MUNICIPAL CORPORATION OF DELHI.

SWAMI DAYANAND HOSPITAL SHAHADARA.

“ SOURCES OF ERROR

IN

SMEAR EXAMINATION FOR A.F.B.”

Presented By. Dr.P.P.Singh.

Laboratory Diagnostic Methods

1. By Direct evidence:

a) Demonstration of AFB in Direct Smear.

b) Demonstration of AFB after concentration.

c) Culture Methods.

d) Histopathological Examination.

1. By Indirect evidence:

a) Routine Haematological

b) Erythrocyte Sedimentation Rate.

c) Serological Methods.

d) Animal inoculation.

1. Selection of the Patient.

2. Preparation of the Patient.

3. Collection of the Sample.

4. Transportation of the Sample.

5. Instrumentation Stage ( Methodology).

6. Reporting of Results.

7. Assessment of the result with condition of the Patient.

Varities of Specimens are:-

a) Sputum.

b) Laryngial Swab.

c) Gestric Aspiration.

d) Plueral/Peritonial fluid.

e) Urine.

f) Transtracheal / Broncheal Aspiration.

g) Bronchioalveolar Lavage.

h) Stool.

i) Cerebro Spinal Fluid.

j) Tissue as Biopsy.

k) FNAC.

COMMONLY USED STAINING METHODS

i. Ziehl Neelson Method.

ii. Kinyoun’s ( Cold Stain ) Method.

iii. Fluorescent Staining Method.

• Auromine – ‘O’ Stain.

• Auramine Rodamine B Stain.

• Acridine Orange as counter Stain.

Slide 20 x 10 mm- 0.01ml area

Loop 3mm only 1% area if 100 oil field

Smear.for AFB

GRADING OF A.F.B. SMEAR

No. of Bacilli. Report.

0 No Acid Fast bacilli found

1 – 2 in Whole Smear Report Number & Report.

3 – 9 in Whole Smear Rare or +

10 or More Fair or ++

1 or more/field Numerous or +++

PROBABILITY OF POSITIVE RESULT DEPEND UPON:-

a. Smear area examined.

b. No. of bacilli in specimen .

c. Type of specimen.

No. of Bacilli. Probability of Positive result

1 0 in 100 field Less than 10%

2 1-2 in 300 field 50%

3 1-9 in 100 field 80%

4 1-9 in 10 field 90%

False Positive Results.

A. Acid Fast Particles other than Tubercular bacilli.

• Food Particles.

• Precipitate of Stains.

• Saprophytic AFB. M.kansasi , Nocardia.

• Spores of Bacillus subtalis.

• Fibers & Pollen - Wool, Cotton, Bamboo etc.

• Scratches on the Slides.

B. Contamination through the transfer of bacilli from one slide to another.

FALSE NEGATIVE RESULTS:-

i. Inadequate Collection.

ii. Inadequate Storage.

iii. Failure to select

iv. Inadequate Staining

a. Too little material on slide so smear is thin.

b. The smear is too thick, so sufficient light cannot pass through it.

c. Overheating of slide.

d. Smear not fixed properly.

e. Staining with carbol fuchsine is too short or over done by boiling.

f. Too intensive counter stain.

Contd.

v. Inadequate Examination of Smear .

• Erratic or too brief Screening.

• Too few fields Examined

• Colour blindness or other visual defects.

vi. Administrative Errors.

• Misidentification.

• Mistake in Labelling.

• False recording or reporting.

Positive Rate of Direct Smear:-

1. Sputum 40 – 55%

2. Laryngial Swab 25 – 50%

3. Gastric Aspiration. 25-50%

4. Fluids 20-25%

5. C.S.F. 15 – 25%

6. Urine. 10 – 15%

7. Tissues Biopsies 6 - 15%

F.N.A.C. 40 –50%

Concentration Methods.

1.Petroff’s method.

2.N.Acetyle L. Cystien (NACL) + 2% NaOH.

3. Dithriothritol + 2% NaOH (Sputolysin).

4. 13% Trisodium Phosphate + Benzalconium Chloride.

5. 1% Cetyle pyredium Chloride + 2% NaCl.

6. 4% Sulphuric Acid.

7. 5% Oxalic acid.

8. Floatation method, using solvent like Xylol.