The value of Tzanck smear test in diagnosis of erosive, vesicular, bullous, and pustular skin lesions

Murat Durdu, MD,a Mete Baba, MD,a and Deniz Sec kin, MDb Adana and Ankara, Turkey See commentary on page 965
Background: Tzanck smear is generally used for the diagnosis of the pemphigus group of autoimmune bullous diseases and mucocutaneous herpesvirus infections. There are only a few studies in the literature investigating its diagnostic value. Objectives: We aimed to investigate Tzanck smear ndings and to determine the diagnostic value of this test in moist (erosive, vesicular, bullous, and pustular) skin lesions. We also aimed to develop an algorithmic approach for the diagnosis of these types of skin lesions according to the Tzanck smear ndings. Methods: Samples were stained with May-Gru nwald-Giemsa and evaluated by the same dermatologist. In some patients, methylene blue and Gram staining or direct immunouorescence examinations were additionally performed. In all of the study cases, after the evaluation of clinical and laboratory ndings (including, when appropriate, potassium hydroxide examination; viral serology; bacterial and fungal cultures; histopathology; direct and indirect immunouorescence; patch testing), the denite diagnosis was established. We also determined the sensitivity and the specicity of certain Tzanck smear ndings. Results: Tzanck smear was performed in a total of 400 patients with moist skin lesions. The sensitivities of multinucleated giant cells and acantholytic cells in herpetic infections, dyskeratotic acantholytic cells and cocci in bullous impetigo, pseudohyphae in candidiasis, acantholytic cells in pemphigus and more than 10 tadpole cells (magnication 3100) in spongiotic dermatitis were 84.7%, 92%, 100%, 100%, and 81.5%, respectively. Limitations: Because Tzanck smears were evaluated by the same dermatologist, no comment could be made regarding the interobserver reliability of this test and how the level of experience with this technique might affect the results. Also, the sensitivity and the specicity of Tzanck smear test ndings for certain diseases could not be calculated because of an insufcient number of patients. Conclusion: The Tzanck smear test is an inexpensive, useful, and an easy diagnostic tool for certain skin diseases. ( J Am Acad Dermatol 2008;59:958-64.)

INTRODUCTION
Cytology is a diagnostic tool used to investigate the characteristics of individual cells. In this method, materials obtained in a variety of ways are transferred
From the Department of Dermatology, Bas xkent University Faculty of Medicine, Adanaa and Ankarab Hospitals. Funding sources: None. Conflicts of interest: None declared. Accepted for publication July 15, 2008. Reprint requests: Deniz Sec kin, MD, Bas xkent University Faculty of Medicine, Department of Dermatology, 5. sokak No. 48, Bahcelievler, 06490, Ankara, Turkey. E-mail: denizs@baskent-ank. edu.tr, deseckin@hotmail.com. Published online October 17, 2008. 0190-9622/$34.00 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.07.059

to a glass slide, stained with various dyes, and then examined under the light microscope.1 As a method for the diagnosis of cutaneous disorders, cytology was first used by Arnault Tzanck in 1947.1,2 Although it was suggested as a simple, rapid, and reliable technique to be used in the diagnosis of many diseases during the following 6 decades, the practice of cytodiagnosis has been limited to a few diseases.2 To date, therefore, only a few studies have examined the dermatological use and diagnostic value of this method.2-19 The majority of these studies have re-lated to herpetic infections,3-7 pemphigus,8,9 cutaneous leishmaniasis,10 and cutaneous neoplasms, especially basal cell carcinoma.11-17 In this prospective study, we aimed to describe the Tzanck smear ndings in patients with erosive,

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vesicular, bullous, and pustular skin lesions and determine the sensitivity and specicity of this test in some of the skin diseases studied. We also intended to develop an algorithmic approach for diagnosis of moist skin lesions using Tzanck smear test ndings.

MATERIAL AND METHODS

Patients Patients with erosive, vesicular, bullous, and pustular skin lesions who applied to the Department of Dermatology, Bas xkent University Faculty of Medicine, Adana Hospital between January 2006 and January 2008 and could not be diagnosed only by dermatological physical examination were included in the study. Demographic, clinical, and laboratory data including patients age, sex, lesion types, characteristic cytologic ndings, and the diagnostic methods used were recorded. The local ethical committee approved the study and informed consents of the patients were obtained. Preparation of Tzanck smears Three dermatologists took the smear materials from skin lesions while 3 nurses stained them. Lesions were rst cleaned with 70% alcohol swab. When there was a vesicle, bulla, or a pustule, the intact roof of the lesion was incised along one side and folded back, and the base of the lesion was gently scraped with a scalpel (No. 15). Scrapings of the erosive lesions were also obtained. If the lesions had crusts, these crusts were carefully removed with sterile forceps, and the base of the lesions was scraped. The cellular materials obtained were then spread as a thin layer onto at least two microscopic slides and air-dried. All of the specimens were stained with May-Gru nwald-Giemsa (Bio-optica, Milan, Italy). For this procedure; air-dried smears were immersed 5 times for 1 second each into MayGru nwald-Giemsa solution A (methyl alcohol), MayGru nwald-Giemsa solution B (eosin solution), and May-Gru nwald-Giemsa solution C (thiazine dye). The slides were then rinsed and air dried. In lesions suspected to be candidiasis, specimens were also stained with methylene blue.18 Upon detection of numerous cocci and bacilli in the smear, Gram staining was additionally performed.18 In specimens showing only acantholytic cells in cytologic examination, direct immunofluorescence microscopic examination was also performed.19 Evaluation of Tzanck smears and denition of the denitive diagnosis In each case, the stained preparations were evaluated under a light microscope (310 and 340 magnication, and 3100 magnication with

immersion oil) by the same dermatologist after clinical examination. Those diagnoses made by this dermatologist were then classied as infectious or immunologic diseases, spongiotic dermatitis, or genodermatoses. In all of the study cases, after the evaluation of clinical and laboratory ndings (including, when appropriate, potassium hydroxide (KOH) examination; viral serology; bacterial and fungal cultures; histopathology; direct and indirect immunouorescence; patch testing with the European standard series), the denitive diagnosis was established. The diagnoses based on clinical and Tzanck smear ndings were then compared with the denitive ones. Cytologic ndings observed on smears were photographed with a Kodak EasyShare Z700 digital camera (Eastman Kodak Company, Rochester, NY), which has a resolution of 1600 3 1200 pixels. Images were stored in compressed (4:1) Joint Photographic Experts Group format. Calculations of the diagnostic value of Tzanck smear ndings We determined the diagnostic value of certain Tzanck smear ndings of the cutaneous diseases observed in more than 10 patients. For this purpose, we calculated the parameters, including sensitivity (true positives 4 [true positives 1 false negatives]) and specicity (true negatives 4 [true negatives 1 false positives]) for the Tzanck smear ndings investigated. For the diagnostic values of multinucleated giant cells and acantholytic cells in herpetic infections, of tadpole cells in spongiotic dermatitis, of dyskeratotic acantholytic cells and cocci in bullous impetigo, of acantholytic cells in pemphigus, and of pseudohyphae in candidiasis; diseases with lesions other than herpetic infections, those other than spongiotic dermatitis, those other than bullous impetigo, those other than pemphigus, and those other than candidiasis, respectively, were used as control groups.

RESULTS
Four hundred patients (217 [54.2%] women and 183 [45.8%] men) were included in the study. The patients ages ranged between 2 and 68 years (mean, 36 years). Of the lesions, 176 (44%) were vesicular, 116 (29%) were erosive, 63 (15.8%) were pustular, and 45 (11.2%) were bullous (Table I). In addition to May-Gru nwald-Giemsa staining, methylene blue and Gram staining was performed in 12 (3%) and 25 (6.3%) patients, respectively, while direct immunouorescence examination was conducted in 28 patients (7%).

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Table I. Categories and number of skin diseases, lesion types, characteristic cytologic findings, positivity of cytology, and diagnostic methods used in the study subjects
Skin diseases No. Lesion types (No.) Cytologic findings Positivity of cytology, No. (%*) Diagnostic methods (No.)

Infections 299 Herpetic infections 249 Vesicle (126), erosion Herpes simplex 157 (74), pustule (49) Varicella zoster 92 Bullous impetigo 25 Bullae (17), erosion (8) Hand, foot, and mouth disease 7 Vesicle (6), erosion (1) Orf 6 Pustule (6) Candidiasis 12 Pustule (8), erosion (4) Immunologic disorders 45 Pemphigus 21 Erosion (7), bullae (12), vesicle (2) Pemphigus vulgaris 14 Pemphigus foliaceus 4 Pemphigus herpetiformis 3 Bullous pemphigoid 9 Bullae (9) Erythema multiforme 15 Erosion (8), bullae (7) Spongiotic dermatitis Allergic contact dermatitis Irritant contact dermatitis Genodermatosis Hailey-Hailey disease Dariers disease Total

Acantholytic and multinucleated giant cells1

Cocci and dyskeratotic acantholytic cells2 Cells with syncytial nuclei1 Guarnieri body1,2 Pseudohyphae2 Acantholytic cells1

211 (84.7) 128 (81.5) 83 (90.2) 23 (92) 6 5 12 21 (100)

Histopathology (83), serology (82), clinical findings (84) Bacterial culture (25) Clinical findings and serology (7) Histopathology (6) Fungal culture (12), KOH preparation (12) Histopathology (30), DIF and IIF (30)

Eosinophils, absence of acantholytic cells1 Necrotic cells, leukocytes, no acantholytic cells2

7 9

Histopathology (15)

54 36 Erosion (12), vesicle (24) Tadpole cells, lymphocyte predominance20 18 Vesicle (18) 2 1 Erosion (1) 1 Erosion (91) 400 Tadpole cells, neutrophil predominance20 Acantholytic cells1 Corps ronds and grains1

29 (80.5) 15 1 1

Histopathology (54), patch test with European standard series (54)

Histopathology (2), DIF and IIF (2)

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DIF, Direct immunofluorescence; IIF, indirect immunofluorescence; KOH, potassium hydroxide. *Stated if the total number is higher than 20.

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Table II. Sensitivity and specificity of some of the cytologic findings

Sensitivity (%) Specificity (%)

Herpetic infections Multinucleated giant cells and acantholytic cells Spongiotic dermatitis More than 10 tadpole cells (3100 magnification) Bullous impetigo Dyskeratotic acantholytic cells and cocci Pemphigus Acantholytic cells Candidiasis Pseudohyphae

84.7

100

81.5

99.3

92.0

100 Fig 1. Intranuclear inclusion bodies (arrows) in multinucleated giant cell. (May-Gru nwald Giemsa stain; original magnification: 31000.)

100 100

43.4 100

After clinical examination of the patients and the evaluation of Tzanck smear tests of the lesions, the dermatologist classied his diagnoses as infectious for 299 lesions (74.8%), spongiotic dermatitis for 54 (13.5%), immunologic for 45 (11.2%), and genodermatoses for 2 (0.5%). The categories and number of skin diseases are shown in Table I.1,2,20 For the denitive diagnosis, the following laboratory examinations were needed: Histopathologic examination in 190 patients (47.5%), viral serology in 89 (22.3%), patch test with European standard series in 54 (13.5%), direct and indirect immunouorescence examination in 32 (8%), bacterial culture in 25 (6.26%), KOH preparation in 12 (3%), and fungal culture in 12 (3%) (see Table I). Cytologic ndings Infectious diseases. Cytologic ndings observed in different disease groups, and the sensitivity and specicity of some of those Tzanck smear ndings are shown in Tables I and II, respectively. Cytologic examination was performed in 299 cutaneous infectious lesions (74.8%). The most common infectious disease group was herpetic infections (62.3%). The characteristic finding for herpetic infections was acantholytic and multinucleated giant cells. The sensitivity and specificity of this cytologic finding for herpetic infections were 84.7% and 100%, respectively (see Table II). Tzanck smear positivity was 100%, 69.2%, and 59.7% in vesicles, pustules, and erosive lesions, respectively. Intranuclear inclusions (Cowdry A body) were observed in only 9 of 92 specimens (9.8%) of herpes zoster (Fig 1). Cytologic examination showed dyskeratotic acantholytic cells, numerous neutrophils and cocci in 23 (92%) of 25 bullous impetigo lesions. In the other two bullous impetigo lesions, only cocci and

Fig 2. Streptocytes (arrow) in pemphigus. (MayGru nwald Giemsa stain; original magnification: 31000.)

numerous neutrophils were observed. Diagnoses were conrmed by bacterial cultures. Guarnieri bodies were observed in 5 of 6 orf lesions. In the other orf lesion, numerous leukocytes, acantholytic and necrotic cells were noted. Immunologic diseases. Acantholytic cells were present in the cytologic examination of all pemphigus lesions. Streptocytes were observed in only one specimen of pemphigus vulgaris (Fig 2). Direct immunofluorescence tests on Tzanck smears showed IgG deposition around the cells in all of the pemphigus lesions. Cytologic examination in 7 of 9 bullous pemphigoid cases showed numerous eosinophils without acantholytic or tadpole cells. Eosinophils were absent in the remaining two cases. In 9 of 15 erythema multiforme cases, necrotic cells and numerous leukocytes were observed. Tzanck smear in 6 cases also showed apoptotic cells characterized by nuclear fragmentation (Fig 3, A), pyknosis (Fig 3, B), and karyorrhexis. Spongiotic dermatitis. Cytologic examination was performed in 54 cases with spongiotic dermatitis (36 allergic contact dermatitis and 18 irritant contact dermatitis). Tzanck smears showed more than 10

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Fig 3. Apoptotic cells in erythema multiforme showing nuclear fragmentation (arrow) (A) and pyknosis (arrow) (B). (May-Gru nwald Giemsa stain; original magnification: 31000.)

tadpole cells and numerous lymphocytes (at 3100 magnication) in 29 (80.5%) of 36 lesions of allergic contact dermatitis. More than 10 tadpole cells and numerous polymorphonuclear leukocytes (at 3100 magnication) were observed in 15 of 18 lesions of irritant contact dermatitis. Genodermatoses. This group included one case with Dariers disease and one case with HaileyHailey disease. Tzanck smears disclosed corps ronds and grains in the lesions of Dariers disease and acantholytic cells in erosive lesions of Hailey-Hailey disease. Direct immunouorescence tests on Tzanck smears were negative in both cases. Mean duration of microscopic analysis of each Tzanck smear was 9 minutes (range, 2-15 minutes). The cost of Tzanck smear test for each patient was V0.5 for May-Gru nwald-Giemsa staining, V0.15 for each methylene blue and Gram stainings, and V10 for direct immunouorescence examination.

DISCUSSION
Tzanck smear ndings in 400 patients with erosive, vesicular, bullous, and pustular skin lesions reported in this study had skin diseases which are known to be diagnosable by this method as noted in the literature.1-17 For herpes simplex and varicella-zoster infections in which acantholytic and multinucleated giant cells are diagnostic, the percentage of positivity of the

Tzanck test for herpes simplex has been reported between 53.1%3 and 86%,7 and positivity of the Tzanck test for varicella-zoster infections has been reported between 64%5 and 79%6 in different studies. In our study, positivity was 81.5% for herpes simplex and 90.2% for varicella-zoster infections. Solomon et al3 found that the percentage of positivity in Tzanck smears prepared from herpetic infections at an earlier stage tended to be higher than the ones prepared at later stages. Namely, the percentage of positivity in vesicles was much higher than that of pustules.3 We also found negative results in herpetic lesions with pustules and crusts. Intranuclear inclusion bodies surrounded by subtle clear halo (Cowdry A body) are characteristic of herpetic infections, but are difficult to find.2 In our study, inclusion bodies were observed in only 9 specimens (9.8%) of herpes zoster. Positivity of acantholytic cells in Tzanck smear test in cases with pemphigus has been reported between 93.3%8 and 100%.9 In addition to typical acantholytic cells, Sertoli rosette cells (consists of cell aggregates with an epithelial cell at the center surrounded by a ring of leukocytes) and streptocytes (chains of white blood cells) may also be observed in cases of pemphigus.2 Acantholytic cells were observed in all of our cases with pemphigus, and streptocytes were observed in only one of our patients. In cases with pemphigus, Tzanck smears can also be used for

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Fig 4. Algorithmic approach for erosive, vesicular, bullous, and pustular skin lesions according to the Tzanck smear ndings. (A, B, D-G, I, J, May-Gru nwald Giemsa stain; C, direct immunofluorescence stain; H, methylene blue stain; original magnifications: A, B, D-H, 31000; C, 3400.) DIF, Direct immunofluorescence; SSSS, staphylococcal scalded skin syndrome.

the direct immunofluorescence test.19 Direct immunofluorescence of Tzanck smears disclosed the typical IgG deposits around the acantholytic keratinocytes in all of our patients with pemphigus. Tzanck smear ndings of bullous pemphigoid and erythema multiforme are nonspecic.1,2 In our 6 cases with erythema multiforme, we observed apoptotic cells characterized by pyknosis, karyorrhexis, and nuclear fragmentation for the first time. Presence of more than 10 tadpole cells in a Tzanck smear at 3100 magnication was dened a diagnostic indicator for spongiotic vesicular dermatitis.21 Pariser21 reported this criterion to be 83% sensitive and 100% specific for their cases with spongiotic vesicular dermatitis. Our study demonstrated that this criterion was 81.5% sensitive and 99.3% specific for spongiotic vesicular dermatitis. Interestingly, we detected dense tadpole cells and acantholytic cells in a Tzanck smear of 3 cases with pemphigus

herpetiformis; direct immunofluorescence of materials obtained from these cases showed IgG deposits around the acantholytic keratinocytes. In a previous study, cytologic examination of contact dermatitis lesions also showed that lymphocytes were the predominant cells in 84% of the cases with allergic contact dermatitis, while polymorphonuclear leukocytes were predominant in 82% of the cases with irritant contact dermatitis. The author concluded that cytology would also be of help in differentiating the two types of contact dermatitis.20 The detection of lymphocyte predominance in 80.5% of our cases with allergic contact dermatitis and polymorphonuclear leukocyte predominance in 83.3% of our cases with irritant contact dermatitis also supports the results of the above-mentioned study.20 According to the Tzanck smear ndings we obtained, we propose an algorithmic approach for erosive, vesicular, bullous, and pustular skin lesions

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(Fig 4). This algorithmic approach is essentially based on the specific Tzanck smear findings reported previously and our results. By performing a Tzanck smear and using the algorithmic approach we propose, a dermatologist can diagnose many skin diseases with erosive, vesicular, bullous, and pustular lesions such as herpetic infections; bullous impetigo; pemphigus; orf, hand, foot and mouth disease; candidiasis; contact dermatitis; and Hailey-Hailey disease. Tzanck smear is an inexpensive and rapid test; the mean cost of Tzanck smear test per patient is only V0.5, and it takes only minutes to get the results. If the Tzanck smear test is performed in a laboratory in the United States, it is essential to comply with the Clinical Laboratory Improvement Amendments of 1988 (CLIA88), which establish the minimum performance standards for all laboratory testing, including specic regulations for quality control.22 The current study has some limitations; namely, as Tzanck smears were evaluated by the same dermatologist throughout the study, no comments could be made regarding the interobserver reliability of the Tzanck smear test and how the level of experience with this technique might affect the results. We think that experience would have an impact on diagnostic accuracy rates. Also, we could not calculate the sensitivity and the specicity of Tzanck smear test ndings for certain diseases because of an insufcient number of patients. We think that special training is needed to make the breadth of diagnoses reported herein, but that practicing dermatologists can learn to use this technique to aid in their practice. In conclusion, Tzanck smear test appears to be a useful and practical diagnostic tool in certain skin diseases. We believe that the number of skin diseases diagnosed by the Tzanck smear test would be increased by its routine use in daily dermatology practice.
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