Slit Skin Smear Staining: Introduction, Preparation, Procedure, Result Interpretation, Application and Keynotes

achaurasiya

4 years ago

Slit Skin Smear Staining Introduction

Slit Skin Smear Staining is a very useful technique to see the presence of the causative organism, Mycobacterium leprae, in the skin smear of a patient’s body since slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. The causative agent of leprosy, also called Hansen’ disease is M. leprae and three cardinal signs of leprosy are as follows-

Hypo or hyperpigmented anaesthetic patch
Peripheral nerve involvement
Positive Acid-Fast Bacilli (AFB)in slit skin smear

In 1963, Wade introduced the SSS method to diagnose leprosy.
A small amount of bloodless tissue fluid by slit and scrape method
Cheap and easy to perform

Slit Skin Smear (SSS) Preparation

Requirements for Slit Skin Smear (SSS)

Surgical Blade No. 15
Surgical Handle No. 3
Clean and grease-free glass slides
Spirit swab
Spirit lamp
Swab stick
Dry cotton
Tincture of benzoin
Matchbox
Slide box
Diamond pencil
Forceps
Gloves

Procedure of Slit Skin Smear

Carefully explain the procedure to the patient.
Prepare clean micro slide.
Adequately clean sites with spirit swabs.
Pinch area to make bloodless.
Cut 5 mm long and 2 mm deep only.
Scrape bloodless tissue fluid.
Spread fluid evenly making approximately 8 mm diameter round smear on the slide.
Make one slide per patient i.e., four smears per slide, one site – one smear (Note- Make sure that you maintain the same sequence of smears on the slide.)
Air dry and fix over spirit-lamp; store in a dry place.
Cover the cut site with a tincture of benzoin and apply a pinch of cotton.

Slit Skin Smear Staining

Ziehl-Neelsen (ZN) Method

Requirements for SSS Staining

Staining Rack
Spirit lamp
Stain droppers
Stains:
1. 1% carbol fuchsin (freshly prepared)
2. 1% acid alcohol or 5% sulfuric acid
3. 1% methylene blue
Slide drying rack
Specimens: It depends on the form of leprosy suspected by the treating physician; the preferred specimens may be. 1. Slit Skin smears from the earlobes, elbows, and knees2. Skin biopsy from edges of active patches3. Nerve biopsy from thickened nerves

Slit Skin Smear Staining Procedure

Set staining rack over lab sink.
Align the slides on the staining rack.
Cover slides with 1% carbol fuchsin and heat gently for 15 minutes.
Wash slides with tap water.
Decolorize slides with 1% acid alcohol (HCl in 70 % Alcohol) for a few seconds or 5% Sulphuric Acid solution for 10 seconds.
Wash slides once more with tap water.
Cover the slides with 1% methylene blue for 1 minute.
Wash slides once more with tap water.
Air dry slides on the drying rack.

Observation of Slit Skin Smear

Scan under 10X objective of a microscope.
Finally, observe under 100X objective (oil immersion).
Read slide systematically in a zigzag fashion.
Each smear is graded according to Ridley’s logarithmic scale as shown below.

Result Interpretaion of Slit Skin Smear Staining

Grading: Ridley’s logarithmic scale

0 = Negative; no AFB in entire smear

1+ = 1-10 AFB in 100 microscopic field

2+ = 1-10 AFB in 10 microscopic field

3+ = 1-10 AFB in 1 average microscopic field

4+ = 10-100 AFB in 1 average field

5+ = 100-1000 AFB in on average field

6+ = >1000 AFB, AFB in 1 average field with clumps (globi)

Bacteriological Index (BI)

An estimation of the number of bacilli in the smear; semi-quantitative method (Ridley 1958)
BI is calculated by adding the index from the various sites and divided by the total number of sites examined
Maximum BI- site is also indicated inpatient report

Morphological Index (MI)

An estimation of solidly stained bacilli in a smear
Count 200 AFB of all morphology and note only solidly stained bacilli among them
- Expressed in Percentage.
- When bacilli are <200, expressed infraction
Characteristics of Solid bacilli:
- Uniformly stained organism
- Round ends
- Length is approximately 5 x width
- Parallel sides

Applications of Slit Skin Smear Staining

To diagnose: All leprosy cases does not show positive to M. leprae (10-50%)
To classify the disease whether multibacillary (MB) or paucibacillary (PB)
- Smear positive cases are MB
- It shows highly positive in LL/BL and weakly/negative in BB/BT cases
- ≥ 4+ at any individual site is treated with 24 months MB-multidrug therapy (MDT) in a referral center
To monitor the efficacy of treatment
To rule out relapse
To have an idea of drug resistance

Limitations of Slit Skin Smear Staining

SSS sensitivity is low toward the tuberculoid pole and thus PB cases of leprosy can be missed.
A negative SSS does not exclude leprosy since it requires a minimum of 10,000 bacilli/g of tissue for reliable detection by Z- N staining.
Smears may be negative in PB leprosy lesions where M. leprae load is scanty.
It is a technician-dependent test and hence a skilled staff needs.
The observations are also subjective.

Keynotes on Slit Skin Smear Staining

SSS has even been low sensitivity (10–50%, depending upon the expertise of laboratory workers), It is still the gold standard for all diagnostic techniques due to specificity of nearly 100%.
Molecular test, PCR assay has been increasingly used as an alternative for its diagnosis due to its higher sensitivity.
The sensitivity of the PCR is higher in the multibacillary patients due to have of a load of bacilli.
The difference between SSS and Fite Faraco methods are as follow-SSS detects AFB in smear taken from dermis and the Fite Faraco method detects AFB in tissue obtained by skin biopsy taken from clinically suspected lesions.
Four common sites for slit skin smears are -Right ear-lobeLeft ear-lobeEdge of the active lesion (if no skin lesion right arm)Edge of the active lesion (if no skin lesion right thigh)
Follow up: Smear is always performed from previous smear site
If any new skin lesion also takes from the edge of the lesion.
SSS smear interpretation-Bacilli are red dots against a blue background whereas viable bacilli are seen as uniformly stained bacilli or solid bacilli having length 4 times greater than the breadth. Bacilli sides are parallel and the ends may be rounded, straight, or pointed. The dead bacilli ( M. leprae) stain irregularly and appear as granular or fragmented. The bacilli may be seen singly, in small groups, or closely packed bunches called globi (as shown above image). Irregular blue-stained structures scattered among the bacilli are the cells of various structures in the skin.