Beruflich Dokumente
Kultur Dokumente
AFB MICROSCOPY
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Learning At the end of the module, the participant will be able to:
Objectives Explain collection of appropriate sputum specimens
Explain processing and registration of sputum specimens
Describe sputum smearing and staining
Describe the features of properly prepared and well
stained smears (ZN, auramine staining)
Describe smear examination and define grading of smears
(ZN, auramine staining)
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AFB Microscopy Module
AFB MICROSCOPY
For diagnostic purposes, one to three sputum specimens must be collected and examined.
The national policy for declaring a smear-positive TB case and for exclusion of a smear-
positive case should be followed.
- Strategy 1: two positive sputa to declare a smear-positive case, three negative
sputa to exclude smear-positive TB
- Strategy 2: one positive sputum to declare a smear-positive case, two negative
sputa to exclude smear-positive TB
For out-patients, the first specimen should be collected at the time symptoms
suspected of TB are identified, this is commonly called a spot specimen.
For hospitalized patients early morning specimens on consecutive days may be
collected.
For patients on treatment, follow-up sputum specimens should be collected at
intervals specified by the NTP. Early morning sputum is recommended.
The patient should be given clear instructions on how to collect the sputum. This includes
- the importance of sputum examination for diagnosis of TB,
- the importance of producing real sputum and not saliva,
- how to produce good sputum (i.e. by repeatedly deeply inhaling and exhaling
followed by cough from as deep inside the chest as possible; if the condition of
the patient allows, fast walking/running around the health facility may help to
initiate a good cough),
- how to open and close the sputum container,
- how to avoid contamination of the outside of the container by carefully spitting
into the container and properly closing the container,
- how to safely deliver the sputum container to the laboratory or assigned person.
A good specimen should be 3-5 ml. It is usually thick and mucoid, but may be fluid and
contain pieces of purulent material. The color varies from opaque white to green; when
blood is present, it may be reddish to brown. Blood-streaked sputum is suitable; however
a specimen that is very bloody should not be examined. The patient should instead be
referred to a medical person for immediate management before sputum smear
examination.
Clear saliva is not acceptable and should only be tested if the patient cannot produce
sputum even after carefully following the instructions. This may be more common when
collecting follow-up specimens. Although not the optimal specimen, saliva can produce
positive results.
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AFB Microscopy Module
If the health facility does not perform smear microscopy, there are a number of options:
- refer the patient to the nearest facility with smear microscopy,
- collect the specimens at the health facility and send these by public transport or an
assigned person to the nearest smear microscopy facility,
- collect the specimens at the health facility, or at assigned points in the
communities, have the smears prepared and fixed by a trained person, then send
the slides by public transport or an assigned person to the nearest smear
microscopy facility.
All specimens requested for AFB microscopy have to be registered in the TB laboratory
register; one line for the diagnostic smears of one patient and one line for each follow-up
(one or two smears). The sputum container should be labeled on the side, and not on the
lid, with the laboratory number and serial number. Also the slides should be labeled with
the laboratory line serial number and with the column number or code.
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AFB Microscopy Module
- place each slide on its container to avoid mixing up slides with the wrong
specimen.
Sputum smearing
To make good quality sputum smears:
- smears should be prepared in a clean area, best covered with waste paper,
- place the slide on the smearing area, and open the corresponding container (and
only this one),
- use an application stick (e.g. of bamboo), or a wire loop, to select and pick up the
thick particles of sputum. If yellowish particles are not present (e.g. follow-up
sputum), pick up a whitish particle or the most mucoid part of the sample,
- prepare the smear in an oval shape in the center of the slide; 2-3 cm in length and
1-2 cm in width. This will allow for reading 100-150 fields (2 by 1 cm smear or 3
by 2 cm smear) in one length,
- spread the sputum on the slide by firmly pressing the stick/wire loop
perpendicular to the slide in small concentric circles. Continue spreading the
smear until an even result with fine streaks and no thick parts has been obtained.
Proper spreading is very important; the smear should be neither too thick nor too
thin. The thickness is correct if the letters can be read when the smear is held 4-5
cm over a piece of printed paper,
- place the stick into a discard container and use a new stick for each specimen,
- if a wire loop is used, dip this in a sand-alcohol bottle. Move the wire loop up and
down to remove excess sputum. Heat the wire loop in a flame until red-hot, and
allow to cool before reusing,
- allow the smear to air-dry at room temperature; do not dry in direct sunlight or
over a flame,
- after it is completely dry, use forceps to pass the slide, holding it upwards, over a
flame 2-3 times for 2-3 seconds each. Alternatively cover the smear with alcohol
and let it evaporate.
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AFB Microscopy Module
- apply filtered 1% carbol fuchsin on the slides (best to filter through a funnel and
paper while pouring the staining solution on the smears),
- gently heat until steaming,
- keep the carbol fuchsin on the slide for at least 10 minutes, rinse with water and
drain,
- apply decolorizing solution for 3-5 minutes, then rinse with water and drain,
- repeat the decolorizing step if too much red remains, rinse with water and drain,
- apply methylene blue 0.1% counter stain for 1 minute (no more than 2 minutes),
rinse with water and drain,
- air dry on slide rack.
SMEAR EXAMINATION
ZN Smears
Examine 1 length of the smear (2cm) or 100 fields with bright field microscope, using
1000x magnification. If less than 10 AFB are found in 100 fields, the number of AFB
should be counted. For high positives, examination of only 20-30 fields is sufficient.
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AFB Microscopy Module
250 x magnification, or < 20 AFB at 400x magnification, are found, the exact number of
AFB should be counted. For high positives, examination of less than 1 length may be
sufficient.
Grading of the smears is presented in the following table (HPF= high power field, or the
view field size at 1000x magnification)
IUATLD/WHO ZN FM FM
scale 1000 x magnification, 200-250x 400x magnification,
100 HPF per length of magnification, 40 fields= 200 HPF
the smear 30 fields= 300 HPF per length of the
per length of the smear
smear
Negative Zero AFB/1 length Zero AFB/1 length Zero AFB/1 length
Scanty 1-9 AFB/1 length 1-29 AFB/1 length 1-19 AFB/1 length
1+ 10-99 AFB/1 length 30-299 AFB/1 length 20-199 AFB/1
length
2+ 1-10 AFB/1 HPF on 10-100 AFB/1 field 5-50 AFB/1 field on
average on average average
3+ >10 AFB/1 HPF on >100 AFB/1 field on >50 AFB/1 field on
average average average
The results should be registered in the TB Laboratory Register (Appendix 1), be recorded
on the sputum examination request form (Appendix 2) and be forwarded to the person
requesting the sputum examination(s).
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AFB Microscopy Module
Appendix 1
Laboratory register for sputum smear microscopy
Year__________
Lab Date Name Sex Age Name of Address Reason for Result of specimen Signatur Remarks
serial M/F treatment new examination * e
No. unit patients diagnosi follow 1 2 3
s** up
(month)
***
* Check the appropriate category from the Request for Sputum Smear Examination
** Add the TB treatment registration number for all cases put on treatment at the center; or the name of the center where they were sent to take
their treatment
*** Write the TB treatment registration number
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AFB Microscopy Module
Appendix 2
Patients name:__________________________________________________
Signature____________________________
The completed form (with results) should be sent promptly to the treatment unit