Sputum Smear Microscopy

in DOTS Programmes

David Dawson
WHO Collaborating Centre
The Prince Charles Hospital
Brisbane Australia

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 DOTS Strategy for TB Control

• government commitment to ensuring sustained

comprehensive TB control activities
• case detection by sputum-smear microscopy
among symptomatic patients
• standardised short-course chemotherapy using
regimens of 6 to 8 months, for at least all
confirmed smear-positive cases.
• regular and uninterrupted supply of drugs
• standardized recording and reporting systems

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 The DOTS Partnership

N.T.P LABS

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 TB suspects

MICROSCOPY

positive negative

review
TB
cases
NOT TB
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Microscopy in DOTS

• specimen collection and transport

• laboratory facilities
• smear preparation
• Ziehl-Neelsen staining
• microscopy

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Specimen Collection
and Transport

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Quality of Final Result
vs Specimen Quality vs Lab Skill

Specimen Lab Skill Result

poor poor poor
poor good poor
good poor poor
good good good

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Sputum Collection Container

• screw-capped
• water-tight
• translucent
• wide-mouthed
• sturdy
• disposable
• 30-40 ml
• sterile
• clean
• labelled
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Specimen Collection
(DOTS Programmes)
Day 1 Day 2

spot early a.m. spot

(clinic) (home) (clinic)
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Specimen Collection and Transport

• use appropriate container

• ensure container is labelled, sealed
• completed request form
• 2-5 ml sputum not saliva
• store in cool location
• transport without delay
• maintain specimen security

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Laboratory Facilities

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 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

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 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

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 TB Microscopy

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 TB Microscopy

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Smear Preparation

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Lab Procedure for TB Microscopy

1. Check patient details

(specimen container vs request form)
2. Enter details in Laboratory Register.
3. Label specimen with lab ref number
4. Check (and record) specimen quality.
5. Prepare smear.
6. Stain, examine, report.

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 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)

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Smear Preparation
(TB Microscopy)

adequate size 1-2 x 2-3 cm

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Smear Preparation for TB Microscopy

1. label slide 2. make smear

4. heat fix 3. air dry

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Ziehl-Neelsen Staining

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Ziehl-Neelsen Stain

General Format

1. Primary Stain (carbol fuchsin)

2. Decolouriser (acid +/- alcohol)

3. Counterstain (methylene blue)

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Principle of ZN Stain - 1

M tuberculosis Proteus sp e.g.

complex waxy cell wall

carbol fuchsin

HEAT

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Principle of ZN Stain - 2

M tuberculosis Proteus sp

carbol fuchsin

decolourise

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Principle of ZN Stain - 3
M tuberculosis Proteus sp

carbol fuchsin

decolourise

counterstain

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Principle of ZN Stain - 4
M tuberculosis Proteus sp

carbol fuchsin

decolourise

counterstain

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 ZN Decolourisation Conditions

„ 3% HCl in ethanol
or ethanol + 5% H2SO4
or 5% H2SO4
or 25% H2SO4

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Attributes of High Quality
Ziehl-Neelsen Preps

semi-uniform transparent film

2 cm by 1 cm
free of stain deposit, artefact
MTB stained deep red
counterstain gives contrast
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 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).

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 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).

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 QA Issues in ZN Staining

1. Use standard, documented method.

2. Use mycobacteria-free water for making
carbol 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?

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Smear Examination

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Examination of ZN Smears
Under oil immersion, examine at
least 100, but up to 300,
useful, representative fields
(minimum time 5 mins)

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 Reporting ZN Results

No AFB No AFB found in 100 fields

x AFB <10 AFB found in 100 fields

1+ AFB 1-10 AFB per 10 fields

2+ AFB 1-10 AFB per field

3+ AFB >10 AFB per field

SPUTUM SMEAR MICROSCOPY Cebu DJD 2002

 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
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002
 Sources of Error in
Acid-fast Microscopy
False POSITIVE
• AFB contamination
Š container
Š slide
Š specimen carryover
Š carbol fuchsin solution
• artefact in specimen/smear
• technician inexperience
SPUTUM SMEAR MICROSCOPY Cebu DJD 2002