# Presentation: 709007

## AFB Smear: Quality Control

- Pawan Angra MS
- Division of Laboratory Systems
- Public Health Practice Program Office
- Centers for Disease Control and Prevention

## AFB-MICROSCOPY:

- All mycobacteria are acid fast, no exception
- -	In high burden countries like India
- AFB in sputum = TB

## Quality Control

- Laboratory arrangement
- Equipment
- Collection and transport of specimen
- Handling of specimen
- Reagents and method
- Reporting of results

## Laboratory Arrangement

- Physical facility
- Arrangement of equipments, supplies for logical and efficient work flow
- Written procedures with valid references
- Records should be retained for two years

## Slide 5

**Notes:**
Laboratory Arrangement

To perform TB microscopy, a laboratory must have three areas:

## Slide 6

**Notes:**
Area 1
 a well lighted space with a sink or basin with running water
for preparation and staining of smears,

## Slide 7

**Notes:**
Area 2
a microscope table, (if there is no electricity, this table should
be placed directly in front of a window) 

## Slide 8

**Notes:**
Area 3
and a table for recording results and slide storage. 

## Laboratory Equipment

- Meet specification
- Validated periodically to ensure proper function
- Written operating and maintenance instructions
- Service and maintenance record - daily, monthly,	six monthly

## Slide 10

**Notes:**
Microscopic Examination

A good quality binocular microscope is
recommended for reading TB smears.

It must be equipped with an electrical light 
source or mirror, an oil immersion or 100X objective, eyepieces of 8-10X magnification and a mechanical stage.

Daily care and cleaning are essential to avoid dust, oil build
up and moisture.

An area of subdued lighting is preferable for reading slides.

However, if no electricity is available, daylight must be used
as a light source and the microscope should be placed in front
of a window.

## Slide 11

**Notes:**
Arrange the following supplies next to the microscope: a
bottle of immersion oil, organic solvent, lens paper, laboratory
register and a slide box for storage.	

Use the 40X objective to focus and determine a suitable
reading area of the slide.

Place a drop of immersion oil on the stained
smear. Let the drop fall freely onto the slide.

Never touch the slide with the oil applicator. This
could lead to carry over of AFB in the immersion oil to
the next slide.

Turn the nosepiece to bring the 100X objective into place.
Now, gently lower the 100X objective.

It should barely touch the oil. Never allow the lens to touch
the slide. This can damage the lens and possibly break the
slide.
While looking through the eye-piece, adjust the immersion
lens slowly and focus until the image on the smear appears.
To fine focus, turn the fine adjustment knob carefully.

## Slide 12

**Notes:**
After completing the reading, remove the oil from the slide
with organic solvent.

When dried, place the slides carefully in a slide storage
container for transfer to a referral laboratory for quality
control. This must be done on a regular basis as determined
by the National Tuberculosis Programme.

## Slide 13

**Notes:**
Before examining the next slide, wipe the immersion lens
with lens paper. This will protect from carry over of organisms
that could create a false positive reading.

Handle specimens carefully!

All sputum specimens are considered potentially infectious.
Save all specimens until the smears have been examined and
recorded; then sterilize and discard them. Disposable
containers must be used only once.

## Calculation of ‘g’

- RPM= 1000 ****g/1.12 x  r
**g = 1.12 x 10****-6 ****x r x RPM**** 2**
- 	Where, g* *= required relative centrifugal force; *r *= radius of the rotor in mm; *RPM *= the number of revolutions per minute required to achieve the necessary *g*-force.

## Collection and Transport

- Written instructions to perform a test
- Adequately filled form
- Request forms should be kept separate from specimen
- Evaluate the quality of specimen
- Discard leaking, broken specimen containers
- Document sample arrival time

## Sample collection

- Use wide mouth, transluscent, screw cap, 50 ml capacity-single use container
- Instruct the patient to produce deep cough and then collect sputum
- Saliva, nasal, nasopharyngeal secretions are poor quality specimen

## Slide 17

**Notes:**
Sputum Specimens

For best recovery of tubercle bacilli, collection
of three specimens is advised   At least one should
be an “early morning” specimen.

The patient should be properly instructed to ensure that the
collected specimen is of sputum, not saliva.

Clear saliva or nasal discharge is not considered a suitable
specimen for TB. Nevertheless they should be processed.
Specific instructions for collection can be found in the TB
program manual.

A good sputum specimen should be 3-5ml in quantity. It is
frequently thick and mucoid, but may be fluid with chunks of
dead tissue. The color may range from opaque white to green.
Bloody specimens will be reddish or brown in color.

Record the visual appearance of the sputum sample on the
lab form.

## Transportation

- Label the closed containers (not the lid)
- Store in a cool place
- Transport ASAP to lab with all paper work
- For smears at least twice a week
- For culture with in 3-4 days, keep refrigerated

## Smear preparation

**Smear preparation**

## Smear preparation

- Label a new, clean slide with patient information with a diamond point stylus
- Using an applicator stick, pick up blood-specked, opaque, grayish, or yellowish cheesy mucus
- Spread it on the slide in 2.0 X 1.0 cm area in correct thickness

## Slide 21

**Notes:**
How can you tell you have the proper thickness?

If it is too thin, the specimen may yield false negative results.
If it is too thick, the smear may wash off the slide during the
staining procedure. 
Newsprint can usually be read behind a
dried smear when it is the proper thickness.
If a wire loop is used, clean it before the next use. Dip the
contaminated loop into a sand and alcohol bottle and move it
up and down to remove solid debris.

Next, heat the wire loop in the flame until red hot. Allow it to
cool before using it again.

Wet slides can create aerosols if disturbed. Place them in a
protected area where they can air dry for 15-30 minutes.

Do NOT flame them to induce drying.
This can also produce aerosols.

## Reagents and Stains

- Assign date of receive and opening of stains and reagents.
- Label name, date of preparation and expiry of stains and reagents.
- Expired and unsatisfactory material should be removed from lab
- Stock rotation- first come first out

## Z-N staining method (WHO)

- Primary stain: Carbolfuchsin solution 0.3%
- Decolourising agent: 3% acid alcohol, or 25% Sulphuric acid
- Counterstain: Methylene blue 0.3%

## Carbolfuchsin

- Purity correction
- 	80% is available dye and 3% is required
-  Corrected weight= 100 X 3 = 3.75 g
- 				         80
- If the available dye is 88% or more no correction is required.

## Slide 25

**Notes:**
Filter carbol fuchsin prior to use. If precipitate is
still detected, discard the stain.

## Decolourising agent

- 3% acid-alcohol
-  Concentrated hydrochloric acid: 3ml
- 95% ethanol (technical grade): 97 ml
-  Carefully add concentrated HCl to 95% ethanol
-  ***Remember always add acid to alcohol***

## Decolourising agent

- 25% Sulphuric acid
-  Conc. sulphuric acid (technical grade): 25 ml
- Sterile distilled water: 100 ml
- Carefully and slowly add concentrated sulphuric acid to water.
***Remember always add acid to water***

## Slide 28

## Staining procedure

- 	Batches of 12
- 1)	Flood with Carbolfuchsin
- 2)	Heat the slides until steam arises. Stain for 5 minutes
- 3)	Drain the stain and wash under gentle stream of water
- 4)	Flood with decolourising solution-3 minutes
- 5)	Drain and wash under gentle stream of water
- 6)	Flood with counterstain for 1 minute
- 7)	Drain and wash under gentle stream of water
- 8)	Air dry smears

## Slide 30

**Notes:**
The staining procedure should be posted above
the staining area.

To avoid cross contamination, never stain AFB smears in a
staining dish.

Now, let’s study the important steps of the staining procedure. 

## Slide 31

**Notes:**
Using forceps, place slides on a staining rack with the smear
side up. Place all slides in uniform orientation.

Slides must not touch each other. This avoids the possibility
of stain running over and causing cross-contamination. Include
a + and - control slide daily for quality control purposes. Never stain more than 12 slides at a time.

## Slide 32

**Notes:**
Cover the entire surface of each slide with carbol fuchsin.
Using a Bunsen burner or cotton and alcohol flame, gently
heat the slides until vapor rises.

Do not allow them to boil or dry. Boiling will
alter the shape of the TB bacilli and could result
in a false negative reading.

Allow the stain to remain on the slides for 5 minutes. Maintain
heat throughout this period. Adequate time is required for
the carbol fuchsin to penetrate and stain the cell wall.

## Slide 33

**Notes:**
Gently wash the stain from each slide with a stream of cold
water until all free stain has washed away. Always rinse gently
so the smear is not washed off of the slide.
Individually tilt each slide to drain off any excess rinse water.
This prevents puddles remaining on the slide which can dilute
the next reagent.

## Slide 34

**Notes:**
Cover each slide with decolorizing solution such as acid
alcohol.

Leave this on the slides for 3 minutes. If under-decolorized,
sputum contents other than the TB bacilli may remain stained.
This could lead to a false positive result.

## Slide 35

**Notes:**
If the positive control slide does not show AFB, either the
staining reagents are defective or the procedure has been
performed incorrectly. When the problem has been identified
and corrected, repeat the staining procedure with new slides
and controls.

Each phase of smear microscopy must be carefully performed.
Errors made during any step can lead to incorrect results.

## Microscopy

- View positive and negative control slides
- Clean OIL after each slide
- Keep samples until reporting

## Reporting

- Results should be reported with in 24 hrs.
- Weekly and monthly analysis of positive results
- Compare with previous trends, investigate any deviation
- Maintain documentation as per requirement

## Some reasons for false positive results:

- Errors in specimen handling or recording information
- Re-use of containers or slides
- Unfiltered fuchsin
- Contaminated immersion oil
- Inadequate decolorization

**Notes:**
Some reasons for false positive results include:

Errors in specimen handling or recording
information
Re-use of containers or positive slides
Unfiltered fuchsin
Contaminated immersion oil
Inadequate decolorization

## Some reasons for false negative results:

- Errors in specimen handling or recording information
- Poor quality sputum
- Excessive decolorization
- Reading less than 100 microscopic fields

**Notes:**
Some reasons for false negative results include:

Errors in specimen handling or recording
information
Poor quality sputum
Excessive decolorization
Reading less than 100 microscopic fields

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