C1: Preparation of Panel Testing Slides with Known Contents

1\. Introduction

This procedure is a self-explanatory laboratory method for producing
multiple test slides from AFB positive and negative samples. Your
laboratory staff should read and understand both the procedure and the
testing protocols before developing test slides. This procedure has been
reproduced/validated in state and national laboratories. If your
laboratory has difficulty in producing slides that meet the requirements
for consistency you should either: 1) review the procedure with special
attention to the steps of heating and re-suspension; or 2) select
patient specimens with less mucus. The sample development procedure
requires materials that are routinely available in a national or
regional reference laboratory in a low-income country. If your
laboratory has continued difficulties with clumping of AFB that prevents
slide to slide consistency, the use of N-acetyl-L cysteine (NALC) may
improve the quality of the slides***.*** Your laboratory should
demonstrate proficiency in producing samples with a minimum of 25-30
slides that are consistent for negative and low numbers of AFB before
proceeding to developing test slide sets.

**NaOH method**

**(ref Dr. Nguyen Ngoc Lan, Pham Ngoc Thach Hospital, Ho Chi Minh City,
Vietnam and Dr. Alex Sloutsky, Massachusetts Dept. Health)**

2\. Materials Required

Note: Processing should be performed in a Biological Safety Cabinet.

50 ml plastic screw cap tubes

40% Formaldehyde

4% NaOH

Vortex

Water bath at 55-60°C

Distilled water

Centrifuge

Slides

**Positive specimen** (fresh specimens, no more than 2 days old, are
preferred)

Amount: 3 ml or more;

AFB load: \>2+ AFB by Ziehl-Neelsen direct smear;

Color: White to light green; *blood stained* specimens should be
avoided;

> Thickness: Watery (less mucous) specimens are preferred to increase
> consistency.

**Negative specimen** (fresh specimens, no more than 2 days old, are
preferred)

Amount: 5 ml or more;

Color: white to green;

Thickness: Watery (less mucous) specimens are preferred to increase
consistency

**Note:** An AFB negative specimen with 20 or more white blood cells per
field is preferred.

3\. Preparation of AFB Positive Stock

a\. Place 3 ml of AFB positive specimen into a 50 ml screw cap plastic
tube. If volume of the specimen is more than 3 ml, aliquot it into
[separate]{.underline} tubes.

b\. Add 1 drop (approx. 50 µl) of 40% Formaldehyde per 1 ml of sputum,
vortex well.

c\. Incubate for 1 hour at room temperature (25- 30oC).

d\. Add 1 ml of 4% NaOH (if the sputum is too thick, add up to 2 ml of
NaOH solution so that the final concentration of NaOH is always 1-2%).

e\. Vortex thoroughly for 4-5 min.

f\. Add up to 20 ml of distilled water, mix well.

g\. Incubate in a water bath for 30 min. at 55-60oC, mix occasionally by
inverting the tube during incubation. If there is no water bath
available, boil a beaker of water, cool to 90-95oC and place the tube in
the beaker for 20-25 min. It is important to maintain the incubation
temperature in the 55-90oC range.

h\. Add distilled water to a total volume of 40 ml, mix by inversion.

i\. Centrifuge @ 3,000 x g for 20 min. at room temperature (25-30oC).

j\. Decant supernatant carefully, add 0.5-1 ml of distilled water to
resuspend pellets. If initial sputum was aliquoted into portions,
pellets from the same specimen are combined, prior to resuspending.

**Note:** It is advisable to avoid specimens containing impurities (food
remains etc.) However if the impurities are still found in the sediment
after it is dissolved in distilled water, filter the specimen through
the gauze and recentrifuge it.

4\. Preparation of AFB Negative Stock

a\. Distribute 3-4 ml aliquots of AFB-negative sputum into 50 ml screw
cap tubes.

b\. [Note:]{.underline} Several good quality negative sputa can be
pooled together and then split into 3 ml aliquots. Sputa should be
checked for AFB prior to pooling.

c\. Add 1 drop ( approx. 50 µl) of 40% Formaldehyde per 1 ml of sputum,
vortex well.

d\. Incubate for 1 hour at room temperature (25-30oC).

e\. Add 1 ml of 4% NaOH (if the sputum is too thick, add up to 2 ml of
NaOH solution so that the final concentration of NaOH is always 1-2%).

f\. Vortex for 2-3 min.

g\. Add up to 20 ml of distilled water, mix well.

h\. Incubate in a water bath for 10 min. at 55-60oC (Note: the negative
specimen should be heated for a shorter period than the positive
specimen to preserve white blood cells). If there is no water bath
available, boil a beaker of water, cool to 90-95oC and place the tube in
the beaker for 5-10 min.

This preparation is used as a diluent in the Dilution Procedure (step
7).

5\. Evaluation of Positive Stock Preparations

a\. If foam has formed on top of the stock solution, pipette the
contents from beneath the foam into a fresh tube.

b\. Using a standard microbiological loop make 2-3 test smears (approx.
1x2 cm in size) from the suspension for evaluation of the stock
preparations.

c\. Use a well leveled surface for drying the smears.

Positive stock: It is optimal to have concentration 50-60 AFB per
microscope field.

6\. Dilution Procedure

a\. Using negative preparation as a diluent make dilutions according to
WHO Guidelines for AFB quantification:

0 AFB/100 fields: negative

1-9 AFB/100 fields: exact \# of AFB required

10-99 AFB/100 fields: 1+

1-10 AFB/field: 2+

\>10 AFB/field : 3+

b\. Choose suitable AFB concentration on a case-to-case basis within
suggested range. For better results, however, it may be recommended
using 20 AFB/field for 3+ smears, 5 AFB/field for 2+ smear, 50 AFB/100
fields for 1+ smears, and 5 AFB/100 fields for "exact" smears.

c\. Make 3-4 ml of each suspension in order to be able to generate
sufficient amount of smears.

d\. For easy calculations both AFB-positive and AFB-negative aliquots
are measured in drops. Calibrate one typical disposable Pasteur pipette
by measuring the number of drops in 1 ml of sputum suspension.
[Note:]{.underline} do not use water for calibration since the amount of
drops may be different from sputum due to the lack of viscosity.

e\. For calculation of the dilution factor use the following formula :

**N =** **( DC / AC ) \* A**

> **where :**

**N** - is amount of drops of positive sputum to be added.

**DC** - is desired AFB concentration.

**AC** - is actual AFB concentration.

**A** - is the amount of drops in a given volume that was estimated
during

calibration.

**Example:** AFB concentration in the stock suspension (AC) is 65
AFB/field and we have to prepare 4 ml (A = 60 drops) of 2+ suspension
(DC=5 AFB/field ).

In this case N = (5 AFB /65 AFB )\*60 drops

> N = 4.6 drops (approx 5 drops). So, 5 drops of the positive prep is
> mixed with 55 (60 - 5 = 55 ) drops of the negative prep.

Procedural notes:

1\. It is important for reading and interpretation of results that
appearance of the smears is more or less consistent, and that is why it
would be beneficial to keep the amount of leucocytes as stable as
possible in various dilutions. In order to achieve this, it is suggested
to dilute negative sputum with distilled water (prior to adding NaOH)
when the amount of leukocytes is relatively high and avoid dilution if
the amount of leukocytes is low.

2\. It would be also useful when making 1+ suspension to consider making
two different concentrations: 50 AFB/100 fields for 1+ smear preparation
and 15 AFB/100 fields for further dilution to "exact" count smear.

7\. Prepare and Validate Batches of Slides

a\. Using diluted stock preparations, prepare slide batches (50-100
slides per batch is recommended). **Note:** If laboratories are
proficient in developing consistent slides, then developing many slides
from fewer samples will help to save time. Heat fixed slides should last
for months if stored in a cool/dry location.

b\. The consistency of each batch of slides must be validated by
selecting a sample of = 6 slides from each batch to be stained and read
by different technicians to document consistency. Some samples that are
produced and tested will not be of sufficient consistency and should be
discarded.

**Apppendix C.1 Form 1:** **Validation Log for AFB Panel testing slide
batches** can be used to record results for the test slides and
determine if consistency standard is acceptable.

**Number of Slides made** The laboratory should record how many slides
were made from each sample to determine how many slides are available
for test slide sets. We recommend that laboratories prepare 50-100
slides so that sufficient slides are available to put duplicate samples
(one stained and one unstained) in test slide sets.

**Date slides made** This is the date that the test slides were
produced. The length of time that slides can be stored without affecting
performance has not been determined, but we estimate that 4-6 months is
practical with proper storage.

**Slide test results** (columns 1-6) Each column represents the number
AFB/100 fields for 6 separate slides selected for the sample and
preferably read by 2-6 different technicians. For high positives (2+ or
3+) the technicians may estimate the number AFB/100 fields by selecting
a sufficient number of representative fields. For low positives (exact
count AFB/100 fields and 1+) and AFB negatives slides the technicians
should read a minimum of 300 fields per slide and record the average
number AFB/100 fields.

**Average/Mean** average is computed from slide test results 1-6 (see
example)

**Standard deviation** The standard deviation is computed from slide
test results 1-6 (see example).

Insert formula

**Consistency** The consistency column result is computed using the
following formula:

> Mean \[M\] minus 2 standard deviations \[SD\]
>
> If M - 2 SD is \> 0 then consistency is true (sufficient)
>
> If M - 2 SD is \< 0 then consistency is false (insufficient)

If the consistency is false---then there is too much variation in the
number of AFB per slide and this sample is not of sufficient consistency
to use in a PT test for a reliable evaluation of performance. This
formula provides an objective evaluation of consistency, but the
laboratory should still review and determine what is acceptable
variation within a sample of slides.

**Report Result** This is the slide test result for all the test slides.
This test result should be representative of the 6 slides tested and the
sample should meet the consistency criteria.

8\. Prepare Panel Testing Sets

Sets of slides with identical composition of positives and negatives can
be made from the prepared batches of slides.

**Appendix C.2:** **Logbook of Test Slide Sets** can be used to select
slide sets and record the original batch numbers and expected results
for a 10 slide panel testing exercise. This form can also be used to
record and evaluate the results from one or more peripheral laboratories
that perform the PT test. Form 2 serves as the official record of
results for multiple slide sets that are sent to different laboratories.

## Alternate Procedure using NALC (ref Dr. Sang Jae Kim, South Korea)

a\. Collection of sputum specimens: sputum specimens with numerous AFB
should be collected from the patients and be stored for not more than 2
days after collection in order to prevent destruction of sputum cells.
Fresh AFB negative sputa also must be selected from the routine
specimens.

b\. Preparation of mucolytic solution: 2% of N-acetyl-L-cysteine is
mixed with an equal amount of 2.9% sodium citrate.2H2O right before use.

c\. Liquefaction of sputum specimen: AFB positive and negative sputum
samples are mixed with an equal amount of mucolytic solution separately
and shake gently to liquefy specimens.

d\. Dilution of AFB positive sputum homogenate: the liquefied
AFB-positive sputum is diluted with varying proportions of AFB-negative
specimen.

e\. AFB counts of sputum dilutions: one drop of each sputum dilution is
spread on a slide with a smear size of 2 cm2 and dry and sterilize in a
hot oven for one hour without scorching. 10 smears are prepared with
each sputum dilution and stained with Ziehl-Neelsen staining method and
count AFB per 1, 10 or 100 microscopic fields. Sputum dilutions whose
average AFB counts fall into "1-9/100 fields", "10-99/100 fields (1+)",
"1-10 per microscopic field (2+)" or "more than 10 per microscopic field
(3+)" are selected and used to prepare as many smear slides as possible.
Negative slides must also be prepared. AFB counts should be rechecked
with randomly selected 10 to 15 slides again after completion of smear
preparation in order to confirm AFB counts of every batch of slides.

C.2 Validation Log for AFB Panel Testing Slide Batches

  -------------- ---------------- -------- --------------- ------- ------- ------- ------- ------- ------------- --------------- ----------------- ------------
                                                                                                                                                   

  **Validation                                                                                                                                     
  Log for AFB                                                                                                                                      
  Panel Testing                                                                                                                                    
  Slide                                                                                                                                            
  Batches**                                                                                                                                        

                                                                                                                                                   

  *** ***        ***Slide                  ***Slide                                                                                                 
                 Preparation***            evaluation***                                                                                           

  **Batch No.**  **Number of      **Date   **Slide test                                                          **Standard**    **Consistency**   **Report**
                 slides made**    Slides   results**                                                                                               
                                  Made**                                                                                                           

                                           **(AFB per 100                                                        **deviation**   **(average minus  **result**
                                           fields)**                                                                             2**               

                                           **1**           **2**   **3**   **4**   **5**   **6**   **Average**   ** **           **stand            
                                                                                                                                 deviations)**     

                                           10              10      50      15      10      11      17.7          16.0            FALSE             1+

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    

                                                                                                                                                    
  -------------- ---------------- -------- --------------- ------- ------- ------- ------- ------- ------------- --------------- ----------------- ------------

C3: Logbook of Test Slide Sets

(Record of a set of 10 slides selected from Form 1)

Central Laboratory administering test
:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Slide Set No. \_\_\_\_\_\_

Date slide set sent to peripheral laboratories:
\_\_\_\_\_/\_\_\_\_/\_\_\_\_\_\_\_

  --------- ------------- --------- ------------ -------------- ------- ------- ------- ------- ------- ------- ------- ------- -------- --------------
                                                                                                                                         

  **Slide   **Stained or  **Batch   **Expected   **Peripheral                                                                            **Comments**
  no.**     unstained**   no.**     result**     Laboratory                                                                              
                                                 Results**                                                                               

  ** **                   ** **     **(from Form **1**          **2**   **3**   **4**   **5**   **6**   **7**   **8**   **9**   **10**   
                                    1.)**                                                                                                

  Slide 1                                                                                                                                 

  Slide 2                                                                                                                                 

  Slide 3                                                                                                                                 

  Slide 4                                                                                                                                 

  Slide 5                                                                                                                                 

  Slide 6                                                                                                                                 

  Slide 7                                                                                                                                 

  Slide 8                                                                                                                                 

  Slide 9                                                                                                                                 

  Slide 10                                                                                                                                
  --------- ------------- --------- ------------ -------------- ------- ------- ------- ------- ------- ------- ------- ------- -------- --------------

**C.4: Panel Testing Recording and Feedback Form**

Peripheral
Laboratory:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Date PT received by your
laboratory\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ (DD/MM/YY)

Date PT results returned to Central Laboratory
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_( DD/MM/YY)

Name of technician reading test smears:
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Note: If more than one technician performs AFB microscopy in the
laboratory, each technician should read all 10 smears and record their
results on a separate form. Technicians should not discuss results or
share forms until all results have been sent back to the central
laboratory. Forms for all technicians should be sent to the central
laboratory for evaluation.

+------------+------------+-------------+----------------+------------+
|            |            | ## Ref      |                |            |
|            |            | erence Labo |                |            |
|            |            | ratory Only |                |            |
+------------+------------+-------------+----------------+------------+
| **Slide    | **Result** | **Expected  | **Error Type** | **Points** |
| Number**   |            | Result**    |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+
|            |            |             |                |            |
+------------+------------+-------------+----------------+------------+

  -------------- --------------- --------------- ------------- -------------
  **Feedback**                                                 

  Total Points:                                  Pass/Fail:    

  HFP            HFN             LFP             LFN           QE

  Recommended                                                  
  Action:                                                      
  -------------- --------------- --------------- ------------- -------------

**C.5: Panel Testing Report of Multiple Laboratories for District
Supervisor & NTP**

  -----------------------------------------------------------------------
  District :

  District Supervisor:

  Test Slide Set:

  Supervising Laboratory:
  -----------------------------------------------------------------------

  -------------- ---------- --------- --------- --------- --------- --------- --------- -------- ----------
  **Peripheral   **Annual   **SPR**   **PT      **HFN**   **HFP**   **LFN**   **LFP**   **QE**   **Total
  Lab**          Volume**             Score**                                                    Errors**

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

                                                                                                 

  **District                                                                                     
  Averages**                                                                                     
  -------------- ---------- --------- --------- --------- --------- --------- --------- -------- ----------

#### SPR :slide positivity rate PT: Panel Testing HFN: High False Negatives HFP: High False Positives LFN: Now False Negatives LFP: Low False Positives QE: Quantitation Errors