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RESIDENT CORNER
Year : 2019  |  Volume : 20  |  Issue : 4  |  Page : 341-344

Forgetting the cardinal sign is a cardinal sin: Slit-skin smear


Department of Dermatology, Venereology and Leprosy, Dr. D Y Patil Medical College and Hospital, Navi Mumbai, Maharashtra, India

Date of Web Publication30-Sep-2019

Correspondence Address:
Dr Aditi Jaiswal
Department of Dermatology, Venereology and Leprosy, Dr. D Y Patil Medical College and Hospital, 503-D, Vijay Building, Nerul, Navi Mumbai - 400 706, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpd.IJPD_74_19

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How to cite this article:
Gautam M, Jaiswal A. Forgetting the cardinal sign is a cardinal sin: Slit-skin smear. Indian J Paediatr Dermatol 2019;20:341-4

How to cite this URL:
Gautam M, Jaiswal A. Forgetting the cardinal sign is a cardinal sin: Slit-skin smear. Indian J Paediatr Dermatol [serial online] 2019 [cited 2019 Nov 14];20:341-4. Available from: http://www.ijpd.in/text.asp?2019/20/4/341/268400




  What Is Slit-Skin Smear? Top


Slit-skin smear (SSS) is a cytodiagnostic technique used as a side laboratory procedure in the diagnosis of various cutaneous dermatoses. It is a safe and easy technique, in which collection of sample is done from a tiny cut in the lesional skin, stained, and examined under a microscope.


  Name the Various Diseases in Which Slit-Skin Smear Can Be Used as a Diagnostic Modality Top


SSS can be used as a diagnostic modality in the following disorders:

  • Leprosy


    1. As a screening procedure for all patients in whom the diagnosis of leprosy is suspected.[1] It is one of the cardinal signs of leprosy
    2. To diagnose relapse of leprosy
    3. To classify leprosy and thereby assess the prognosis.


  • Cutaneous mastocytosis – SSSs made from cutaneous lesions of mastocytoma stained with toluidine blue or Giemsa stain can identify mast cells characterized by the presence of metachromatic granules in their cytoplasm[2]
  • Cutaneous leishmaniasis – SSS made from cutaneous lesions of leishmaniasis shows leishmania donovani bodies either intracellularly within mononuclear macrophages or as extracellular structures.[3]



  Describe the Historical Aspects of Slit-Skin Smear Top


In 1884, Patrick Manson used squeeze and pierce method to diagnose leprosy. Later Alvarez surgically extracted and grinded the nodules to make smears.[4] Muir used skin clip method to prepare smears. Finally, in 1963, Wade introduced the SSS method to diagnose leprosy.[5]


  Explain the Role of Slit-Skin Smear in the Initial Who Classification of Leprosy Top


SSS was an essential component of multidrug therapy (MDT) program in the beginning. The WHO (1981) classified leprosy as multibacillary (MB) or paucibacillary (PB) on the basis of SSS positivity, in which patients with a bacteriological index (BI) of more than 2 were treated as MB and the rest as PB.[6] However, due to the lack of interest and expertise among the staff and field workers for performing SSS, Georgiev and McDougall (1988) suggested to abandon this test in leprosy control programs.[7] The WHO in 1988 simplified the leprosy classification and included all smear-positive cases in the MB group and all smear-negative cases in the PB group.[8] However, with the development of a highly simplified operational classification of leprosy as PB or MB based on the number of skin lesions and nerves involved, SSS is no longer mandatory for the diagnosis of leprosy in leprosy control programs.[9]


  What Are the Sites from Which Samples Are Collected for Slit-Skin Smear in Leprosy? Top


  • It is recommended to take smear from two sites under aseptic conditions, i.e., earlobe and active (erythematous or infiltrated) lesion[1]
  • It can also be taken from sites with high probability of harboring bacilli, i.e., forehead, chin, elbow, knees, dorsal surface of finger, and buttocks.


The same sites are used for follow-up smears and in relapsed cases along with new relapse lesions.

Note that smears from the forehead, cheeks, chin, buttocks, or nasal mucosa are no longer recommended for cosmetic and practical reasons.[1]


  Explain the Procedure for Slit-Skin Smear Top


Strict aseptic precautions must be taken when performing SSS as it is an invasive procedure.

The procedure is explained to the patient and consent is taken.

Preparation of slide

  1. Clean the selected site with spirit and betadine
  2. Pinch the skin between the thumb and index finger of the left hand to make it avascular. It is difficult to exert sufficient pressure with this method resulting in bloody smear and multiple slits. This difficulty can be overcome using a chalazion clamp, curved artery forceps, or sponge holding forceps[10]
  3. Make an incision with a Bard–Parker blade No. 15 approximately 5-mm long and 2-mm deep in the skin. The blade is then turned 90°. Scrape the bottom and sides of the slit to obtain sufficient material for smear. Care should be taken, so that there is no blood in the specimen as this may interfere with staining and reading. If bleeding is noted, wipe the blood away with cotton
  4. Mount the material onto a sterile and labeled slide, making a circle from inside to outside of approximately 8 mm in size. Tissue is collected from all the required sites on the same slide
  5. Press the cut surface of the skin with a piece of cotton wool to stop bleeding and seal the part with tincture benzene
  6. Allow the smear to air-dry and fix it by passing the slide two to three times over a flame. Heating should be adequate. Too little heat may not fix the smear and it may wash out. Overheating can lead to charring and cracking of smear.


Staining of slide

  1. Primary staining: The smear is stained using Ziehl–Neelsen (ZN) method. Flood the slide with 1% carbol fuchsin stain and heat it till slight fuming occurs. Don't let the stain boil. Stain is left for 3–5 min and then wash the slide until run-off water is colorless
  2. Decolorizing: Cover the slide with 1% acid–alcohol (1% HCL and 70% alcohol) or 5% sulfuric acid for 10 s which is followed by rinsing[1]
  3. Counter-staining: Pour 2% methylene blue onto the slide for 1 min. Wash under running water and let it dry
  4. Now examine 100 fields of slide under ×100 oil immersion lens.



  How Is Slit-Skin Smear Interpreted? Top


  • Bacilli are seen as red dots against a blue background. Viable bacilli are seen as uniformly stained bacilli or solid bacilli (S) having length 4 times greater than the breadth. Their sides are parallel and the ends may be rounded, straight, or pointed. The dead bacilli stain irregularly and appear as granular (G) or fragmented (F)[Figure 1], [Figure 2], [Figure 3][9]
  • The bacilli may be seen singly, in small groups or closely packed bunches called globi [Figure 1]
  • Irregular blue-stained structures scattered among the bacilli are the cells of various structures in the dermis.
Figure 1: Solid (S) bacilli stained homogeneously pink over blue cytoplasm by slit-skin smear

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Figure 2: Ziehl–Neelsen stain showing globi in a lepromatous leprosy patient

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Figure 3: Granular (G) bacilli, i.e., dead bacilli seen on Ziehl–Neelsen stain

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  What Indices Can Be Studied With Slit-Skin Smear? Top


SSS can used to study the BI, morphological index (MI), and solid, fragmented, and granular (SFG) index.

Bacteriological index

The total number of bacilli in a smear is the BI and it includes both living and dead bacilli.

Recording of bacteriological index

Simple method (Dharmendra's method)

  • Many bacilli (+++)
  • Moderate numbers (++)
  • Few bacilli (+)
  • No bacilli (−).[11]


Ridley's logarithmic scale for bacteriological index

  • 6+ Many clumps of bacilli in an average field (over 1000)
  • 5+ 100–1000 bacilli in an average field
  • 4+ 10–100 bacilli in an average field
  • 3+ 1–10 bacilli in an average field
  • 2+ 1–10 bacilli in 10 fields
  • 1+ 1–10 bacilli in 100 fields
  • The presence of globi (cigar bundle appearance of bacilli) indicates high BI[12]
  • BI is a function of cellular immunity because the dead bacilli will be removed from the site by scavenging cells of the host. BI in skin smears starts falling after 1 year of MDT roughly as 0.6–1.0 log per year and continues even after stopping the treatment and this is the basis for fixed duration MDT.[9]


Morphological index

MI is the percentage of solid-staining bacilli (living bacilli) among the total bacilli (solid + nonsolid). It indicates if a patient's leprosy is active or inactive.

A total of 200 single bacterial elements are counted carefully, noting whether they are regular and solid staining or fragmented or granular. The MI is then calculated as percentage of solid-staining rods.[4]

MI is a function of chemotherapy. A fall in the MI indicates improvement and thereby effective treatment and an increase in MI is a sign of worsening.

It is a more sensitive parameter of therapeutic failure, noncompliance, drug resistance, or relapse.[9]

MI is expressed as percentage.

MI falls rapidly to 0 within 5 weeks following treatment with rifampicin.[9]

Ridley's solid, fragmented, and granular index

The bacilli are divided into three classes:

  1. Solid-staining unbroken rods (S)
  2. Fragmented, i.e., bacilli in which the acid-fast substance is interrupted at one or more points (F)
  3. Round granules either in line or in clumps (G).[13]


Solid (S), fragmented (F), and granular (G).

A value is assigned to the bacilli of each class in a smear:

  • 2 if bacilli are numerous (over 20% of all bacilli)
  • 1 if few bacilli (1%–20%)
  • 0 if <1%.


Thus, the relative proportion of bacilli in the 3 classes SFG (in this order) are represented by one of the permutations of 2-1-0. SFG index varies from 0 (no solid organisms) to 10.

Of the three indices described, BI is used as a routine as it is simple and easy.

MI and SFG indices need high standards of fixation, staining, and microscopy. They are, therefore, reserved for research purpose only.


  What Is the Sensitivity and Specificity of Slit-Skin Smear? Top


SSS has a low sensitivity (10%–50%, depending on the expertise of the laboratory workers), but it is highly specific (100%).[14]

Because SSS depends on the bacterial load, it is highly sensitive in the diagnosis of lepromatous, borderline lepromatous, and histoid leprosy, but its sensitivity is low at the tuberculoid end (TT and BT).[9]


  How Often Should Slit-Skin Smear Be Taken during and After the Completion of Multidrug Therapy? Top


Ideally, all patients should have one SSS examination before the start of MDT to decide whether to give PB or MB treatment.[9]

With fixed duration treatment regimens, SSS is not needed either to stop treatment or to follow-up the patients after completion of MDT.


  What Are the Limitations of Slit-Skin Smear? Top


  • Because the sensitivity of SSS is low toward the tuberculoid pole, PB cases of leprosy can be missed
  • It must be remembered that a negative smear does not exclude leprosy. It requires a minimum of 10,000 bacilli/g of tissue for reliable detection by ZN staining. Smears may be negative in PB leprosy lesions where Mycobacterium leprae are scantly present[15]
  • It is a technician-dependent test and depends on training of the staff
  • The observations are also subjective.



  What Is the Relevance of Slit-Skin Smear in the Current Era? Top


Despite the announcement of leprosy elimination in India, new cases of leprosy are being reported regularly from different parts of the country. Hence, the role of SSS cannot be undermined despite its various limitations and it remains a gold standard for the diagnosis of leprosy until newer, more sensitive diagnostic tools such as polymerase chain reaction become available for routine testing.[16]

  • It is useful in the diagnosis of paucilesional MB cases and thereby treat them adequately
  • SSS also helps determine the prognosis (e.g., lepra reactions are common in patients with high BI)
  • Relapse versus reactions.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Groenen G, Saunderson P, Ji B, ILEP Medico Social Commission. ILEP Learning Guide Three: How to do a Skin Smear Examination for Leprosy. London: International Federation of Anti-Leprosy Associations; 2003.  Back to cited text no. 1
    
2.
Harman M, Akdeniz S, Mizrak B. Slit-skin smear in diagnosis of cutaneous mastocytomas. Indian J Dermatol Venereol Leprol 2010;76:187-9.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Bhargava A, Ramesh V, Verma S, Salotra P, Bala M. Revisiting the role of the slit-skin smear in the diagnosis of Indian post-kala-azar dermal leishmaniasis. Indian J Dermatol Venereol Leprol 2018;84:690-5.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Sehgal VN, Joginder. Slit-skin smear in leprosy. Int J Dermatol 1990;29:9-16.  Back to cited text no. 4
    
5.
Abu-Ahmed H, Belehu A, Stoner G, Touw J, Atlaw T. Selection of sites for slit-skin smears. Lepr Rev 1979;50:283-7.  Back to cited text no. 5
    
6.
WHO Expert Committee on Leprosy. A Guide to Leprosy Control. Geneva: World Health Organization; 1980.  Back to cited text no. 6
    
7.
Georgiev GD, McDougall AC. Skin smears and the bacterial index (BI) in multiple drug therapy leprosy control program: An unsatisfactory and potentially hazardous state of affairs. Int J Lepr Other Mycobact Dis 1988;56:101-4.  Back to cited text no. 7
    
8.
World Health Organization (WHO)WHO Expert Committee on Leprosy: 6th report, WHO Technical Report Series, no. 768, GenevaWorld Health Organization 1988.  Back to cited text no. 8
    
9.
Mahajan VK. Slit-skin smear in leprosy: Lest we forget it! Indian J Lepr 2013;85:177-83.  Back to cited text no. 9
    
10.
Deshmukh KM, Sharma YK, Kumar A. The use of forceps for creating sustained pressure for slit skin smear. J Am Acad Dermatol 2019. pii: S0190-9622(19)30332-9.  Back to cited text no. 10
    
11.
Jopling WH, McDougall AC. Handbook of leprosy. 4th edn. Oxford: Heinemann Professional Publishing, 1988.  Back to cited text no. 11
    
12.
Ridley DS. Bacterial indices. In: Leprosy in Theory and Practice. Cochrane RG, Davey TF, editors. Bristol: John Wright and Sons Ltd.;1964. p. 620-2.  Back to cited text no. 12
    
13.
Ridley DS. The SFG (solid, fragmented, granular) index for bacterial morphology. Lepr Rev 1971;42:96-7.  Back to cited text no. 13
    
14.
Report of the International Leprosy Association Technical Forum. Diagnosis and classification of leprosy. Lepr Rev 2002;73:S17-26.  Back to cited text no. 14
    
15.
Banerjee S, Biswas N, Kanti Das N, Sil A, Ghosh P, Hasanoor Raja AH, et al. Diagnosing leprosy: Revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis. Int J Dermatol 2011;50:1522-7.  Back to cited text no. 15
    
16.
Premalatha P, Renuka IV, Meghana A, Devi SI, Charyulu P, Sampoorna G. Utility of bacillary index in slit skin smears in correlation with clinical and histopathological alterations in Hansen's disease: An attempt to revive a simple useful procedure. Ann Med Health Sci Res 2016;6:181-4.  Back to cited text no. 16
[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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