10.5826/dpc.1203a101
Introduction:  Few studies have been conducted on the use of Direct Immunofluorescence (DIF) on skin/mucosal scraping smear for diagnosis of pemphigus disease; however, the diagnostic value of DIF on the smear has not been fully evaluated.
Objectives: to assess the sensitivity and specificity of DIF on skin/mucosal smear for diagnose of pemphigus in the patients presenting with mucocutaneous erosive lesions.
Methods: A total of 89 patients including 40 males and 49 females aged between 23 and 80 years old with various bullous disorders were enrolled in the study. For definite diagnosis, all the patients were subjected to lesional biopsy for pathological studies and perilesional biopsy for DIF studies. In all the cases, skin/mucosal scraping  smears were prepared from the perilesional healthy skin/mucosa and were stained with immunofluorescence conjugated anti-IgG.
Results:Of 89 patients, 56 (63%) patients were diagnosed with pemphigus. Immunodeposits favoring the pemphigus were demonstrated in the 46 smears of 56 cases of pemphigus (sensitivity of 82%). No case with other types of bullous disease had positive DIF on the smear (specificity of 100%). 
Conclusion: The findings of the study showed that the sensitivity of DIF on the smear is not high enough to allow us replacing the conventional DIF with smear-DIF for diagnosis of pemphigus, while the specificity of 100% would allow the   unequivocal identification of a subset of patients with pemphigus.
text/xml
2160-9381
1983
en
Nikoo Mozafari
https://creativecommons.org/licenses/by-nc/4.0
Dermatology Practical & Conceptual
smear
Can immunofluorescence on Tzanck smear be used as a substitute for direct immunofluorescence on skin biopsy in diagnosis of pemphigus disease? 
Text.Serial.Journal
https://dpcj.org/index.php/dpc
Original Article
2022-07-28
2021-08-23
2022-07-28
2022-09-15
e2022101
Dermatol Pract Concept
Introduction:  Few studies have been conducted on the use of Direct Immunofluorescence (DIF) on skin/mucosal scraping smear for diagnosis of pemphigus disease; however, the diagnostic value of DIF on the smear has not been fully evaluated.
Objectives: to assess the sensitivity and specificity of DIF on skin/mucosal smear for diagnose of pemphigus in the patients presenting with mucocutaneous erosive lesions.
Methods: A total of 89 patients including 40 males and 49 females aged between 23 and 80 years old with various bullous disorders were enrolled in the study. For definite diagnosis, all the patients were subjected to lesional biopsy for pathological studies and perilesional biopsy for DIF studies. In all the cases, skin/mucosal scraping  smears were prepared from the perilesional healthy skin/mucosa and were stained with immunofluorescence conjugated anti-IgG.
Results:Of 89 patients, 56 (63%) patients were diagnosed with pemphigus. Immunodeposits favoring the pemphigus were demonstrated in the 46 smears of 56 cases of pemphigus (sensitivity of 82%). No case with other types of bullous disease had positive DIF on the smear (specificity of 100%). 
Conclusion: The findings of the study showed that the sensitivity of DIF on the smear is not high enough to allow us replacing the conventional DIF with smear-DIF for diagnosis of pemphigus, while the specificity of 100% would allow the   unequivocal identification of a subset of patients with pemphigus.
Nikoo Mozafari
2022/07/28
10.5826/dpc.1203a101
e2022101
Skin Research Center, Shahid Beheshti University of Medical Sciences; Department of dermatology, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2160-9381
smear
en
e2022101
Aithal V, Kini U, Jayaseelan E. Role of direct immunofluorescence on Tzanck smears in pemphigus vulgaris. Diagn Cytopathol. 2007;35(7):403-407. DOI: 10.1002/dc.20657. PMID:17580352.
Can immunofluorescence on Tzanck smear be used as a substitute for direct immunofluorescence on skin biopsy in diagnosis of pemphigus disease? 
https://dpcj.org/index.php/dpc/article/download/1983/1679
Can Immunofluorescence on Skin/Mucosal Scraping
 Smear for Pemphigus Diagnosis Substitute Direct
      Immunofluorescence on Skin Biopsy?
 Mohammad Shahidi Dadras 1, Zahra Asadi Kani 1, Behnaz Hamedani 1, Sahar Dadkhahfar 1,
                        Nazy Rastgoo1, Nikoo Mozafari 1,2


1 Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2 Department of dermatology, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran.



Key words: pemphigus, direct immunofluorescence, sensitivity, specificity, smear
Citation: Shahidi Dadras M, Asadi Kani Z, Hamedani B, Dadkhahfar S, Rastgoo N, Mozafari N. Can Immunofluorescence on skin/
mucosal scraping smear for pemphigus diagnosis Substitute Direct Immunofluorescence on Skin Biopsy? Dermatol Pract Concept.
2022;12(3):e2022101. DOI: https://doi.org/10.5826/dpc.1203a101
Accepted: October 24, 2021; Published: July 2022
Copyright: ©2022 Shahidi Dadras et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-
NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use,
distribution, and reproduction in any medium, provided the original authors and source are credited.
Funding: None.
Competing interests: None.
Authorship: All authors have contributed significantly to this publication.
Corresponding author: Nikoo Mozafari, MD, Tajrish Sq, Skin Research Center, Shohada-e-Tajrish Hospital, Shahrdari St, Tehran, Iran
Telephone number: (98) 21-22741507. E-mail address: Nikoo_MD@yahoo.com




  ABSTRACT             Introduction: Few studies have been conducted on the use of Direct Immunofluorescence (DIF) on
                       skin/mucosal scraping smear for diagnosis of pemphigus disease; however, the diagnostic value of DIF
                       on the smear has not been fully evaluated.
                       Objectives: The present study was carried out to assess the sensitivity and specificity of DIF on skin/
                       mucosal smear for diagnose of pemphigus in the patients presenting with mucocutaneous erosive
                       lesions.
                       Methodology: A total of 89 patients including 40 males and 49 females aged between 23 and 80
                       years old with various bullous disorders were enrolled in the study. For definite diagnosis, all the pa-
                       tients were subjected to lesional biopsy for pathological studies and perilesional biopsy for DIF studies.
                       In all the cases, skin/mucosal scraping smears were prepared from the perilesional healthy skin/mucosa
                       and were stained with immunofluorescence conjugated anti-IgG.
                       Results: Of 89 patients, 56 (63%) patients were diagnosed with pemphigus. Immunodeposits favoring
                       the pemphigus were demonstrated in the 46 smears of 56 cases of pemphigus (sensitivity of 82%). No
                       case with other types of bullous disease had positive DIF on the smear (specificity of 100%).
                       Conclusion: The findings of the study showed that the sensitivity of DIF on the smear is not high
                       enough to allow us replacing the conventional DIF with smear-DIF for diagnosis of pemphigus, while the
                       ­specificity of 100% would allow the unequivocal identification of a subset of patients with pemphigus.




Original Article | Dermatol Pract Concept. 2022;12(3):e2022101                                                                           1
Introduction                                                      skin/mucosa and were stained with immunofluorescence
                                                                  conjugated anti-IgG.
Pemphigus as a rare autoimmune blistering disease is charac-
terized by the widespread flaccid blisters and erosions on the    Preparation of the Smears and Application of DIF
skin and mucous membranes [1]. The hallmark of pemphi-            on the Samples
gus is finding the immunoglobulin G (IgG) autoantibodies          For preparing the smears, the perilesional skin or mucosa
against the cell surface of keratinocytes. Pemphigus is clas-     adjacent to the fresh blister or erosion was first anesthetized
sified into subtypes based on the main autoantigen involved       through the intradermal lidocaine injection and then, they
in the pathogenesis of disease [1]. Detection of IgG auto-        were gently scraped using the small curette. Then, the ob-
antibodies raised against the cell surface of keratinocytes is    tained cellular materials were spread as a thin layer onto
considered as the gold standard for diagnosis of pemphigus.       at least two glass slides and were air-dried. The prepared
Pemphigus can be differentiated from other vesiculobullous        smears were sent to the Department of Pathology for stain-
or pustular diseases through detection of these autoantibod-      ing. Smears were incubated with fluorescein isothiocyanate
ies [2]. Direct Immunofluorescence (DIF) examination is the       (FITC)-conjugated goat antihuman IgG (CEDARLANE, lot
most reliable and sensitive diagnostic test used for all forms    number: 7201111401) for 30 minutes in a moist chamber
of pemphigus. DIF is able to show the IgG and C3 deposi-          at 37degree temperature. The sections were then washed in
tion around the epithelial cells, confirming the diagnosis of     Phosphate-buffered saline (PBS) (2washes of 15 min each),
pemphigus [3].                                                    mounted in buffered glycerol, and examined under fluores-
    Durdu et al have demonstrated that the keratinocyte cells     cent microscope.
obtained by Tzanck smear can be used as a substrate for               Detection of the ring-shaped deposition of IgG on the
DIF studies [4]. Obtaining the Tzanck smears is less invasive     individual acantholytic cells or the net-like intercellular fluo-
than skin or mucosal biopsy and would be useful in the pem-       rescence pattern when sheet of cells were present was consid-
phigus patients with conjunctiva involvement or inaccessible      ered positive for diagnosis of pemphigus (Figure 1).
oral lesions that cannot be biopsied easily [5]. In this tech-        The immunofluorescence (IF)-stained samples were stud-
nique, preparation of the samples is much more rapid than         ied independently by 2 of the authors. They were unaware of
the conventional DIF and there is no need for specialized         the results of the conventional DIF.
equipment such as the cryostat.

                                                                  Calculation of the Diagnostic Value of IF -Stained
Objectives                                                        Skin/Mucosal Scraping Smears
                                                                  The parameters including sensitivity (ie the percentage of
The current study was performed to investigate the useful-        patients with positive conventional DIF whose IF-stained
ness of DIF on skin scraping smears obtained from intact          smear was positive), specificity (ie the percentage of patients
perilesional skin in the patients with bullous/erosive disor-     with negative conventional DIF whose IF-stained smear was
ders in order to evaluate the use of skin/mucosal smears as       negative), Positive Predictive Value (PPV) (ie the percentage
an alternative to skin or mucosal biopsies for diagnosis of       of patients with positive IF-stained smear whose conven-
pemphigus.                                                        tional DIF was also positive) ,and Negative Predictive Value
                                                                  (NPV) (ie, the percentage of patients with negative IF-stained
                                                                  smear whose conventional DIF was also negative) were cal-
Methods
                                                                  culated to determine the diagnostic value of the IF-stained
Patients                                                          smear (Table 1). Kappa coefficient was calculated to evaluate
The study protocol was approved by the Institutional Ethics       the concordance of the IF-stained smear and conventional
Committee and an informed written consent was obtained            DIF, and the P value of less than 0.05 was considered as sta-
from all the patients.                                            tistically significant.
    A total of 89 consecutive patients with erosive, vesicular,
bullous or pustular skin, or mucosal lesions were included
                                                                  Results
in this study. Demographic, clinical and laboratory data
including patients age, gender, and lesion location were re-      Totally, 89 patients (40 males and 49 females) aged between
corded using a questionnaire. For definite diagnosis, all the     23- 80 years were included in this study. Table1 shows the
patients were subjected to lesional biopsy for pathological       characteristics of the patients and their diagnosis based on
studies and perilesional biopsy for DIF studies. In all the       the histopathological and conventional DIF results. Classical
cases, smears were prepared from the perilesional healthy         suprabasal acantholysis at the lesional biopsy and immune



2                                                                      Original Article | Dermatol Pract Concept. 2022;12(3):e2022101
Figure 1. Positive direct immunofluorescence examination of skin scraping smear in a patient with pemphigus vulgaris shows immunoglobin
G deposition around the individual acantholytic cells (A) and those in groups (B) (400×).



                         Table 1. Characteristics of Patients with Bullous/Erosive Lesion
 Age, years                        46.60±12.16,
 Mean ±SD,                            (23-80)
 range
 Gender, N(%)       Female                         49 (55%)
                    Male                           40 (45%)
 Diagnosis             Diagnose of patient with bullous          Intraepidermal Positive DIF on        Positive DIF on perilesional
                      disease based on histopathology of            perilesional punch biopsy             scraping smear N(%)
                              lesional samples, N                          sample N(%)
                    Pemphigus                          56                   56 (100%)                           46 (82%)
                        Vulgaris                       54                       54                                  44
                        Folliaceous                     2                        2                                   2
                    Bullous pemphigoid                  6                     0(0%)                               0(0%)
                    TEN                                 3                     0(0%)                               0(0%)
                    Erythema multiforme                 3                     0(0%)                               0(0%)
                    Fixed drug eruption                 7                     0(0%)                               0(0%)
                    Herpes zoster                       3                     0(0%)                               0(0%)
                    chicken pox                         3                     0(0%)                               0(0%)
                    Bullous impetigo                    2                     0(0%)                               0(0%)
                    Bite reaction                       1                     0(0%)                               0(0%)
                    Acute eczema                        3                     0(0%)                               0(0%)
                    Sweet syndrome                      1                     0(0%)                               0(0%)
                    Pustular psoriasis                  1                     0(0%)                               0(0%)
 Site of obtaining smear or biopsy for IF study N(%)
                    Oral mucosa                    19 (21%)                     16                                  12
                    Extremities                     9 (10%)                      2                                   2
                    trunk                          40 (45%)                     18                                  14
                    Scalp                          21 (24%)                     20                                  18

DIF = Direct Immunofluorescence; IF = Immunofluorescence; SD = standard deviation; TEN = Toxic Epidermal; Necrolysis.


deposition compatible with the diagnosis of pemphigus                    IF on the smear was positive in 46 (82%) patients with
(intercellular lattice-like pattern) were demonstrated in the        pemphigus. IF on the smear had a sensitivity of 0.82 (95%
IgG–stained perilesional biopsies of 56 cases.                       Confidence Interval [CI] 0.72-0.92), a specificity of 1.00



Original Article | Dermatol Pract Concept. 2022;12(3):e2022101                                                                        3
    Table 2. Distribution of Frequency of Dif on Tzank Smear and Conventional Dif on Skin/Mucosa
                             Biopsy in Patients with Bullous/Erosive Lesions.
                                            Result of conventional DIF (golden criteria for pemphigus diagnosis)
 Results of DIF on Tzank smear               Positive, N(%)                 Negative, N(%)                   Total, N(%)
 Positive, N(%)                                 46 (82%)                        0 (0%)                        46(55%)
 Negative, N(%)                                 10 (18%)                      33 (100%)                       43(45%)
 Total N(%)                                    56 (100%)                      33 (100%)                      89 (100%)

Kappa = 0.773 P < 0.001
DIF = Direct Immunofluorescence.



(95% CI 1.00-1.00), a PPV of 1.00 (95% CI 1.00-1.00), and        cell or in the intercellular region in the case of cell clumps,
a NPV of 0.77 (95% CI 0.64-0.89). A significant concor-          compatible with positive IF pattern of pemphigus [4].
dance was found between the results of IF-stained smears             Although, the IF examination of skin scraping smear
and those prepared by the conventional DIF for diagnose of       seems a simple and practical cytological technique for diag-
pemphigus (Kappa= 0.773, P <0.001). Table 2 presents the         nosis of pemphigus, there is a limited evidence on the relative
data on IF-stained smears and those prepared by the conven-      sensitivity and specificity of DIF on the smear compared to
tional DIF.                                                      the DIF on skin biopsy as a gold standard.
                                                                     According to the review of the literature, there are a few

Conclusions                                                      related studies with divergent results in this context. Durdu
                                                                 et al have reported about the typical IgG deposit around
Lesional skin/mucosal scraping called Tzanck smear is gen-       the acantholytic keratinocytes in the Tzanck smears of all
erally used for diagnosis of the Herpes Simplex Virus infec-     (100%) the 20 patients with pemphigus [4]. Nonetheless,
tions [6]. The presence of acantholytic cells accompanied        Aithal et al have shown that among 12 pemphigus patients
by multinucleated giant cells is a characteristic cytological    with positive DIF on the skin biopsy, only 6 of them (50%)
finding for diagnosis of herpetic infections [6]. This method    had positive DIF on the Tzanck smear [8].
has also been suggested as a simple and rapid technique to           In the current study, the result of DIF examination on
be used in diagnosis of pemphigus disease [6]. Cytological       the smear was positive in 46 (82%) of the pemphigus pa-
examination of the smears obtained from scraping of floor        tients (out of 56 patients). Ten patients had negative results.
of the blisters in the pemphigus patients has shown the pres-    The larger sample size or technical issue in the IF staining of
ence of typical acantholytic cells (or Tzanck cells). These      the smears might explain observing these 10 false negative
cells are not pathognomonic for the pemphigus and are            results.
commonly observed in other types of bullous disease such             It also could be attributed to the fact that the smears
as Hailey-Hailey disease and herpetic infections [4,6]. The      were taken from the healthy perilesional skin and not from
cyto-diagnosis is not extensively used due to low specificity    the blister floor. In scraping of the intact skin, collected
of this technique in diagnosis of the pemphigus. Positivity of   keratinocytes are mostly from the superficial epidermal
                                                                 ­
acantholytic cells in the cases with pemphigus has been re-      layers where immune depositions are partly or completely
ported by 96.7%-100% while, the specificity of acantholytic      absent in the subset of patients with pemphigus vulgaris.
cells for pemphigus has been reported by 43.3%-60% [4,7].        In pemphigus vulgaris, due to the difference in the relative
This means that, if we rely on the use of Tzanck smear alone,    amount of desmoglein 3 in the epidermal layers, occasionally
40%-60% of the cases presented with erosive and bullous          the fluorescence may be limited to or more intense in the
eruptions would falsely be diagnosed as pemphigus. Then,         lower levels of the epidermis [2].
for definite diagnosis of pemphigus, autoantibodies raised           According to the results, the sensitivity and specificity of
against the epithelial cell membrane have to be detected by      DIF on skin/mucosal smear for diagnosis of pemphigus were
applying the DIF staining [3].                                   equal to 82% and 100%, respectively. This sensitivity was
     DIF analysis of the perilesional skin biopsy is the most    not high enough to allow us replacing the conventional DIF
accurate approach for diagnosis of pemphigus , showing IgG       on skin biopsy with DIF on skin/mucosal smear, for diag-
deposits on the surface of keratinocytes [3] . The direct im-    nosis of pemphigus. In other words, approximately 20% of
munofluorescence test on Tzanck smears has been proposed         pemphigus patients would be missed if we rely on IF staining
as a simple alternative to skin biopsy for diagnosis of pem-     on the smear alone. Nonetheless, the observed specificity of
phigus [4]. DIF examination of a Tzanck smear shows bright       100% allows an extremely high level of confidence to diag-
green fluorescence at the cell margins of single acantholytic    nose the pemphigus in the case of positive DIF on the smear.



4                                                                     Original Article | Dermatol Pract Concept. 2022;12(3):e2022101
    One limitation of this study is that the only DIF was used        3. Giurdanella F, Diercks G, Jonkman M, Pas H. Laboratory diag-
as a gold standard to differentiate pemphigus cases from                   nosis of pemphigus: direct immunofluorescence remains the gold
                                                                           standard. Br J Dermatol. 2016;175(1):185-186. DOI: 10.1111/
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                                                                           bjd.14408. PMID: 26798993.
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if, it is negative then, a biopsy must be taken for conven-                 3083.2010.03740.x. PMID: 20553359.
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Original Article | Dermatol Pract Concept. 2022;12(3):e2022101                                                                              5
