# Presentation: 003380

##  Sputum Smear Examination by Fluorescence Microscopy

**C.N. PARAMASIVAN**
**Tuberculosis Research Centre **
**Indian Council of Medical Research **
**Chennai**

## Fluorescence Microscopy

- More than 50 smears examined/day
-  Continuous availability of power
- More sensitive. where small No. of bacilli are present
- Majority of FM +ve samples are also +ve by culture
- Does not yield more false positive than ZN
- Doubtful smears to be re examined by ZN

## Reagents for staining

**Auramine-phenol solution**
**Auramine O    ****	****	****    25 g**
**Ethanol****	****             3000 ml**
**Phenol****	****	****   ****	****  250 g**
**Distilled water          ****	****5300 ml**
**Suspend 25 g auramine-O in 3000 ml of ethanol in a 5-litre conical flask. Add a magnet and place on a magnetic stirrer until solution is complete.**
**Dissolve 250 g phenol in 5300 ml of distilled water.**
**Mix the phenol solution with alcoholic auramine solution.**
**Store in an amber colored bottle at room temp. for up to 3 mths.**
**Filter before use.**

## Reagents for staining

**Acid-alcohol for decolorization**
**	****	****Sodium chloride****	****	****    20 g**
**	****	****Hydrochloric acid, A.R.****	****    20 ml**
**	****	****Distilled water****	****	****	****  500 ml**
**	****	****Ethanol****	****	****	****1500 ml**
-  **Dissolve sodium chloride in distilled water**
** ****Add conc. hydrochloric acid, mix thoroughly.**
** ****Add alcohol**
** ****Can be stored at room temperature for 3 months.**

## Reagents for staining

**Counter stain**
**	****	****Potassium permanganate****	****     1 gm**
**	****	****Distilled water****	****	****	****1000 ml**
**Dissolve and store in an amber colored bottle**
**Stays at room temperature for up to 3 months.**

## Staining procedure

- Place the slides on a staining rack, with the smeared side facing up, the slides not touching each other
- Flood the slides with freshly filtered auramine-phenol. Let stand for 7-10 min.
- Wash well with running water
- Decolorize with acid-alcohol for 1-2 min.
- Wash as before with water and slope the slides to air dry
- Counter stain with 0.1% KMNO4 for 30 seconds

## Staining procedure - Precautions

**Avoid under-decolorization. AFBs are difficult to over-decolorize since the decolorization procedure with acid-alcohol is relatively milder than the 25% H****2****SO****4**** used in Z-N.**
**Thick smears:**** Interfere with decolorization, and counter stain. Mask the presence of AFB and tendency to flake, resulting in loss of smear material and possible transfer of material to other slides**
**Strong counter stain:**** May mask the presence of AFB**
**Re Staining**** : Smears examined by FM may be restained by Z-N to confirm findings. However, Z-N stained smears cannot be used for FM**
**Fading:**** Stained smears may fade on exposure to light. To be stored wrapped in brown or black paper and kept away from light**

## EXAMINATION PROCEDURE 

**The mercury vapor lamp : ****It takes about 10 min. to reach full intensity**
**Examination:**** Using the low power objective ****(100-150x)**** first examine a known pos. slide to ensure that the microscope is correctly set up**
**Appearance:****  Bacilli appear as slender bright yellow fluorescent rods, standing out clearly against a dark background**
**Positive:****   ****Minimum of 4 AFB in the entire smear.**
**Negative:   Less than 4 bacilli  ( No.of bacilli to be recorded)**
**Morphological Confirmation:**** With a high power objective ****(400-600x)**** - To be done with all doubtful smears as well as scanty positives. **
**Examination:**** At least three horizontal sweeps on the entire smear. **
**Gradation:**** Grade positive smears into three degrees of positivity using the high power objective**

## QUANTIFICATION OF FLUOROCHROME SMEAR RESULTS

|    
ZN
  1000x | 
REPORT |                 FM Magnification |  |  |
| --- | --- | --- | --- | --- |
|  |  | 250x | 450x | 630x |
|   0 | NO.AFB | 0 | 0 | 0 |
| 
1-9/100FIELD | 
EXACT NO | DIVIDE
  by 10               by 4                by 2 |  |  |
| 10-99/FIELD | 1+ |  |  |  |
| 1-10/FIELD | 2+ |  |  |  |
| >10/FIELD | 3+ |  |  |  |

## FM Grading

| 
No. of bacilli per HPF | 
Grade  |
| --- | --- |
| 
Less than 6 per field | 
1+ |
| 
6-100 bacilli per field | 
2+ |
| 
More than 100 per field or large clumps | 
3+ |

## SMEAR AND CULTURE RESULTS OF 1480 SPUTUM SPECIMENS

| 

CULTURE POSITIVE | SMEAR |  | SPECIMENS |  |
| --- | --- | --- | --- | --- |
|  |     FM  | ZN | No. |   % |
|  | POS | POS | 600 | 40.5 |
|  | POS | NEG | 160 | 10.5 |
|  | NEG | POS |   18 |   1.2 |
|  | NEG | NEG | 174 | 11.8 |
| SUBTOTAL |  |  | 952 | 64.2 |
| 
CULTURE NEGATIVE | POS | POS |  10 |  0.7 |
|  | POS | NEG |  12 |  0.8 |
|  | NEG | POS |    6 |  0.4 |
|  | NEG | NEG | 500 | 33.8 |
| SUB TOTAL |  |  | 528 | 35.7 |
| TOTAL |  |  | 1480 | 100 |

- W. Githui *et al* 1993 East Afr Med J pp293

## COMPARISON OF FM & ZN WITH CULTURE

|  |            FM |  |  |           ZN |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  |  +         |    - | TOTAL |   + |    - | TOTAL |
| 
CULTURE  +
                   -
   TOTAL | 441 | 214 |  655 | 433 | 222 |  655 |
|  | 15 | 713 |  728 | 14 | 714 |  728 |
|  | 456 | 927 | 1383 | 447 | 936 | 1383 |

## RELATIONSHIP OF STAINING METHOD TO QUANTIFIED SPUTUM SMEAR RESULTS

| Smear Quantity | Fluorescence |  | ZN |  |
| --- | --- | --- | --- | --- |
|  | Number |      % | Number |     % |
| Scanty           (+) | 180      | 23.7 | 50 | 8.1 |
| Moderate     (++) | 250 | 32.9 | 249 | 39.3 |
| Heavy        (+++)    | 330 | 43.4 | 325 | 52.6 |
| Total | 760 | 100 | 618 | 100 |
| Scanty p< 0.001 |  |  |  |  |

## FM & ZN comparison in culture positive cases

|  Examined by | Specimens pos. |
| --- | --- |
|    FM           ZN |    No.          % |
|      +             +
     +              -
      -             +
      -              - |   405          62
    36            6
    28            4
  186          28
  |

**Total Culture positive**

**655**

**100**

## Consistency of smear results in duplicate specimens from ZN +ve pts examined by FM

**Tamil Nadu**

**North Arcot**

**Raichur**

**N=1375**

**N=635**

**N=617**

## Spot & collection specimens: smear & culture results of various combinations

| 
   Type of specimen(s) | Positive by |  |
| --- | --- | --- |
|  | Smear % | Culture % |
|    1 spot
   1 collection
   2 spot
   1 spot + 1 collection
   2 collection
   2 spot + 1 collection
   1 spot + 2 collection
   2 spot + 2 collection | 66
77
76
81
83
84
84
85 | 91
93
94
97
97
98
99
95 |

## Fluorescence Microscopy

**Advantages:**
-   Usage of low power objective
-   Size with a 25x objective  : 0.34 mm2
-   Oil Immersion                   : 0.02 mm2
-   Covers15 times as many fields compared to ZN
-   Same area can be covered at a shorter time
-   No. of slides that can be examined per day : about 100
-   Examination of slides for 1 min. by FM equivalent to 4 min.by ZN

## Fluorescence Microscopy

**Advantages**
- Covers an increased area of smear field
- An increased contrast between stained bacilli & background
- Heating not required
- Immersion oil not needed
- May be appropriate in the diag. of HIV + assoc.TB
- With low bacillary content
- Early diagnosis of pulm.TB -  Low bacillary content

## Fluorescence Microscopy

**Disadvantages**
- Handling & maintenance of optical equipment require advanced skill
- Periodic replacement of bulbs
- Continuous supply of power.
- High cost of Microscope and maintenance

## Fluorescence MicroscopyCost Effectiveness:

|  | Light Microscope |      FM |
| --- | --- | --- |
| Equipment | $ 4000x3
 | $10000 |
| Tech. Salary | $1200x3 | $  1200 |
| Total | $15600 | $11200
 |

- Definitive role to play in a high-volume laboratory

- Fluorescence Microscopy