ROLE OF DIRECT IMMUNOFLUORESCENCE ON TZANCK SMEAR IN PEMPHIGUS VULGARIS.
Pemphigus is a group of autoimmune, intraepithelial, chronic blistering skin diseases characterised by loss of normal intercellular adhesion. It occurs due to the formation of autoantibodies, mainly IgG & or C3 against epithelial adhesion molecules, desmosomes. [1,2]
There are three major types of pemphigus, which vary in severity-pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus. [1,2] Pemphigus vulgaris and foliaceus differ in the level of intraepithelial blisters-in vulgaris suprabasal, and in foliaceus subcorneal bullae are formed. 
Pemphigus vulgaris (PV) is the most common pemphigus disorder. It affects mainly middle-aged adults, both sexes equally. It is characterised by autoantibodies against desmoglein-3. It shows a geographical predilection-Mediterranean, South Asia and Jewish region are affected more. 
Clinical features are cutaneous or mucosal blisters. Oral lesions precede skin lesions in more than 50% of the patients and presents as blisters which rupture rapidly resulting in painful erosions. Buccal mucosa, lips and soft palate are the most commonly affected sites. [2,3,4] Diagnosis of PV is based on clinical, histopathological and immunological correlation. [1,2]
Skin biopsy is done for tissue diagnosis . A proper histopathological examination can form the diagnosis in accordance with the location and morphology of blisters. Demonstration of immunoglobulins in the spinous cell junctions by direct immunofluorescence (DIF), from perilesional biopsy (Within 1 cm of the lesion) is often used for a complete diagnosis of PV. 
Immunofluorescence is a histochemical laboratory staining technique used for demonstrating the presence of antibodies bound to antigens in tissues or circulating body fluids. This technique is used to supplement clinical and histopathological findings in PV and other vesiculobullous diseases. They help in the early diagnosis, treatment and monitoring of the disease activity in PV. 
Pemphigus vulgaris has a relentless course, unless timely identified and its immunological progression checked, it can lead to a fatal outcome. So early diagnosis of the condition is imperative to prevent complications. DIF can be done both on tissue biopsies and in cytology smears for confirming the diagnosis. 
Tzanck smear combined with DIF is used as an effective and simple tool, in the rapid diagnosis of PV. [5,6,7] The Tzanck smear has the advantages of being easy to perform, inexpensive, not requiring a specialised laboratory, and causing negligible trauma and discomfort to the patient. 
Objectives of The Study
To assess the role of direct immunofluorescence (DIF) on Tzanck smear taken from mucosal or cutaneous lesions in pemphigus vulgaris and compare it with DIF done on skin biopsy.
MATERIALS AND METHODS
Diagnostic test evaluation.
18 months (March 2017-August 2018).
Department of Pathology, Govt. Medical College, Kottayam.
Sample Size:  Sample size N = Z[[alpha].sup.2]x sensitivity (1-sensitivity) /[d.sup.2] x P Z[alpha] = 1.96 at 95% CI p = prevalence of pemphigus vulgaris in India = 1.8 d = precision/ allowable error So, sensitivity of imprint smear in previous study=40% Taking allowable error as 10%. Sample size, N = Z[[alpha].sup.2]sensitivity (1-sensitivity) = [(1.96).sup.2] x 40x60 = 51.22 [d.sup.2] x P 100 x 1.8
Taking sample size as 51.
Calculated sample size is 51. As the annual number of patients newly diagnosed as pemphigus per year is below 30 in the Department of Dermatology, Government Medical College Kottayam, this study included all newly diagnosed cases of pemphigus.
All clinically diagnosed cases of pemphigus vulgaris were included following a histopathological confirmation.
1. Previously treated pemphigus vulgaris patients.
2. Other pemphigus group of diseases.
3. Patients who are not willing to take part in the study.
Clinically suspected cases of PV attending the skin OPD were evaluated. Study was performed from oral scrape smears procured from clinically diagnosed cases of pemphigus vulgaris attending the dermatology department during the study period. Two scrape smears were taken from oral erosions and air dried and sent immediately to the Department of Pathology. One was stained with May-Grunwald-Giemsa (MGG) stain and detailed cytological analysis done. If oral/mucosal lesions were not present skin erosions are scraped, smeared and evaluated.
The other air-dried smear for DIF staining was stained with fluorescein conjugated rabbit antihuman IgG and with C3 (Dako) for 30 min. Then the smear was rinsed in PBS solution three times for 5 min each, mounted in buffered glycerol and examined immediately under the immunofluorescence microscope. If the smears were positive for immunofluorescence the pattern of staining by the acantholytic cells were noted. The skin biopsy specimens of these patients were received in 10% formalin solution and DIF on skin biopsy was done. Results of DIF on Tzanck smear were correlated with DIF on biopsy, which is the gold standard. Histological diagnosis was made and correlated with Tzanck smear findings.
Written informed consent from each patient was taken prior to the procedures.
Data Management and Analysis
The data was entered in Microsoft excel and further statistical analysis was done using SPSS software (version 20).
1. Sensitivity, specificity, positive predictive value and negative predictive value of Tzanck smears in the assessment of IgG and C3 immunofluorescence was compared with the same in histopathology.
2. Non-parametric test (Kendall tau b) for
* Correlation of IgG fluorescence in Tzanck smear as compared to skin biopsy.
* Correlation of C3 fluorescence in Tzanck smears as compared to skin biopsy.
The level of significance was indicated by correlation coefficient (Between 0 and 1).
Diagnostic test evaluation was done on 30 cases of pemphigus vulgaris presented to Department of Pathology, Government medical college, Kottayam during the study period of 18 months (March 2017-August 2018).
* DIF for IgG and C3 were performed on the Tzanck smears from these cases and the results were compared with the DIF for IgG and C3 done on perilesional skin biopsies.
* The mean age of the present study population was 49 and minimum age was
14 years and maximum was 74 years.
* 53% of the study population were females.
* Majority of study population (70%) had duration of illness not exceeding 6 months.
* Oral mucosa was the initial site involved in majority of cases (70%) followed by skin (7%).
* Initial lesions were erosions in 67% of patients and vesicle in the remaining cases (33%).
* The disease process was generalized in 63% of cases and localized in 37% cases.
* Skin lesions were present in 80% of the cases, with predominant trunk involvement.
* Erosions were predominant lesion (80%) followed by vesicles (20%).
* Oral mucosa was involved in 96% of cases, genital mucosa in 40%, nasal mucosa in 30%.
* Tzanck smear cytology showed acantholytic cells in all the cases with neutrophils as the predominant inflammatory cell.
* Histopathological evaluation of skin biopsies showed suprabasal clefting in all the cases with many showing acantholytic cells and row of tombstone appearance.
* DIF on perilesional skin biopsies showed fishnet positivity for IgG in 83% of cases and for C3 in 70% of cases.
* DIF on Tzanck smears showed fishnet positivity in epithelial keratinocytes for IgG in 80% of cases and for C3 in 60% of cases.
* Sensitivity for IgG was 92% and for C3 was 76%, on Tzanck smear.
* Specificity for IgG was 80% and for C3 was 77%, on Tzanck smear.
* Positive predictive value for IgG was 95% and for C3 was 88%, on Tzanck smear.
* Negative predictive value for IgG was 66% and for C3 was 58%, on Tzanck smear.
* The correlation coefficient between the expression of IgG and C3 on Tzanck smear and that on the skin biopsy was 0.670 for IgG and 0.505 for C3.
* P value for IgG was <0.0004 and for C3 was<0.008, which is statistically significant.
In the present study the study population consisted of 16 females (53.33%) and 14 males (46.67%). The slight female preponderance is comparable to study conducted by Srinath et al . Study conducted by Aithal et al  showed equal distribution among both sexes.
In the present study 70% of cases presented within 6 months of onset of disease. This is comparable to the study conducted by Aithal et al  in which 75% of cases presented within 3 months of onset of disease.
In the present study 70% of cases had oral mucosa as their initial site of involvement. This is comparable to the study conducted by Suliman et al  in which 57% of cases had oral mucosa as their initial site of involvement.
Skin was involved in 80% of cases in the present study. This is comparable to studies conducted by Srinath et al.  Suliman et al  and Aithal et al,  which reported skin involvement in 86%, 85% and 75% of cases, respectively.
The predilection of various site involvement in the present study were as follows-Trunk (73%), extremities (66 %), face (63 %), scalp (60 %).
This is comparable to study conducted by Srinath et al  which showed involvement as follows- trunk (73%), extremities (46%), face (53%) and scalp (33%).
This is also comparable to a study conducted by Suliman et al  which reported extremities and trunk as the most common sites of involvement followed by scalp.
Erosions (80%) were predominant lesions in the present study followed by vesicles, bullae and pustules. This is comparable to study conducted by Suliman et al  which reported erosions as the predominant lesions followed by ulcers and vesicles.
Oral mucosa was the most common site involved in the present study with 96% of the patients having oral lesions. This is comparable to studies conducted by Srinath et al  and Suliman et al  which reported oral mucosal involvement in 100% and 90% of cases, respectively.
The characteristic cytological finding in Tzanck smear in cases of pemphigus vulgaris are the presence of acantholytic cells. Cytological examination of Tzanck smears, by Giemsa stain is in itself a very sensitive and rapid test to diagnose PV. It is also an easier technique when compared with biopsy, to sample multiple sites as well as poorly accessible sites like the retro molar area, but the findings on Tzanck alone are not pathognomonic for PV, because the characteristic acantholytic cells could also be observed in other subtypes of pemphigus.
In the present study acantholytic cells were present in Tzanck smears in all 30 cases (100%). This was comparable to the studies conducted by Srinath et al  and Durdu et al , both of which reported 100% positivity for acantholytic cells, but the study done by Shailaja et al  showed only 50% positivity for acantholytic cells.
In the present study neutrophils were the predominant inflammatory component in Tzanck smears. This is comparable to a study conducted by Aithal et al  which also reported neutrophils as the predominant inflammatory component in Tzanck smears.
Histopathological examination of skin biopsy revealed suprabasal cleft in 100% of cases and acantholytic cells in 90% of cases in the present study. This is comparable to studies conducted by Srinath et al  and Kabir et al  which showed suprabasal cleft with acantholytic cells in 100% and 87 % of cases, respectively. Also comparable to study conducted by Suliman et al  which showed suprabasal cleft in 90% of cases.
Even though the Tzanck smears are highly sensitive, they are not specific for pemphigus. To improve the specificity, DIF is performed on Tzanck smears which makes it a useful diagnostic tool in the early diagnosis of pemphigus. Present study showed DIF positivity for IgG in 24/30 Tzanck smears studied (80%). In a study done by Kabir et al  DIF on Tzanck smears showed positivity for IgG in 13/15 cases (86%). Study done by Durdu et al  showed IgG positivity in 14/14 Tzanck smears (100%). Proportion of clinically diagnosed cases of pemphigus vulgaris showing positivity for IgG by DIF on Tzanck smears in studies conducted by Acosta et al  and Varma et al  were 76% and 77% respectively.
The present study was conducted on 30 cases of Pemphigus Vulgaris patients who presented in the Department of dermatology and whose Tzanck smear samples and perilesional skin biopsies were concurrently received in the Department of Pathology Government medical college Kottayam during the period from March 2017-August 18. DIF was done on Tzanck smear for IgG and C3 and it was compared with DIF done on their corresponding histopathology sections.
The mean age of the present study population was 49. Minimum age was 14 years and maximum was 74 years. Majority belonged to age groups of 30-40 and 40-50 with 6 patients, i.e., 20% each. Mean age is comparable to study conducted by Aithal et al  and Yaeen et al. 
1. DIF on Tzanck smear from mucosal or skin lesion showed a positivity of 80% for IgG and 60% for C3.
2. The corresponding perilesional skin biopsies on DIF showed a positivity of 83% for IgG and 70% for C3.
3. Hence the correlation coefficient between the expression of IgG and C3 on Tzanck smear and that on the skin biopsy was assessed and was found to be 0.670 for IgG and 0.505 for C3.
Based on the correlation coefficient, DIF on Tzanck smear can be considered a reasonably good supportive diagnostic test for pemphigus vulgaris and may be recommended after larger series of similar studies are performed.
I express my sincere and heartfelt gratitude to Dr. Sankar S., Professor and Head of Department of Pathology, Dr. Sheeja S, my guide and Dr. Mary Vineetha, my co-guide and Dr. Ginju, Dr. Geethanjali and Mr. Josin Mathew for extending their invaluable help in furnishing my dissertation.
 Kambil SM, Madavamurthy P. Immunobullous disorders: clinical histopathological and immunofluorescence study of thirty-six cases. Muller J Med Sci Res 2014;5(2):134-8.
 Suliman NM, Astrom AN, Ali RW, et al. Clinical and histological characterization of oral pemphigus lesions in patients with skin diseases: a cross sectional study from Sudan. BMC Oral Health 2013;13:66.
 Arpita R, Monica A, Venkatesh N, et al. Oral pemphigus vulgaris: case report. Ethiopian Journal of Health Sciences 2015;25(4):367-72.
 Mohan KH, Pai S, Rao R, et al. Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol 2008;74(4):415-9.
 Aithal V, Kini U, Jayaseelan E. Role of direct immunofluorescence on Tzanck smears in pemphigus vulgaris. Diagnostic Cytopathology 2007;35(7):403-7.
 Yaeen A, Ahmad QM, Farhana A, et al. Diagnostic value of Tzanck smear in various erosive, vesicular and bullous skin lesions. Indian Dermatol Online J 2015;6(6):381-6.
 Prabhala S, Deshpande AK, Reddy M, et al. Study of Tzanck smears over a period of six months. Journal of Evidence Based Medicine and Healthcare 2015;2 (10):1365-71.
 Kabir AK, Kamal M, Choudhury AM. Clinicopathological correlation of blistering diseases of skin. Bangladesh Med Res Counc Bull 2008;34(2):48-53.
 Durdu M, Baba M, MD, Seckin D. The value of Tzanck smear test in diagnosis of erosive, vesicular, bullous and pustular skin lesions. J Am Acad Dermatol 2008;59(6):958-64.
 Acosta AE, Hietanen J, Ivanyi L. Direct immunofluorescence on cytological smears in oral pemphigus. Br J Dermatol 1981;105(6):645-51.
 Verma KK, Khaitan BK, Singh MK. Antibody deposits in Tzanck smears in pemphigus vulgaris. J Cutan Pathol 1993;20(4):317-9.
Arun Jose (1), Sheeja S. (2), Mary Vineetha (3), Sankar S. (4)
(1) Junior Resident, Department of Pathology, GMC, Kottayam, Kerala, India.
(2) Associate Professor, Department of Pathology, GMC, Kottayam, Kerala, India.
(3) Assistant Professor, Department of Dermatology, Venereology and Leprosy, GMC, Kottayam, Kerala, India.
(4) Professor and HOD, Department of Pathology, GMC, Kottayam, Kerala, India.
'Financial or Other Competing Interest': None.
Submission 20-12-2018, Peer Review 16-01-2019, Acceptance 22-01-2019, Published 28-01-2019.
Dr. Arun Jose, Junior Resident, Department of Pathology, Government Medical College, Gandhinagar, P. O. Kottayam-686008, Kerala, India.
Caption: Figure 1. Oral Lesions-Pemphigus Vulgaris
Caption: Figure 2. Histopathoiogy-Suprabasai Bullae Showing Tombstone Appearance--H&E--100X
Caption: Figure 3. Oral Tzanck Smear Showing Acantholytic Cells-Giemsa-- 400X
Caption: Figure 4. DIF Skin Biopsy--Fishnet Positivity for IgG (200X)
Caption: Figure 5. DIF Skin--Fishnet Positivity for C3-200X
Caption: Figure 6. DIF Tzanck Smear--Fishnet Positivity for C3-200X
Caption: Figure 7. DIF Tzanck Smear--Fishnet Positivity for IgG-200X
Caption: Figure 8. DIF Tzanck Smear-Fishnet Positivity for IgG-400X
Table 1. Comparison of Mean Age with Other Studies Study Mean Age (Years) Present Study 49 Aithal et al  47.35 Yaeen et al  35.1 Table 2. Comparison of Gender Distribution with Other Studies Study Males (%) Females (%) Present Study 46.67 53.33 Srinath et al  46.67 53.33 Aithal et al  50 50 Table 3. Comparison of Skin Involvement with Other Studies Study Skin Involvement (%) Present Study 80 Srinath et al  86.67 Suliman et al  85.71 Aithal et al  75 Table 4. Comparison of Various Site Involvement with Different Studies (%) Study Trunk extremities face Scalp Present Study 73 66 63 60 Srinath et al  73 46 53 33 Table 5. Comparison of Oral Involvement with Various Studies Study Oral Involvement Present Study 96% Srinath et al  100% Suliman et al  90% Table 6. Comparison of Cytological Positivity of Tzanck Smears Study Presence of Acantholytic Cells Present Study 100% Srinath et al  100% Durdu et al  100% Shailaja et al  50% Table 7. Comparison of Histopathological Findings with Other Studies Study Suprabasal Cleft Acantholytic Cell (%) (%) Present Study 100 90 Srinath et al  100 100 Kabir et al  87 87 Suliman et al  90 Table 8. Comparison of DIF Positivity for IgG On Tzanck Smears Study IgG Positivity Present Study 80% Kabir et al  86% Durdu et al  100% Acosta et al  76% Varma et al  77%
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|Title Annotation:||Original Research Article|
|Author:||Jose, Arun; Sheeja, S.; Vineetha, Mary; Sankar, S.|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Jan 28, 2019|
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