. <ul><li>1-9 AFB in 100 fields Exact number </li></ul><ul><li>10-99 in 100 fields AFB seen + </li></ul><ul><li>1-10 in 50 fields AFB seen ++ </li></ul><ul><li>> 10 in 20 fields AFB seen +++ </li></ul><ul><li>NO bacilli No AFB seen </li></ul>
  17. 38. Thank you for your kind attention

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