Laboratory Diagnosisof Tuberculosis by SputumMicroscopy
The
hand
book
Paci
fic Is
land
Cou
ntrie
s
Richard LumbIvan Bastian
AcknowledgmentsThis edition of The Handbook, for Pacific Island Countries, was produced with financial assistance from the South Australian TB Services within the Royal AdelaideHospital Department of Thoracic Medicine, and the Australian Tuberculosis and Chest Association.
The authors wish to thank the following for their valuable input and support:
• Dr Armand van DeunBacteriology ConsultantUnionAntwerp, Belgium
• Mr David DawsonTB Laboratory ConsultantBrisbane, Australia
• Mr John ElliotPacific Paramedical Training CentreWellington, New Zealand
• Ms Linda KuoDepartment of Health ServicesRichmond, California, USA
• Ms Kay WithnallLaboratory ConsultantDarwin, Australia
• Prof Barrie Vernon-RobertsIMVS
Laboratory Diagnosis of Tuberculosis by SputumMicroscopy
The
hand
book
Page 1LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Published by:Institute of Medical and Veterinary ScienceFrome Road Adelaide South Australia 5000www.imvs.sa.gov.au
All rights reserved. No part of this book may be reproduced ortransmitted in any form or by any means without the written permissionof the publisher. The authors assert their moral rights in the work.
ISBN 0-9750285-2-9
ProductionProject EditorMark Fitz-Gerald
Technical EditorsRichard Lumb, Ivan Bastian
Manuscript EditorsMark Fitz-Gerald, Richard Lumb
IllustrationsKerry Reid
DesignSue Dyer Design
© 2005 Institute of Medical and Veterinary Science
Laboratory Diagnosis of Tuberculosis by SputumMicroscopy
The
hand
book
Page 2 LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
7 SummaryFalse-negatives – Consequences– PreventionFalse-positives – Consequences– Prevention
8 AppendicesSpecimen containers DocumentationLaboratory layoutSafetyZiehl-Neelsen reagent preparationQuality Control The microscopeTrouble shooting – Staining– MicroscopyPatient information
Contents
Foreword
Introduction
1 Symbols and warnings
2 Sputum collectionSpot-morning-spotHospital patientsSafe collectionPre-collection patient adviceHow to collect a specimenSpecimen qualityRejection criteriaRegistrationStorage and transport
3 Smear preparationWhat you needMaking a smear
4 StainingWhat you need The Ziehl-Neelsen method
5 ExaminationReading smearsAppearance of acid fast bacilli
6 ReportingHow to report
4
6
7
88889
1010101112
141415
181820
222224
2525
2626
26
2727283031343738
454752
Page Page
Page 3LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Foreword
Tuberculosis is a treatable disease that
affects many individuals and almost all
communities. The well-being and
productivity of a country may be affected
severely by this insidious disease.
The World Health Organization developed the Directly ObservedTreatment, Short course (DOTS) strategy for tuberculosis (TB) controlwhich has been adopted by many National Tuberculosis Programmes(NTPs). The DOTS strategy recommends direct smear microscopy as the most effective tool for the diagnosis of TB and for monitoring patientresponse to treatment. An effective TB control programme thereforedepends upon laboratories providing accurate, reliable and timelydetection of infectious cases.
In 2004, in collaboration with Indonesian colleagues, The Handbookwas developed and translated into Bahasa. 15,000 copies were printedand distributed throughout the country. It has subsequently receivedwidespread acceptance and generated interest in other countries.
This version has been revised for Pacific Island Countries many of which are isolated and have relatively high rates of TB in widely-dispersed communities. These conditions present unique challenges forTB control and the provision of laboratory services. The Handbookis intended to assist the NTPs of Pacific Island Countries to achieveexcellence in sputum smear microscopy.
MR RICHARD LUMB
DR IVAN BASTIAN
Page 5LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
The purpose of this Handbook is to teach technicians how to safely collect, process and examine sputum specimens for the laboratory diagnosis of tuberculosis (TB). It should be used together with ‘Quality Assurance of Sputum Microscopy in DOTSProgrammes – Guidelines for Pacific Island Countries’ (WHO).
Sputum microscopySputum smear microscopy is one of the most efficient tools for identifying people with infectious TB.
Smear-positive patients are up to ten times more likely to be infectious than are smear-negative patients.
The purpose of sputum microscopy is to:• Diagnose people with infectious TB• Determine the infectivity of the disease• Monitor the progress of treatment• Confirm that cure has been achieved
Consistent and accurate laboratory practice helps to save lives and improves public health.
Risk of InfectionRisk of infection for laboratory technicians is very low during smear preparation.
A higher risk of infection exists when talking to TB patients before specimens are collected.
In contrast, doctors and nurses working in TB wards and clinics have a much higher risk of becoming infected with TB.
Personal safetyWhen performed correctly sputum examination will not place laboratory technicians at increased risk of developing TB.
Introduction
Page 6 Introduction LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Failure to follow these instructions may harm your health or cause immediate damage to equipment
Failure to follow these instructions may affect test outcomes, or cause equipment damage over time
Correct – the preferred way to do something
Do not do this
You should wear gloves for this procedure
You should wear a gown for this procedure
Wash your handsAlways wash your hands after preparing sputum smears and before leaving the laboratory
This substance is toxic
This substance is corrosive
Symbols and warnings 1
�
✗
8
Symbols and warnings Page 7LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
2 Sputum collection
Spot-morning-spotThree sputum specimens are recommended for the laboratory diagnosis of TB. For theconvenience of the patient collect specimens using the spot-morning-spot principle.
Spot Day – 1• Collect the first specimen when the patient presents to the clinic.• Give the patient a labelled sputum container for the next morning’s sputum collection.
Morning Day – 2• Patient collects early morning sputum and brings it to the clinic.
Spot Day – 2• Collect the third specimen when the patient returns to the clinic with the morning
specimen.
Hospital patientsIf the patient is in hospital, collect a sputum specimen each morning on three consecutive days.
Safe collectionTransmission of TB occurs because infectious droplets are released into the air when a diseased patient coughs.
Collect specimens outside so that infectious droplets are diluted in an open,well-ventilated area
To reduce the possibility of laboratory staff becoming infected:
Tell the patient to cover their mouth when coughing.
Collect sputum outside the laboratory, preferably outside the building well away from other people.
Do not collect sputum specimens in:• Laboratories• Toilet cubicles• Waiting rooms• Reception rooms• Any poorly ventilated area
��✗
Page 8 Sputum collectionLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Sputum collection 2Pre-collection patient advice
If dentures are present,remove them and rinsemouth with water.
• Check the Specimen Request Form• Fill in any missing details • Tick Diagnosis or Follow-up
Tell the patient the bestspecimen comes from the lungs.Saliva or nasal secretionsare unsuitable.
• Clearly label the sputum container with the patient’s name and date of collection
• Label the container, never the lid
��
�
✗
✗
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
RESULTS (Laboratory to complete)
Laboratory Register Number
Collection Date Specimen
Examined by (signature) Date
SEND COMPLETED FORM AND RESULTS
TO THE TREATMENT UNIT PROMPTLY
Appearance Results (check one)
BloodStained
MucoPurulent Saliva neg 1-9 + ++ +++
1
2
3
DiagnosisorFollow up
�
Sputum collection Page 9LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
How to collect a specimen
Do not stand in front of the patient during collection
Instruct the patient to:1. Inhale deeply 2 to 3 times, breathe out hard each time2. Cough deeply from the chest3. Place the open container close to the mouth to collect the sputum4. Screw the lid on tightly
Several attempts may be necessary to obtain a good quality specimen.
If the specimen is poor:1. Keep the most mucoid-purulent sample 2. Discard all the other samples
Specimen quality
Good quality specimenMucoid
Good quality specimenPurulent
Blood stained Poor quality specimens are thin and watery or composed largely of bubbles
� � � ✗
2 Sputum collection
✗
Rejection criteria• Broken or leaking specimen containers• Specimen in inappropriate/non-sterile container • Specimen container details do not match the Specimen Request Form• The specimen has been collected into a fixative (e.g. formalin)• The specimen has been collected into a tissue
Page 10 Sputum collectionLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Patient Informationpage 52
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
RESULTS (Laboratory to complete)
Laboratory Register Number
Collection Date Specimen
Examined by (signature) Date
SEND COMPLETED FORM AND RESULTS
TO THE TREATMENT UNIT PROMPTLY
Appearance Results (check one)
BloodStained
MucoPurulent Saliva neg 1-9 + ++ +++
1
2
3
Name in full
Laboratory Register
LabRegister
No.Date Age
SexM/F
Complete address(for new patients)
Name ofreferring Health
Centre
Reason forexamination*
MicroscopyResults
Signature Remarks
Diagnosis Follow-up 1 2 3
* If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
Laboratory Register
RegistrationRegister the specimen before processing
Sputum collection 2
1. Check patient details on container match the Specimen Request Form2. Transfer patient details from the Specimen Request Form to the Laboratory Register 3. Write the Laboratory Register Number (LRN) on the side of the specimen container4. Write the LRN on the Specimen Request Form5. For Follow-up patients write the Patient Identity Number (PIN) in the Remarks Column
of the Laboratory Register
For each patient, use the same LRN and the numbers 1, 2, 3 to identify the: • Spot (1)• Morning (2)• Spot (3) specimens.
Saliva specimens must be reported on the Specimen Request Form.
2
4
Specimen Request Form
1
3
757/1
5
Sputum collection Page 11LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
In remote settings, sputum specimens need to be transported to a microscopy laboratory.
StorageTo preserve specimen quality:• Prior to dispatch store specimens in a refrigerator
(do not freeze), or keep as cool as possible• Protect from sunlight
What you need• Strong specimen containers (see page 27)• Permanent marker to write details on the side of the container• Plastic, ziplock bag for each specimen• Transport box• Master list of specimens• Sputum request forms
Specimen containers must be strong enough to withstand transportation effects
Approved secondary packaging (transport box) must:• Be leak proof and strong• Contain absorbent material, bench roll etc• Be kept out of sunlight• Keep request forms separate from sputum specimens
Packing checklistIs the sputum container clearly labelled with• Patient name• Date of collection• Spot (1) Morning (2) Spot (3)
Always label the container never the lid (see page 9)
• Are Request Forms completed correctly?• Are Request Forms packed separately from specimens?
Prepare a Master list that contains the details for each specimen being transported
Ensure the Master list contains the name and address of the laboratory sending the specimens
Check that the number of specimens equals that on the Master list
Transport• Check your local regulations for transport by air• Dispatch stored specimens twice weekly
2 Sputum collection Specimen storage and transportSp
ecim
en s
tora
ge a
nd tr
ansp
ort
Page 12 Sputum collectionLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
5
Sputum collection Page 13LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Sputum collection 2Specimen storage and transport
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
2
3
6
4
���
1
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
8
7
Keep upright
Absorbent paper
Packing specimensPut several layers of absorbent paper in the bottom of the shipper.
1. Check each specimen container is labelled with– Patient name– Date– Spot (1), Morning (2), Spot (3)
2. Seal each container in a separate bio-hazard bag3. Cross check specimens against Request Form4. Put sealed bio-hazard bags into the shipper5. Put Request Forms and Master list into a separate sealed bag6. Pack shipper to prevent movement7. Add sealed bag containing Request Forms last8. Seal and address the shipper
Keep coolStore uprightDeliver urgently
Sputum smears must be prepared promptly after collection.
To effectively prepare smears, you will need:
A solid bench with a non-absorbent surface that can be disinfected
3 Smear preparation What you need
Never reuse sputum smear slides
Applicator stickBamboo/wooden applicator sticks are better because they: • Separate purulent material from saliva faster• Pick up more sputum than wire loops• Are easier to handle• Are faster to use
Wire loopsSome technicians prefer wire loops because they can be reused however they:• Are more time consuming• Collect a smaller sample volume • Are less efficient
New, clean glass slides
Discard bucket containing disinfectant (e.g. 5% Phenol)
Bamboo/wooden applicator sticks or wire loop
Alcohol/sand trap forcleaning loops
Spirit lamp
Slide rack for drying smears
Page 14 Smear preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Handle slides by edges only
Sputum specimen with purulent portions within saliva
Making a smear
Never put more than one sputum specimen on each slide
Write the LRN and the 1, 2, or 3 identifier on the frosted end of each slide using a lead pencil
For clear slides use a diamond pen
Do not mix purulent/bloodstained portions with saliva/mucous
Select only purulent orbloodstained portions ofsputum
Ziehl-Neelsen stained smear – purulent
1
2
Ziehl-Neelsen stained smear – saliva
Smear preparation 3
B
A
A BMore acid fast bacilli
(AFB) will be found in the purulent portions
of a specimen.
Smear preparation Page 15LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Add Laboratory CodeNumber (LCN) ifapplicable
LRN Spot
Smear the specimen in the centre of the slide, covering2cm by 1cm
�
✗
Discard the applicatorstick into discard bucket after use, do notflame, do not reuse
To clean a wire loop• Insert loop in sand trap and rotate• Flame the loop to red-hot and allow to cool
Always clean and sterilise a wire loop between each specimen
Retain all specimens untilresults are reported
3 4
65
1cm
2cm
Making a smear3 Smear preparation
Wash your hands after smear preparation
Page 16 Smear preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Dry smears on a slide rack, out of direct sunlight
When dry, heat fix thesmears:• ensure the smear is
facing upwards • pass 3 times through
the flame of a spirit lamp.
Overheating willdamage the bacilliCorrectly prepared smear
�
Stained smears resulting from poor smear preparation
Too thick
✗
Too thin
✗
Not centred and too small
✗
Multiples
✗
87
9
Making a smear Smear preparation 3
Smear preparation Page 17LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
To stain smears using the Ziehl-Neelsen method you will need:
Slide rack to support slides over sink or bucket
Forceps
Slide rack for drying stained slides
Spirit burner
Water
Timer
What you needStaining 4
Page 18 StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Staining
Carbol fuchsin 0.3 %
3% HCI in ethanol or 25% H2S04
You will require 2 – 3 volumes of decolouriser for eachvolume of stain.
Methylene blue 0.3 %
What you need 4
Staining Page 19LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Staining 4
Cover each slide completely with carbol fuchsin
21
• Heat each slide from below until steam rises, always keep the flame moving
• Stop heating when steam rises
Do not boil
3
Do not splash adjacent slides
6
• Gently rinse each slide with a stream of water• Tilt each slide to drain off excess water
5
4
Place the slides smear upwards, in LRN order, on a staining rack over the sink or basin, about a finger-width apart
Ensure the slides are level
Leave the heated stain on the slides – minimum 5 minutes
A longer time is OK provided the stain does not dry on the slide
✗
The Ziehl-Neelsen method
Page 20 StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Staining 47
Add decolourising solution to the slide and leave for 3 minutes
9
Cover each slide with methylene blue for 30 seconds only
11
• Air dry away from direct sunlight• Do not dry slides using blotting paper
10
• Gently rinse each slide with water• Do not splash other slides • Tilt each slide to drain off excess water
12Do not examine slides until they have dried
A correctly stained smear
8
• Gently rinse each slide with water• Do not splash other slides • Tilt each slide to drain off excess water
The Ziehl-Neelsen method
Staining Page 21LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
AFB’s are stained red. Some males are red/green colour-blind, hence all male techniciansshould be tested for colour blindness.
Smears must be consistently and systematically examined to ensure a representative area of the smear is reported.
Use the 10X objective lens to find a suitable purulent area to examine:• It should have more inflammatory cells than
epithelial cells• For uniformly poor quality specimens, find an area
relatively free of epithelial cells
Inflammatory cells (high power) – look for areas like this
2
• Check the smear is facing upwards• Apply one drop of immersion oil• The drop must fall freely onto the smear so that the oil
applicator does not become contaminated with TB organisms
Never allow the oil applicator to touch the slide
3
Carefully rotate the 100X oil objective lens over the slide
Reading smears
Avoid areas containing epithelial cells (low power)
Examination5
✗
�
Page 22 ExaminationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
1
5Examination
4
Carefully adjust the fine focus until cells are sharp
Never allow the lens to touch the glass slide
Direction of traversing the stained slide
Examine at least 100 high power fields before recording a negative result You should take approximately 5 minutes to read a negative smear
6
Gently wipe the oil from the slide with the edge of a clean piece of tissue paper
Avoid contamination, always use a clean piece of toilet paper
Store the slides in LRN order as they will be needed for External Quality Assessment (EQA)
Do not write the result on the slide
There is no need to treat slides with xylol
Wipe the lens gently with tissue paper to remove immersion oil after you have finished examining a batch of slides
Reading smears
5
7
8
✗✗
�
Examination Page 23LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
• Viewed with an oil immersion lens, AFB are red, slender rods, sometimes with one or more granules
• Tubercle bacilli may occur singly, as V-shaped forms, or as clumps of bacilli
Typical morphological characteristics of Mycobacterium tuberculosis
Where possible all new positive smears should be reviewed by another technician
single bacilli V-shaped forms
clumps of bacilli
Examination5 Appearance of acid fast bacilli (AFB)
Page 24 ExaminationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
RESULTS (Laboratory to complete)
Laboratory Register Number
Collection Date Specimen
Examined by (signature) Date
SEND COMPLETED FORM AND RESULTS
TO THE TREATMENT UNIT PROMPTLY
Appearance Results (check one)
BloodStained
MucoPurulent Saliva neg 1-9 + ++ +++
1
2
3
What you see What to report
No AFB in 100 fields Negative for AFB
1 – 9 AFB in 100 fields Report what you see
10 – 99 AFB in 100 fields 1+
1 – 10 AFB per field, check 50 fields 2+
More than 10 AFB per field, check 20 fields 3+
Reporting 6How to report
1. Check the smear LRN matches the Specimen Request Form2. Write the results on the patient’s Specimen Request Form3. Date and sign the Specimen Request Form4. Record the results in the Laboratory Register immediately after reading
the smear. Use red pen for Positive results5. Return the completed Specimen Request Form to the doctor or clinic
Do not give results to the patients as lost reports may delay treatmentDo not write the results on the slide as they are needed for QC checking
Name in full
Laboratory Register
LabRegister
No.Date Age
SexM/F
Complete address(for new patients)
Name ofreferring Health
Centre
Reason forexamination*
MicroscopyResults
Signature Remarks
Diagnosis Follow-up 1 2 3
* If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
Date Sign
757/1
757/1
1
3
4
5
Record Results
Return to doctor or clinic
2
Laboratory Register
Specimen Request Form
Reporting Page 25LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
The number of AFB found indicates how
infectious the patient is,hence it is important
to record exactly what you see.
Be consistent and accurate in all your work, lives depend on you
False-negatives – consequences• Patients with TB may not be treated resulting in on-going disease, disease
transmission, or death• Intensive phase treatment may not be completed, resulting in inadequate treatment and
potential drug resistance
Prevention• Label sputum containers, slides and laboratory forms accurately• The specimen must contain sputum not saliva• Select purulent material to make the smear• Smear preparation – centred, not too thick or thin, 2cm x 1cm in size• Heat carbol fuchsin until steaming• Do not boil during fixation• Stain with carbol fuchsin – minimum 5 minutes• Do not overheat the carbol fuchsin• Decolourise until no more carbol fuchsin is released, maximum 3 minutes• Counterstain – maximum 30 seconds• Keep the microscope well maintained and the lenses clean• Perform regular QC on stains and reagents• Check the slide LRN matches the Specimen Request Form before recording the result
Don’t rush – examine a smear for 5 minutes minimum before recording a negative
False-positives – consequences• Patients are treated unnecessarily• Treatment may be continued longer than necessary• Medications will be wasted
Prevention• Ensure technicians can reliably recognise acid-fast bacilli• Label sputum containers, slides and laboratory forms accurately• Always use new unscratched slides• Use bamboo/wooden sticks once only• Do not allow carbol fuchsin to dry on the smear• Decolourise adequately• The oil applicator must not touch the slide• Keep the microscope well maintained, the lenses clean, store appropriately• Perform regular QC of stains and reagents• Check the slide LRN matches the Specimen Request Form before recording the result
7 Summary
False-negative meansreported negative but truly
smear-positive
False-positive meansreported positive but truly
smear-negative
Page 26 SummaryLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Ideal specimen container
Specimen containers Appendices 8
Unacceptable containers
✗ ✗ ✗ ✗
�Clear break-resistant plastic
�Wide-mouth �Multi-thread screw cap
�Single-use
�Clean
�Can be written on with a permanent marker pen
OpaqueToo small
Extra insert lidScrew cap inadequate
Too smallScrew cap inadequate
ColouredScrew cap inadequate
Specimen containers Page 27LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Specimen Request FormThis example shows the type of information required on a Specimen Request Form.
8 Appendices Documentation
NATIONAL TUBERCULOSIS CONTROL PROGRAMME
REQUEST FOR SPUTUM EXAMINATION
Treatment unit Date
Contact person Phone
Patient name
Date of birth Age Sex � M � F
Other names
Home address
Island
Examination for � Diagnosis � Follow-up
Patient Identification Number
RESULTS (Laboratory to complete)
Laboratory Register Number
Collection Date Specimen
Examined by (signature) Date
SEND COMPLETED FORM AND RESULTS
TO THE TREATMENT UNIT PROMPTLY
Appearance Results (check one)
BloodStained
MucoPurulent Saliva neg 1-9 + ++ +++
1
2
3
Page 28 Specimen Request FormLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Documentation Appendices 8Laboratory RegisterThis example shows the type of information required on a Laboratory Register
Name in full
Laboratory Register
LabRegister
No.Date Age
SexM/F
Complete address(for new patients)
Name ofreferring Health
Centre
Reason forexamination*
MicroscopyResults
Signature Remarks
Diagnosis Follow-up 1 2 3
* If sputum is for diagnosis, put a tick () mark in the space under “Diagnosis”If sputum is for follow-up of patients on treatment, write Patient’s Identity Number (PIN) in the space under “Follow-up”.
The Laboratory Register Numbering SystemThe LRN begins at number 1 at the start of each year. It increases by one with each patient, until the end of the year.
Do not return to LRN Number 1 at the end of each day, week or month
Laboratory Register Page 29LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
The basic requirements for a sputum microscopy laboratory include:
8
• A table/bench to prepare smears• Sink or plastic basin to stain smears• A table to examine smears• A table for paperwork• Basin for hand washing• Adequate lighting• Non-skid flooring• An area for receiving specimens• Good ventilation
Appendices Laboratory layout
Biological safety cabinets (BSC)• Preparing sputum smears does not require a BSC• Only laboratories performing culture and drug susceptibility testing need
a functioning BSC
Never use a clean air cabinet as it can blow TB organisms into the laboratory
Microscopy bench Records bench
Bench for incomingspecimens
Sliding window toreceive specimens
Win
dow
open
s to
exte
rior
Storagecupboards
Window
Smearpreparation
area
Sink
Handwashingbasin
Waste bin
Gown rack
Page 30 Laboratory layoutLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Transmission of TB occurs through micro-aerosols called infectious droplets that contain tubercle bacilli. Good laboratory methods aim to minimise formation of droplet nuclei.
Assume all specimens are potentially infectious
General laboratory safety• Never smoke, eat or drink in the laboratory• Do not wear gowns/coats or gloves outside the laboratory• A surgical mask provides no worthwhile protection for technicians• Wash hands regularly, especially after removing gloves, and always before
leaving the laboratory • Do not allow unauthorised people within the laboratory area• Do not perform work for which you are not trained• Always follow the safety procedures
GownsThe laboratory should provide a supply of clean gowns• Hang your gown in a designated area when not in use• Change gowns on a regular basis• Replace immediately if contaminated
Gloves• Disposable gloves should be worn when handling specimens• Do not wear gloves for microscopy, report writing or during staining
If gloves are not available, you can still prepare sputum smears but you mustwash your hands after processing is completed
Disinfection methods
Surfaces Spills Prepare
Phenol 5% � � Every 2 days
Alcohol 80% v/v � ✗ Weekly
Hypochlorite 5%* ✗ � Every 2 days
*Corrosive to metal
How to clean up spills1. Put on a gown and gloves2. Place a paper towel or cloth over the spill area and liberally apply phenol solution3. Leave covered – minimum 15 minutes4. Clean up the contaminated material and put into the waste container5. Clean with a final wash using 80% v/v alcohol6. Wash your hands after the clean up is complete
Safety Appendices 8
Safety Page 31LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Waste disposal
Locate the burning drum away from people in an open area as the fumes are toxic
8 Appendices Safety
1
Add phenol to a lined bucket
Burn bucket contents weekly
3
2
Tighten caps, add specimencontainers and contents of the discard bucket to the phenol bucket
4
When cool bury burning drum contents at least1.5 metres deep
Page 32 SafetyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
8
Poor posture – feet unsupportedGood posture – supporting your feetstraightens your back
Poor posture – microscope too lowand feet not flat
Good posture – raise the microscopeto help straighten your back and keepyour feet flat on the floor
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Safety Appendices
Ergonomics
Safety Page 33LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Record each batch of prepared reagent in a Reagent Preparation Workbook including:• Reagent name• Signature of the technician who prepared it• The date of preparation• The results of QC testing
Reagent Preparation Workbook
8 Appendices Ziehl-Neelsen reagent preparation
Ready to use reagents must also be QC tested, and the results recorded in the Reagent Preparation Workbook.
Date Reagent QC Results Initials
Positive Negative
25/11/03 Carbol fuchsin 0.3% � � RL
25/11/03 3% HCl/alcohol � � RL
Page 34 Ziehl-Neelsen reagent preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Wash your hands after preparing reagents
Carbol fuchsin – 0.3%
Phenol crystals and vapour are corrosive, toxic and may cause burns Use care, prepare in a well ventilated area
Grade
Basic fuchsin powder 3.0g Certified
Ethanol (or methanol) 100ml Technical
Phenol crystals* 50g *use colourless not tinted crystals Analytical
Distilled water 900ml
Preparation1. Add 100ml of alcohol (or methanol) to a one litre glass flask 2. Add 50g of phenol crystals and dissolve3. Add 3.0g of basic fuchsin powder4. Mix well with gentle heating until dissolved5. Add distilled water to make one litre6. Label the bottle – “0.3% carbol fuchsin”, date and initial7. Store in a dark bottle in a cupboard at room temperature (expiry 6 months)8. Prior to use filter stock solution into the staining bottle
Ziehl-Neelsen reagent preparation Appendices 8
Label0.3% carbol fuchsinDateInitial
Filter stock solution before use
Ziehl-Neelsen reagent preparation Page 35LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
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Decolourising solution
Always add the acid to ethanol or water. Solutions will generate heat.
3% HCI ethanol Grade
Concentrated hydrochloric acid (HCl) 30ml Technical
95% ethanol 970ml Technical
25% H2SO4
Concentrated sulphuric acid (H2SO4) 250ml Technical
Distilled water 750ml
Preparation1. Carefully add the HCl to the ethanol or the H2SO4 to the water2. Label the bottle “3% HCl in ethanol” or “25% H2SO4”, date and initial3. Store in a dark bottle in a cupboard at room temperature (expiry 12 months)
Counterstain
Methylene blue chloride 3.0g
Distilled water 1000ml
Preparation1. Dissolve the methylene blue chloride in distilled water2. Label the bottle – “0.3% methylene blue”, date and initial3. Store in a dark bottle in a cupboard at room temperature (expiry 12 months)4. Prior to use filter stock solution into the staining bottle
8 Appendices Ziehl-Neelsen reagent preparation
Filter stock solution before use
Label0.3% Methylene blueDateInitial
Page 36 Ziehl-Neelsen reagent preparationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Preparing positive control slides1. Obtain a 2+ or 3+ smear-positive specimen2. Label slides as positive controls (include preparation date)3. Prepare 50 smears and air dry4. Heat fix and store in dark, dry place5. Use within 12 months of preparation
Preparing negative control slidesPrepare smears from any smear-negative sputum (not saliva) specimen.
Quality Control procedureEach weekStain and examine one smear-positive and one smear-negative control slide on the firststaining run of each week:• Record the result in the Laboratory Register• Store the control slides in sequence with completed slides
When using new reagents• Stain and examine one smear-positive and one smear-negative using new reagents• Record the result in the Reagent Preparation Workbook
Unacceptable results• Positive control has poor staining or no visible AFB• Negative control has AFB or AFB-like objects present• Background is not properly decolourised• Stain deposit is present on the QC slides
Report all unacceptable results to the laboratory supervisor immediately
Quality Control Appendices 8
Ziehl-Neelsen reagent preparation Page 37LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
The microscopy area should be:• Free from dust• On a sturdy level platform• Away from centrifuges and refrigerators• Away from water, sinks or chemicals to avoid splashes or spills• Ergonomically correct work position (see page 33)
8 Appendices The microscope
Binocular eye pieces
Diopter ring adjustment
Nose piece Voltageregulator
Coarsefocus
Finefocus
Lenses
Stage
Condenser diaphragm
Field diaphragm
Centering screws
Power On/Off
Stage Ymovement
Stage Xmovement
Page 38 The microscopeLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Setting up the microscopeFor binocular microscopes with pre-centred and fixed condensers:1. Set the variable voltage regulator to minimum2. Turn the power on3. Slowly adjust until the desired light intensity is reached4. Place a stained slide onto the stage5. Rotate the nose-piece to the 10X objective6. Bring the smear into focus with the coarse and fine adjustment knobs
Always use the focusing adjustment knobs to lower the stage away from the lens
7. Adjust the interpupillary distance until the right and left images merge
The microscope Appendices 8
11. Place one drop of immersion oil onto the smear and rotate the 100X objective into place
8. Focus the image with the right eye by looking into the right eye-piece and adjusting with the fine focus knob
9. Focus the image with the left eye by looking into the left eye piece and turning the dioptre ring
10. Open the condenser iris diaphragm so that the field is evenly lit
The microscope Page 39LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
12. Focus using the fine adjustment knob13. Use the variable voltage regulator to achieve a comfortable illumination14. Once the smear has been read, rotate the 100X objective away, locate the 10X objective
over the slide, and then remove the slide15. When finished, reset the voltage regulator to a minimum, and turn the power off16. At the end of each day, use lens paper, muslin cloth, or fine tissue paper to carefully
remove immersion oil from the 100X lens, cover the microscope, or put it in the microscope box and return to the humidity controlled cupboard
Do’s and Don’ts• The 100X objective is the only lens requiring immersion oil• Keep immersion oil away from other lenses• Immersion oil must have medium viscosity and a refractive index (RI) greater than
1.5. Any synthetic, non-drying oil with an RI > 1.5 is suitable (refer to manufacturer’s instructions).
• Do not use cedar wood oil as it leaves a sticky residue on the lens• Do not use liquid paraffin because it has a low refractive index resulting in
an inferior image
Immersion oil – a simple test
8 Appendices The microscope
Glass rod ‘disappears’RI > 1.5
Glass rod still visiblebelow the surface RI < 1.5
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Good immersion oil Poor immersion oil
Page 40 The microscopeLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Maintenance
Xylol produces irritant fumes
Cleaning lenses
Some cleaning agents will damage lenses over time
Cleaning Agent Long term use Infrequent use
Manufacturer’s recommendation � �Ethyl ether/alcohol (80/20) � �Alcohol ✗ �Benzene/petrol ✗ �Acetone/ketones ✗ �Xylol ✗ ✗
• When ever possible use the cleaning fluid recommended by the manufacturer• Use a minimum amount of cleaning fluid, never dip a lens into cleaning fluid• Lens paper is best for cleaning optical surfaces as it does not scratch the lens• Alternatives are muslin cloth, silk, or fine quality toilet paper• Do not use ordinary paper or cotton wool to clean lenses• Keep the microscope covered when not in use• Keep the eye-pieces in place• Fungus or dust may enter through holes where objectives in the nose-piece are missing
The microscope Appendices 8
Cover holes from missing objectives
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The microscope Page 41LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
8 Appendices The microscope
• If the image appears hazy with black dots, check for dust or dirt on the lenses (eye-pieces, objectives, condenser and illuminator lens). If:– the black dot moves when the eye-piece is rotated, then the dust is on the eye-piece– the black dot moves when the slide is moved, then it is on the slide– these two are ruled out, then assume the dust is on the objective (if inside the
objective, it appears as dots; if on the outside, then as a hazy image)• Dust can be removed using a camel-hair/artist brush or by blowing over the lens
with an air brush
A simple air brush made using a pasteur pipette andrubber bulb
Light source• Never touch the glass bulb surface as skin oils will burn, reducing light intensity • Use lens paper to hold the bulb when inserting into the microscope
Mechanical parts• Never disassemble the microscope• Stiffness of movement may be due to an accumulation of dust in the sliding channel,
or in the rack and pinion• Remove the dust with an air brush or artist brush, clean with a solvent such as petrol,
then polish and apply high-quality silicone grease to lubricate the moving parts• Stiffness may be due to bending of some part. The up and down movement of the
mechanical stage will loosen over time. Both problems need to be assessed by a service engineer
Use a tissue, do not touch the globe with your fingers
Page 42 The microscopeLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Fungal growth
The microscope Appendices 8
Fungus growing inside the eyepiece tube
Fungus growing inside the microscope head piece
• Fungus grows on the lenses, the eye-piece tube and prisms causing the microscope image to become hazy and unclear
• To check for fungus turn the microscope on:– Rotate the 10x objective into the light path – Take out both eyepieces, look down the eyepiece tubes for fungus
• To prevent fungal growth, the microscope should be kept in a warm cupboard.– A cupboard with a tightly fitting door, heated by a light globe (maximum 40W),
located at the top of the cupboard near to the microscope head– Always leave the cupboard light on, even when the microscope
is not in the cupboard– Check the temperature inside the cupboard is at least 5°C warmer than room
temperature– Microscopes must be kept in the cupboard even if the laboratory is air-conditioned
The microscope Page 43LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
8 Appendices The microscope
• Where power is unreliable, store the microscope in its box (or tight fitting cover) with silica gel, salt, or rice (~100 grams) placed in an open pot on the microscope stage.– Replace the salt when it begins to look wet– Replace the rice when it is no longer dry and crisp– Silica gel changes colour from blue to pink when it is unable to absorb
any more moisture– Dry gel by placing in a hot air oven or heating in a saucepan until the
blue colour reappears
Warming box for microscope storage
Placement of desiccating agent
• If proper storage not available, keep the microscope in the shade and with good air circulation.
Page 44 The microscopeLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Trouble-shooting Staining Appendices 8Well stained slides
Problem Cause RemedySmear too pink Insufficient decolourisation Decolourise for longer
Acid/alcohol concentration For commercial reagents, check less than 3% with NTP
For in-house reagents, recheck stain preparation and QC results
Acid/alcohol reagent has expired Replace reagentor been stored in direct sunlight Store stain bottle in the dark
Carbol fuchsin (CF) has dried Check smears are level over sinkon smear Add sufficient CF
Smear too thick Prepare new smear
When correctly stained this slide looks like A above
A
Trouble-shooting Staining Page 45LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
8 Appendices Trouble-shooting Staining
Problem Cause RemedyPale acid-fast bacilli CF concentration <0.3% For commercial reagents,
check with NTP For in-house reagents, recheck stainpreparation and QC results
CF insufficiently heated Heat CF to steaming
CF staining time less than 5 minutes Stain for a minimum of 5 minutes
Smear overheated during preparation Pass over flame 3 times, 1-2or staining seconds each time
Stop heating when CF steams
CF reagent has expired or stored Replace reagentin direct sunlight Store stain bottle in the dark
Over decolourised with 3% Decolourise for 3 minutes HCl/ethanol maximum
Problem Cause RemedyCounterstain too dark Excessive staining time Do not exceed 30 seconds
Inadequate washing step after Extend washing stepcounterstaining
Methylene blue concentration For commercial reagents, check too strong with NTP
For in-house reagents, recheck stainpreparation and QC results
Smear too thick Prepare new smear
Problem Cause RemedyDeposit on slide Stains not filtered Filter stains prior to use
Soot deposit on underside of smear Clean with a moist tissue
Page 46 Trouble-shooting StainingLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Problem Cause RemedyLight flickers or does Loose plug or connection Check wall sockets, transformer, not turn on power supply
Loose light bulb Reinstall the bulb – Do not touch bulb with fingers
Dirty bulb contacts Replace bulbErratic voltage supply Use a voltage stabiliserFaulty on-off switch Replace the switchFuse blown or transformer blown Replace the fuseDiscoloured bulb/burnt out Replace the bulb – Do not touch
bulb with fingers
Problem Cause RemedyUneven illumination Field of view partially blocked Rotate the nose-piece until it
clicks into positionIris diaphragm is almost closed Recalibrate microscopeor condenser is not alignedDirty lenses Gently wipe the lenses with lens
paper/soft cloth. If the trouble persists clean with lens paper soaked in the recommended lens cleaning fluid
Heavy fungal growth on lenses Clean the lens using lens cleaningfluid as recommended by the manufacturer
Trouble-shooting Microscopy Appendices 8
Problem Cause RemedyExcessive image contrast Iris diaphragm is almost closed Open diaphragm
Trouble-shooting Microscopy Page 47LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Problem Cause RemedyUnclear image with glare Iris diaphragm too far open Close the iris diaphragm to make
the opening smaller
Problem Cause RemedySpecimen focused at Slide upside down Turn it over10x but not at higher magnification
Problem Cause RemedySpecimen goes out of focus Slide is not flat on the stage Clean the stage and underside more than usual at high of slide magnification
Problem Cause RemedyMechanical stage is loose Poor tension adjustment on the Adjust tension with tension or stiff mechanical stage adjustment device
Solidified lubricants Microscope requires service
Problem Cause RemedyMechanical stage cannot Lock set too low Adjust to proper height and be raised lock
8 Appendices Trouble-shooting Microscopy
Page 48 Trouble-shooting MicroscopyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Problem Cause RemedyOil immersion objective does Is oil being used? Apply immersion oilnot give a clear image
Light source collector lens dirty Clean using lens paper and cleaning fluid
Poor quality immersion oil Use quality immersion oil(low refractive index) (see page 40)
Surface of the lens is dirty Clean lens with lens paperIf oil/fungus inside the objective, replace lens
Water on slide Air dry slidesBubbles in immersion oil Remove oil from slide and
carefully reapply oilOil inside lens Clean or replace lens
Trouble-shooting Microscopy Appendices 8
Trouble-shooting Microscopy Page 49LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
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Problem Cause RemedyCracked objective lens Lens has been dropped Replace lens
Lens forced into slide or stage Replace lens
8 Appendices Trouble-shooting Microscopy
Problem Cause RemedyDust/dirt visible in the field Dust on the collector lens of the Clean all surfacesof view light source
Dust on the top-most lens of the Clean all surfaces condenserDust on the eye-piece Clean all surfaces
Page 50 Trouble-shooting MicroscopyLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
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Problem Cause RemedyHeadaches/incomplete Eye-pieces are not matched Use matched eye-piecesbinocular vision
Improper adjustment of interpupillary Adjust the interpupillary distancedistanceDioptre adjustment was not done Adjust dioptre settings
Problem Cause RemedyFuse blows frequently Fuse incorrectly rated Replace with correctly rated fuse
Unstable line voltage Use voltage protection device
Problem Cause RemedyRegular or semi regular The glass slide is scratched Learn to recognise glass artefactscrescent shapes that maybeconfused for AFBs
Trouble-shooting Microscopy Page 51LABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
Trouble-shooting Microscopy Appendices 8
8 Appendices Patient information
Page 52 Patient informationLABORATORY DIAGNOSIS OF TUBERCULOSIS BY SPUTUM MICROSCOPY
• Your doctor/nurse has sent you to the laboratory because they suspect that you may have the symptoms of tuberculosis (TB).
• To diagnose TB three sputum specimens are needed and they will be collected:1. At first presentation2. Next morning before breakfast3. When you return to the laboratory tomorrow
• Good quality specimens from the lungs are required not saliva or nasal secretions.
• Rinse your mouth out with water if you have recently eaten, or if you have dentures (remove them first).
• To obtain a good quality sputum specimen:1. Inhale deeply 2-3 times, breathe out hard each time2. Cough deeply from the chest3. Place the open container close to your mouth to collect the specimen4. Screw the lid on tightly when done
• You may be asked to repeat the collection to obtain a good quality specimen
It is important that you provide three, good quality specimens
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The specimens must be from the lungs, not saliva or nasal secretions.
If you have recentlyeaten, or have dentures(remove them) thenrinse your mouth outwith water.
Please cover your mouth when coughing!
Institute of Medical and Veterinary ScienceQuality Pathology supporting Training and Research