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SPUTUM SMEAR MICROSCOPY DJD 2002Cebu

Sputum Smear Microscopy

in DOTS Programmes

David Dawson

WHO Collaborating Centre

The Prince Charles HospitalBrisbane Australia


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DOTS Strategy for TB Control

government commitment to ensuring sustained

comprehensive TB control activities

case detection by sputum-smear microscopyamong symptomatic patients

standardised short-course chemotherapy usingregimens of 6 to 8 months, for at least all

confirmed smear-positive cases. regular and uninterrupted supply of drugs

standardized recording and reporting systems


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The DOTS Partnership

N.T.P LABS


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TB suspects

MICROSCOPY

positive negative

NOT TB

TBcases

review


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Microscopy in DOTS

specimen collection and transport

laboratory facilities

smear preparation

Ziehl-Neelsen staining

microscopy


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7/36SPUTUM SMEAR MICROSCOPY DJD 2002Cebu

Specimen

poor

poor

good

good

Lab Skill

poor

good

poor

good

Result

poor

poor

poor

good

Quality of Final Result

vs Specimen Quality vs Lab Skill


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Specimen Collection

(DOTS Programmes)

spot spot

Day 1 Day 2

early a.m.

(clinic) (clinic)(home)



Specimen Collection and Transport

use appropriate container

ensure container is labelled, sealed

completed request form

2-5 ml sputum notsaliva

store in cool location

transport without delay

maintain specimen security



Laboratory Facilities



Laboratory Facilities

(for TB Microscopy)

defined, secure work area effective ventilation

electric microscope (x 1000) tidy uncluttered work bench

level staining sink microscopy bench

secure storage



Biosafety in TB Microscopy infection risk in microscopy is LOW

smear preparation carries highest risk

effective ventilation removes aerosol

staff must be educated in biosafety

personal protective devices (?? value)

disinfectants vs MTB (phenolics)

staff health should be monitored



TB Microscopy



Smear Preparation



Lab Procedure for TB Microscopy

1. Check patient details

(specimen containervs

request form)2. Enter details in Laboratory Register.

3. Label specimen with lab ref number4. Check (and record) specimen quality.

5. Prepare smear.6. Stain, examine, report.



QA Issues in Smear Preparation

1. Check, record, report specimen quality.

2. Keep lab register accurate, current.

3. Label slide before making smear.

4. Ensure smear prepared is representative

of the specimen.

5. Use gentle heat for fixing smear.(overheating destroys acid-fastness)



Smear Preparation

(TB Microscopy)

adequate size 1-2 x 2-3 cm



Smear Preparation for TB Microscopy

1. label slide 2. make smear

4. heat fix 3. air dry



Ziehl-Neelsen Staining


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Ziehl-Neelsen Stain

General Format

1. Primary Stain (carbol fuchsin)

2. Decolouriser (acid +/- alcohol)

3. Counterstain (methylene blue)


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M tuberculosisM tuberculosis ProteusProteus spsp e.g.e.g.

carbol fuchsin

HEAT

Principle of ZN Stain - 1

complex waxy cell wall


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M tuberculosisM tuberculosis ProteusProteus spsp

carbol fuchsin

decolourise



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M tuberculosisM tuberculosis Proteus spProteus sp

carbol fuchsin

decolourise


counterstain


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M tuberculosisM tuberculosis Proteus spProteus sp

carbol fuchsin

decolourise


counterstain


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3% HCl in ethanolor ethanol + 5% H2SO4

or 5% H2SO4

or 25% H2SO4

ZN Decolourisation Conditions


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semi-uniform transparent film

2 cm by 1 cm

free of stain deposit, artefact

MTB stained deep red

counterstain gives contrast

Attributes of High Quality

Ziehl-Neelsen Preps


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Quality Assurance Issues

Labels on reagent bottles should show:

1. Name of reagent.

2. Preparation date.3. Where prepared and by whom.

4. Expiry date (6 months for ZN).5. Date/s of QC checks (for CF).


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Quality Assurance Issues

Labels on reagent bottles should show:

1. Name of reagent.

2. Preparation date.3. Where prepared and by whom.

4. Expiry date (6 months for ZN).5. Date/s of QC checks (for CF).


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QA Issues in ZN Staining

1. Use standard, documented method.

2. Use mycobacteria-free water for makingcarbol fuchsin.

3. Perform QC on all reagents before use.(Results of QC must be documented.)

4. Leave hot CF in contact 5-10 mins.

How often should control slides be run?


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Smear Examination


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Examination of ZN Smears

Under oil immersion, examine at

least 100, but up to 300,useful, representative fields

(minimum time 5 mins)


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Reporting ZN Results

No AFB No AFB found in 100 fields

xAFB 10 AFB per field

S f E i


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Sources of Error in

Acid-fast Microscopy

False NEGATIVE poor specimen quality

faulty smear preparation

poor quality carbol fuchsin

carbol fuchsin exposure too short

technician inexperience

S f E i


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False POSITIVE

AFB contamination container slide specimen carryover carbol fuchsin solution

artefact in specimen/smear

technician inexperience

Sources of Error in

Acid-fast Microscopy

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