LEADING ARTICLE

Am J Clin Dermatol 2009; 10 (3): 141-152 1175-0561/09/0003-0141/$49.95/0

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Reintroducing the Tzanck Smear

Brent Kelly1 and Tally Shimoni2
1 Department of Dermatology, University of Texas Medical Branch, Galveston, Texas, USA 2 School of Medicine, University of Texas Medical Branch, Galveston, Texas, USA

Abstract

The Tzanck smear is a rapid and simple technique that can be performed in the clinic or doctors office with minimal patient discomfort or cost. It is known to most dermatologists as a rapid test for diagnosing herpes virus infections. Its use, however, can be more widely applied, including in the diagnosis of pustular diseases of the newborn, cutaneous infections, vesiculobullous diseases, and non-melanoma skin cancers. Material is gently scraped from the base of a vesicle, blister, or pustule or directly from the lesion or tumor. Typically, the material is allowed to air dry after which it can then be stained with a variety of stains, including Giemsa, toluidine blue, and methylene blue. The Tzanck smear should not be used to replace more standard diagnostic techniques, but rather as an adjunct to the physical examination.

The Tzanck smear is known to most dermatologists as a rapid test for diagnosing various herpes virus infections. Its use, however, can be much more broadly applied and the test may be a valuable office tool for a variety of skin conditions. This simple, quick, and reliable test utilizes cytology, i.e. the evaluation of single cells. Interpretation of single cells is often difficult without the correlating tissue architecture that is easily obtained by the more common dermatologic diagnostic procedure, the skin biopsy. In certain circumstances, however, the rapid information that can be obtained by the Tzanck smear may not only lead to the correct diagnosis, but may enable the clinician to initiate therapy and perhaps improve disease outcome.[1] George Papanicolaou was credited with the first use of exfoliative cytology for the detection of uterine/cervical cancer in 1943. A few years later, in 1947, Arnault Tzanck described a simple technique to evaluate vesiculobullous diseases. He worked closely with his colleagues Darier and Civatte in an era long before immunofluorescence became available as a means of distinguishing between blistering disorders.[1] Now, over half a century later, interest in clinical cytology in dermatologic practice is mostly limited to evaluation of herpes infection with the Tzanck smear, superficial fungal infections with potassium hydroxide, and scabies detection with mineral oil preparations. Cytology, it seems, has not become a major influential factor in todays clinical practice. The reasons for this are probably multi-factorial and complex. Certainly, the

ease with which biopsies can be obtained is a factor. Also, more technologically advanced diagnostic tests such as immunostaining, polymerase chain reaction (PCR), and immunofluorescence are now available; however, while these are certainly useful, they are also quite expensive. In addition, cytology may not be emphasized or adequately taught in dermatology and dermatopathology training programs. Indeed, no dedicated sections to cytology or tissue smears can be found in the most commonly used dermatopathology textbooks.[2-4] There is also an interest in the development of new technology that provides non-invasive diagnostic evaluations, for example, confocal in vivo microscopy. These advances, however, are not widely available and their clinical utility is still unknown. As our patients desire inexpensive, non-invasive but reliable results, we should re-evaluate and re-introduce ourselves to a cheap, rapid, non-invasive, simple, and precise test the Tzanck smear. 1. Technique
1.1 Preparation

The Tzanck smear is a rapid and simple technique that can be performed in the clinic or doctors office with minimal patient discomfort or cost. Samples should be taken from an unroofed vesicle or blister by gently scraping the base of the lesion. The material obtained is subsequently smeared on to a

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clean microscopic slide and allowed to air dry. Cytodiagnosis of suspected malignant lesions requires removal of any crust from ulcerated tumors. If the tumor is not ulcerated, the lesion should be incised with a sharp, pointed scalpel. The material is obtained by scraping a blunt scalpel along the base of the lesion and the tissue obtained is then pressed between two slides.[5]
1.2 Fixation

The cytologic specimen obtained can be placed in a fixative to preserve the sample and prevent artifacts developing during the drying process.[5] Most commonly, however, the specimen is allowed to air dry prior to staining.
1.3 Staining
Fig. 1. Epidermal multinucleated giant cells are characteristic of herpes virus infections. Example of multinucleated giant cells in a Tzanck smear of the vesicle contents of herpes simplex virus infection lesions.

Several different staining methods are available, of which the Giemsa stain is the most commonly employed. Giemsa stain is a solution containing azure II-eosin, glycerin (glycerol), and methanol (methyl alcohol).[5] This stain is poured over the smear, allowed to sit for 15 minutes, and subsequently washed with sterile water. When examined under the microscope, the stained nuclei vary in color from reddish blue to purple to pink and the cytoplasm stains blue. Other stains include hematoxylin and eosin, Wright, methylene blue, Papanicolaou, and toluidine blue. Rapid processing can be achieved with toluidine blue, which requires only 60 seconds for staining.

2. Applications
2.1 Cutaneous Infections
2.1.1 Herpes Virus Infections

Herpes viruses encompass a large group. The most common herpes viruses causing cutaneous diseases include herpes simplex (types I and II) and varicella zoster.[6] Cytology of the base of the vesicles/blisters does not allow differentiation between the two. Nor does it differentiate between recurrent and primary lesions. It will, however, typically reveal multinucleated syncytial giant cells that exhibit nuclear molding, so that the nuclei fit together like a jigsaw puzzle (figure 1).[1] The cells appear as if they have been inflated (ballooning degeneration), and can grow tremendously in size, sometimes reaching a diameter of 6080 mm.[7] Previous studies confirmed that positive Tzanck preparations have a high correlation ith positive viral cultures (94.1%), demonstrating this method to be an accurate, rapid tool for the diagnosis of cutaneous herpes simplex.[8]
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The diagnosis of herpes simplex virus (HSV) infection can also be confirmed by viral culture, PCR, direct fluorescence antibody, and type-specific serologic tests.[9] However, the overall sensitivity of viral cultures is only approximately 50%.[10] Moreover, the diagnostic yield is highest in the early stages of the disease. Thus, the culture should optimally be obtained within 4 days of the appearance of an active lesion since viral shedding is most significant at this time. Viral isolation rates are also higher in primary compared with recurrent genital herpes, particularly in the setting of asymptomatic recurrences with subclinical shedding.[11] Real-time HSV PCR assays have recently emerged as a method to confirm HSV infection. Compared with viral cultures, PCR has been demonstrated to be more sensitive. In fact, one study demonstrated that HSV DNA could be detected in ulcerative lesions on 15 of 17 days compared with only 3 of 17 days by viral isolation.[12] However, the cost of the assay, which substantially exceeds that of the Tzanck smear and viral-isolation culture techniques, limits adoption of real-time HSV PCR as the primary diagnostic tool. Thus, although further development of this assay now allows differentiation of HSV type 1 and HSV type 2 using real-time TaqMan PCR techniques,[13] it is seldom employed because of the excessive cost. Although various serologic assays have been used for diagnosis of HSV infection, including complement fixation, passive hemagglutination, ELISA, and radioimmunoassay, these tests have limited applications. To illustrate, a review of serologic commercial immunoassays for HSV antibodies found that the overall sensitivity and specificity was only approximately 70%.[14]
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The assays failed to identify the correct subtype of HSV in 2637% of patients.
2.1.2 Superficial Fungal Infections

Dermatophyte infections are a common diagnosis in any dermatologic practice. The potassium hydroxide method for fungal evaluation is commonly employed and its diagnostic value is not debated.[1] However, it can be difficult to evaluate the morphology of the fungal elements. Often, these elements are difficult to separate from the surrounding scale debris or previously applied medicated creams. Occasionally, a direct stain, such as the Tzanck smear, can be helpful. Since fungal scrapings do not adhere well to a microscopic slide, Barr[1] has described obtaining appropriate material for examination using plastic tape strippings. In this manner, scrapings are made in the regular fashion, but left adherent to the lesion. The tape is then applied to the lesion, removing the abraded scrapings, which are then stained using Paragon Multiple Stain for 60 seconds. The sticky surface of the tape is then applied to a clean microscopic slide for direct examination. This technique will allow some interpretation to be made regarding the morphology of hyphae, pseudohyphae, and spores.[1] In addition, the preferential staining of these elements creates a marked contrast from the background scale and debris (figure 2). Giemsa-stained smears can differentiate between Candida and geophilic strains of dermatophytes, both of which can present with pustules or large bullae. Dermatophyte infections demonstrate segmented, branched hyphae. Candida infection reveals typical oval budding yeasts with septate hyphae and psuedohyphae (elongated, filamentous cells connected endto-end).[15]

facial papules diagnosed as cryptococcosis by direct smear.[18] This was later confirmed by histology and tissue culture. Cryptococcosis is an important fungal infection, particularly in immunosuppressed patients. It represents the third most common invasive fungal infection in organ transplant patients.[19] Cutaneous cryptococcosis almost always represents disseminated disease,[20] which bears a high mortality rate. Thus, early detection and intervention are critical. Recognition of disseminated cryptococcosis may pose a challenge because of its varying presentation. More often than is generally appreciated, it appears to present with papules or ulcers, with a molluscum or cellulitis-like appearance, sometimes even resembling panniculitis.[21] A rapid bedside test such as the

2.1.3 Deep Fungal Infections

In 1986, Lesher and Kight[16] reported a case of a 36-year-old HIV-positive (human T-lymphotropic virus III+) woman with scattered facial papules. A Tzanck/direct smear revealed negatively staining budding yeasts singly and in clusters morphologically consistent with Histoplasma. This fungus was later confirmed by a histologic biopsy specimen. Two previous case reports described disseminated cryptococcosis diagnosed by the Tzanck smear. In 1984, Borton and Wintroub[17] reported a case of a 31-year-old man with AIDS and herpetiform lesions on the face. A Tzanck smear was performed to evaluate possible herpes virus infection, but instead of revealing multinucleated syncytial giant cells, the smear revealed multiple encapsulated organisms. Another case report described a 60-year-old renal transplant recipient with multiple
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Fig. 2. Superficial fungal infections. (a) Clinical photograph showing an erythematous, scaly plaque with some pustules. (b) Although a potassium hydroxide preparation is quite helpful in diagnosis, the presence of hyphae or pseudohyphae can also be easily identified using Giemsa-stained smears.
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direct smear may be ideal for identifying organisms (figure 3). It is important to realize, however, that the organism may not be obtainable from a superficial scraping because the manifestations of cutaneous cryptococcosis are protean.[22] Certainly, the direct smear should not replace the more standard testing of biopsy, tissue culture, and serologic evaluations. However, as demonstrated in the reported cases, it can be helpful for making an early diagnosis and perhaps improving outcome if therapy is quickly initiated. Civila et al.[23] demonstrated that the direct smear could identify most lesions of cutaneous sporotrichosis. In 36 of 42 patients evaluated, asteroid bodies with club-shaped, peripheral hyaline bodies could be identified. Indeed, these

bodies could be identified on many occasions from the pus alone without the addition of Giemsa stain. Blastomycosis, caused by Blastomyces dermatitidis, is yet another systemic mycosis that can be diagnosed with the aid of a direct smear. Three clinical forms exist: primary pulmonary blastomycosis, systemic blastomycosis, and primary cutaneous inoculation blastomycosis. Principally, blastomycosis manifests as a pulmonary infection that involves the skin after dissemination. Rarely, skin lesions can also follow percutaneous inoculation of the pathogen.[24] Hematogenous spread from the lungs produces systemic blastomycosis. In this form, the skin is the most commonly affected extra-thoracic organ, involving approximately 50% of disseminated cases. Skin lesions are usually solitary and begin as inflammatory nodules that subsequently break down to form granulomatous ulcers and plaques. The borders are typically raised. If a crusted edge is removed, a granulomatous base studded with pustules is revealed. Direct examination of a smear of pus from a lesion is a rapid way to diagnose blastomycosis. After the crust has been lifted from a lesional border, swabbed material from a micropustule is smeared on a glass slide. Necrotic tissue crushed between two slides can also be used. Microscopic examination of such material will demonstrate double-refractile walls of yeast and single, broad-based buds. This technique can lead to rapid diagnosis and treatment of a condition which, if untreated, can reactivate years later.
2.1.4 Leishmaniasis

Fig. 3. Cryptococcus infection. (a) Clinical photograph showing multiple umbilicated, molluscum-like papules. (b) Tzanck smear showing multiple encapsulated organisms with narrow-based budding, demonstrating Cryptococcus infection.
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Although detection of parasite DNA from lesions by PCR is the most sensitive method of diagnosing both cutaneous and mucosal leishmaniasis, this testing technique is not always feasible in developing countries or institutions without proper laboratory equipment. In practice, it is sensible to diagnose cutaneous leishmaniasis microscopically by identifying amastigotes in biopsies, scrapings, or Giemsa-stained lesion smears. In fact, the Tzanck smear is the most powerful diagnostic tool in the initial stages of this mucocutaneous protozoan infection because Leishman-Donovan bodies can be detected with the May-Gru nwald-Giemsa stain.[7] These bodies appear as blue ellipsoid bodies, 24 mm in length, with an eccentric nucleus and a minor kinetosome at the opposite pole. Wrights cells or large macrophages, which contain numerous protozoa within the weakly basophilic cytoplasm, are also evident. While serum antileishmanial antibodies can be detected with standardized and sensitive assays, it is less costly and more rapid to perform a smear of the material from the ulcer base for prompt interpretation. Ram rez et al.[25] demonstrated that direct microscopic examination of dermal scrapings obtained from the
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bottoms of ulcers in 115 patients had a sensitivity of 90.4%. Moreover, other parasitologic diagnostic methods, such as culture and histopathologic examination of biopsies, were shown to be less sensitive (67.5% and 64.3%, respectively). When microscopic examinations include samples from both the margins of lesions and from the base of the ulcers combined, the sensitivity of diagnosis may rise to 94%. Thus, dermal scrapings can be an effective primary procedure for diagnosis of cutaneous leishmaniasis.
2.1.5 Molluscum Contagiosum

Most often, the characteristic clinical appearance of molluscum contagiosum lesions allows for rapid identification. Occasionally, however, the appearance can be quite atypical, especially in immunosuppressed patients.[26] Molluscum contagiosum can often imitate a deep fungal infection or a neoplasm (figure 4a). Expressing the core or scraping the top of a suspected lesion can reveal Henderson-Patterson inclusion bodies on direct smear. These deeply basophilic, oval bodies are characteristic (figure 4b).
2.1.6 Hand, Foot, and Mouth Disease

Hand, foot, and mouth disease (HFMD), a viral exanthem, mainly affects children <10 years of age. Most cases are attributed to enterovirus, most notably Coxsackie virus A16, and manifest as 35 mm-diameter, lancet or rhomboid-shaped vesicles with a grayish surface surrounded by erythema. Although a clinical diagnosis is adequate in most cases, the virus may be isolated from vesicle, throat, and stool specimens. Rarely, severe neurologic sequelae and death may occur. Enterovirus is highly contagious, especially among children. Thus, prompt diagnosis with a vesicular smear may be beneficial. Moreover, the lesions secondary to HFMD are at times difficult to differentiate from aphthous ulcers. Cytology of oral vesicles suggesting HFMD demonstrates a pattern of moderately hypertrophic and multinucleated epithelial cells. Aphthous ulcers, on the other hand, reveal necrotic epithelial cells intermingled with neutrophils.[7]
2.2 Pustular Diseases of the Newborn

Fig. 4. Molluscum contagiosum. (a) Clinical photograph showing umbilicated lesion of molluscum contagiosum, which is somewhat atypical in this patient with AIDS. (b) Tzanck smear revealing the presence of diagnostic intracytoplasmic molluscum bodies (Henderson-Patterson bodies), the largest known inclusion bodies (3035 mm). These are virus-transformed keratinocytes, appearing as ovoid, deeply basophilic bodies with a hyaline, homogeneous structure surrounded by a membrane.

Pustular diseases arising within the first few weeks of life are common. They can range from benign and self-limited to life threatening. Prompt and precise diagnosis is essential so that early intervention can be instituted if necessary. This group of diseases may represent one of the most important applications of the Tzanck/direct smear. Van Praag et al.[27] suggest that the Tzanck smear should always be the first test performed given its
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ease, speed, and sensitivity. These authors also point out that since infants are at such an increased risk of bacterial, viral, and fungal infections, evaluations should be carried out in all patients with pustular eruptions. Of the following neonatal pustular diseases, most of the data on Tzanck/direct smear use are anecdotal. To our knowledge, prospective studies or other data on the sensitivity and specificity for this test do not exist.
2.2.1 Erythema Toxicum Neonatorum

Erythema toxicum neonatorum is a benign, self-limited, and common condition, seen in 3050% of all infants and of unknown
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etiology. The eruption is typically composed of erythematous macules, papules, and pustules or wheals distributed most commonly on the face and trunk. The lesions usually appear after 2472 hours and can manifest as evolving crops that wax and wane over several days to weeks.[28] The clinical appearance is usually diagnostic, but often the eruption may resemble more serious diseases such as herpes virus infections, bacterial infections, and scabies. A Tzanck/direct smear will reveal a dense accumulation of eosinophils as seen in figure 5. Erythema toxicum neonatorum will resolve spontaneously, and no treatment is necessary.[24] Thus, a rapid Tzanck smear should be the first test in the diagnostic work-up to spare a healthy neonate from an invasive evaluation for sepsis or administration of potentially harmful antibacterial therapy.
2.2.2 Acropustulosis of Infancy and Transient Neonatal Pustular Dermatosis

Acropustulosis of infancy and transient neonatal pustular melanosis both demonstrate abundant neutrophils, few eosinophils, and absent bacteria on the direct smear. Differentiating between the two is based on their clinical presentation. Both conditions are typically benign and self-limited. Acropustulosis of infancy occurs primarily in African American male infants and is typically a recurrent, pruritic, vesicular, and pustular eruption on the hands and feet.[27] Transient neonatal pustular melanosis is also commonly seen in African Americans, but is characterized by multiple 1- to 5-mm pustules that can occur at almost any location. They progress to either a hyperpigmented macule or rupture leaving a collarette of scale. Both acropustulosis and transient neonatal pustular melanosis are selflimited diseases and no specific therapy is recommended since they resolve spontaneously.[24]
2.2.3 Bullous Impetigo

Fig. 5. Erythema toxicum neonatorum. (a) Clinical photograph of a neonate with an erythematous papular and pustular eruption. (b) Smears of pustules of erythema toxicum neonatorum showing plentiful eosinophils.

Certain phage groups of Staphylococcus aureus produce exfoliative exotoxins. When this infection is localized, rapidly enlarging bullae typically in intertriginous areas develop, a condition known as bullous impetigo. The bullae are extremely flaccid and rupture easily, leaving a shallow erosion surrounded by a rim of scale. However, the lesions re-epithelialize quickly and thus do not result in scarring. A direct smear will reveal abundant neutrophils and bacteria (Gram-positive cocci).[24] Pneumonia, septicemia, and osteomyelitis are associated complications if the condition is allowed to progress, making early intervention critical.[29]
2.2.4 Congenital Herpes

Congenital herpes virus infection varies from asymptomatic infection to encephalitis and is most commonly transmitted
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from vesicular lesions in the birth canal. Indeed, HSV isolation at the time of labor is the most important risk factor associated with infantile herpes infection with an odds ratio of 346 (95% CI 125, 956).[30] While only 50% of infants demonstrate clustered cutaneous lesions with an erythematous base 24 hours after birth, rapid diagnosis and initiation of treatment are critical to prevent further progression or complications, such as seizures, pneumonia, respiratory distress, and aseptic meningitis. A prompt smear of the vesicular material is useful for ruling out bacterial sepsis and incontinentia pigmenti, and for diagnosing the herpetic infection. Given that the mortality of congenital herpes exceeds 15% in infants with encephalitis, and increases
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to 57% if the infection is disseminated, rapid diagnosis and initiation of treatment with acyclovir (aciclovir) are essential to minimize neonatal complications.[31]
2.2.5 Congenital and Neonatal Candidiasis

smear negative for the characteristic findings of HSV and demonstrating many eosinophils could steer medical professionals in the correct direction and prevent inappropriate use of acyclovir in patients with incontinentia pigmenti.

Candida species (most often Candida albicans) represent the most common fungal infection in neonates. Two clinical forms are classically described: congenital candidiasis, which is an intrauterine infection; and neonatal candidiasis, acquired by passage through an infected birth canal. For both forms, manifestations may range from localized skin eruptions with no systemic findings to diffuse skin disease with life-threatening systemic infection. Clinical features, direct smear examination of specimens, and appropriate cultures are useful for differentiating the lesions from other more common dermatoses of the neonatal period.[32] To illustrate, a case report by Drut and Drut[33] demonstrated the usefulness of a cytologic study of skin scrapings from an 8-day-old newborn who presented with a generalized maculopapular rash. A direct smear demonstrating mycelia and ovoid spores of fungal organisms aided in rapid diagnosis of candidiasis and initiation of treatment.

2.3 Cutaneous Malignancies

2.3.1 Basal Cell Carcinoma

2.2.6 Congenital, Self-Healing Langerhans Cell Histiocytosis

Congenital, self-healing Langerhans cell histiocytosis is a rare cause of pustules in the neonate. There are anecdotal cases in the literature suggesting that direct smear can be helpful in the diagnosis of this condition. Colon-Fontanez et al.[34] described two cases of a crusted, vesicular eruption initially thought to be herpes virus infection. The direct smear was negative for multinucleated giant cells, but after confirmation of the true diagnosis of Langerhans cell histiocytosis by histology, the authors reviewed their smears and found predominant histiocytes with reniform indented nuclei and abundant cytoplasm.
2.2.7 Incontinentia Pigmenti

Incontinentia pigmenti, an X-linked dominant syndrome, is a multi-system disease characterized by cutaneous, ophthalmologic, and neurologic abnormalities.[35] Because the disease progresses in a manner that mimics the erythematous, linearly distributed vesicles of herpes virus, the two diseases can be grossly indistinguishable. In fact, Faloyin et al.[36] report a case of a female infant noted to have two linear crops of vesicles on her right arm and leg, suggesting herpes viral infection, yet all laboratory tests and cultures of the blister fluid were negative for HSV. Biopsy, on the other hand, showed eosinophilic spongiosis with dyskeratotic epithelial cells. Thus, a Tzanck
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Basal cell carcinoma (BCC) represents the most common skin cancer in Caucasians. Its characteristic clinical appearance leads most dermatologists to the correct diagnosis. Typically, however, a biopsy is obtained and patients must defer treatment until the diagnosis is confirmed. Often, there is a temptation to treat the cancer on the day of the initial visit prior to obtaining biopsy results. Thus, it would be ideal to have a rapid screening test that would give an immediate diagnosis and allow therapy to begin without delay. There is some evidence that the direct smear may be useful in this regard. Findings on the direct smear typically include clusters of deeply basophilic, atypical basal cells. The nucleus-to-cytoplasm ratio is high with the nucleus occupying around 80% of the cell. A rim of basophilic cytoplasm surrounds this nucleus. Often, the architectural feature of peripheral palisading of the basaloid cells can be seen in these clusters[1] (figure 6). Several publications have drawn attention to the high sensitivity of diagnosis of BCC using direct smears. Bakis et al.[37] summarized these studies in a meta-analysis published in 2004. These investigators pointed out that examining cutaneous exfoliative cytology as a diagnostic tool for clinically suspected BCCs in the eight studies (1261 BCCs) included in the metaanalysis had a combined sensitivity of 97% (95% CI 94, 99) and a specificity of 86% (95% CI 80, 91). This high sensitivity, if indeed it is accurate, certainly makes an argument for the possibility of use of the direct smear as an effective screening test for BCC. However, Bakis et al.[37] also emphasized that the poor methodology of many of the studies included in the metaanalysis undermined the statistical results. For example, in many of the studies, it is not clear if the evaluators were blinded to the clinical or correlating histologic information. Variations in observer interpretations were also not addressed in many of the studies. In addition, it is not clear how patients were recruited in six of the eight studies included in the meta-analysis. Despite the lack of prospective studies, it does seem that the direct smear may be an attractive, plausible screening test based on the data available. One of the many benefits of using such a test includes its cost effectiveness. Transportation difficulties, days off work, and scheduling conflicts are issues that make
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nant melanoma. The scraping may affect the prognostic information accorded to the lesion by its histology, i.e. ulceration, Breslows depth.
2.3.2 Squamous Cell Carcinoma

Malignant squamous keratinocytes tend to take a wide variety of forms and shapes.[1] However, the two distinctive cytologic features reported include the tendency of the cells to be arranged individually (isolated cells with no clusters) and pleomorphism. This includes nuclear atypia, increased and abnormal mitoses, and multiple nuclei. Acantholytic cells may also be apparent.[2] As with BCCs, there are no prospective, randomized, blinded studies evaluating the utility of the Tzanck/direct smear for squamous cell carcinoma (SCC). Memije et al.[38] reported data for 30 patients with lesions clinically suspicious for SCC. Direct smear confirmed SCC in 26 patients, which closely correlated to the histologic diagnosis (27 patients positive for SCC). In only one case did the direct smear not correlate with the histologic diagnosis. It may be more difficult to obtain sufficient cellular material for cytologic evaluation from hyperkeratotic SCCs compared with other SCCs. Indeed, direct smear may be more appropriate for diagnosis of ulcerated, soft, and non-keratotic lesions. However, in the study by Memije et al.,[38] the material was obtained from the lesion border or the base of the biopsy.
2.3.3 Mammary and Extramammary Paget Disease

Fig. 6. Basal cell carcinoma (BCC). (a) Clinical photograph of BCC. (b) BCC smear with Giemsa stain showing darkly stained cells with palisading nuclei at the periphery.

return visits difficult. A rapid screening test may minimize these matters, allowing treatment to be initiated at the first visit. Moreover, being less invasive and scarring than a biopsy, this technique may be ideal for the cosmetically sensitive patient. Patients with multiple suspicious lesions may also benefit from use of the direct smear in that they may be spared multiple biopsies that may be quite complicated. The limitations of the direct smear should also be mentioned. Although studies evaluating this technique and its correlation with histologic subtype (nodular, superficial, infiltrative, morpheaform, etc.) are currently lacking, it seems unlikely that they would be distinguishable on direct smear because evaluating the architecture is not possible with cytology. Evaluating a pigmented lesion with a direct smear may be problematic if the lesion in question is revealed to be malig 2009 Adis Data Information BV. All rights reserved.

Anecdotal case reports suggest that Paget cells may be easily seen on a direct smear.[39,40] Thus, cytology may aid in the differentiation between this significant disease and other trivial conditions such as psoriasis or eczema.[7] These cells appear larger than keratinocytes with a vacuolated cytoplasm and hypertrophic nucleus. They can occur singly or in groups. Moreover, stains for epithelial mucin, such as periodic acid Schiff stain, can aid in diagnosis by staining most Paget cells.[5]

2.3.4 Erythroplasia of Queyrat

Direct smear of material from erythroplasia of Queyrat shows polyhedral, spindle-shaped, and round cells with poikilokaryosis or nuclear polymorphism relating to size, shape, and staining. The smear alone is practically diagnostic for this intraepithelial carcinoma.[7]
2.3.5 Melanoma

Given the current standards of using histologic parameters for determining prognostic factors for melanoma (e.g. Breslows
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depth, Clarks level, ulceration, mitotic rate, lymphoid response, vascular invasion), it is difficult to imagine any scenario where obtaining a direct smear would be advised. Distorting the biopsy specimen in any way may lead to inaccurate histologic assessment. However, findings of malignant melanoma on a direct smear have been reported and include pleomorphic cells characteristic of the histologic subtype, typically with abundant pigment. Barr[1] suggests that one application of the direct smear for melanoma is in the setting of obvious disseminated disease. A rapid direct smear may confirm the melanoma and allow any experimental therapy to proceed.
2.3.6 Zosteriform Metastases

Tumors may mimic varicella-zoster virus morphologically. In fact, Manteaux et al.[41] reported on two patients with undifferentiated adenocarcinoma and breast cancer who presented with cutaneous metastases spreading in a dermatomal distribution mimicking herpes zoster. The unusual scenario of zosteriform metastases would be something to consider in patients known to have a pre-existing primary cancer. Since metastases to the skin have a wide spectrum of clinical appearances, and immunosuppressed patients are often infected with herpes virus, the Tzanck smear may help distinguish infection from metastasis. However, a biopsy should be used for definitive diagnosis, since discohesive malignant cells in the vesicles can be misinterpreted as large herpetic balloon cells on direct smear.

2.4 Vesiculobullous Diseases

Long before the modern development of electron microscopy and immunofluorescence, Tzanck developed criteria for the diagnoses of several cutaneous diseases by examination of cytology.[42] Since Tzancks original description of his fundamental method for examining a number of vesiculobullous diseases was published in 1948,[43] numerous research articles attempting to expand on his techniques have been published. None of these modifications has proven more reliable than or as simple as the method initially employed by Tzanck himself to diagnose vesiculobullous diseases, particularly pemphigus vulgaris and bullous pemphigoid.
2.4.1 Pemphigus Vulgaris

Fig. 7. Pemphigus vulgaris. (a) Clinical photograph showing mucosal involvement in a patient with pemphigus vulgaris. (b) Smear of pemphigus vulgaris revealing multiple acantholytic cells (Tzanck cells). A Tzanck cell is a large round keratinocyte with a hypertrophic nucleus, hazy or absent nucleoli, and abundant basophilic cytoplasm. The basophilic staining is deeper peripherally on the cell membrane (mourning edged cells) due to the tendency of the cytoplasm to become condensed at the periphery, resulting in a perinuclear halo.

Lesions of pemphigus vulgaris will often reveal multiple acantholytic squamous cells. Increased nucleus-to-cytoplasm ratio, an eosinophilic cytoplasm, and a more intense basophilic peripheral rim (the mourning edge) characterize these cells
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(figure 7). One study verified the reliability of the Tzanck test as >92% accurate in detecting oral pemphigus through significant cytomorphologic findings.[44] Thus, cytomorphologic studies may be useful in screening suspected cases of oral pemphigus vulgaris in conjunction with an immunocytologic test to provide the definitive diagnosis.
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Pemphigus vegetans has similar findings but is more likely to show increased inflammatory cells, especially eosinophils.
2.4.2 Dermatitis Herpetiformis

Dermatitis herpetiformis is characterized by a pruritic papular-vesicular eruption that eventually coalesces. Diagnosing dermatitis herpetiformis requires a high degree of suspicion as this disease is a true chameleon with many clinical faces and variable presentations. Diagnosis is most readily achieved by direct fluorescence studies of perilesional skin displaying granular IgA deposits in dermal papillae.[45] However, a direct smear can prove to be useful for ruling out other pathologies associated with intense pruritus and grouped papulovesicular rash, including cutaneous HSV.

2.4.3 Toxic Epidermal Necrolysis versus Staphylococcal Scalded Skin Syndrome

Another practical application suggested for the direct smear is its ability to distinguish toxic epidermal necrolysis (TEN) from staphylococcal scalded skin syndrome (SSSS). Although SSSS is typically a disease of children, it can also be seen in adults, especially in those with chronic renal insufficiency or the immunosuppressed.[46] Diffuse skin sloughing can be quite dramatic and a rapid diagnosis is often required. Skin biopsies with a hematoxylin and eosin stain or even frozen section can be time consuming. The direct smear can provide a rapid assessment with the findings correlating to the level of skin split. SSSS, a subcorneal/intraepidermal disease, will reveal few epidermal cells with minimal to no inflammation. TEN, on the other hand, is a sub-epidermal disease that will show necrotic or degenerating basal cells and inflammation.[47] The direct smear should not be used as a substitute for histologic examination, but merely as a rapid bedside test to help guide therapy in these patients. To our knowledge, there are no reports of use of immunofluorescence on the direct smear of the scraped lesion.
2.4.4 Darier Disease and Hailey-Hailey Disease

The direct smear shows acantholytic cells, but would typically require a biopsy for definitive diagnosis, which is characterized by a dilapidated brick wall appearance.[50] Darier disease (keratosis follicularis) also has an autosomal dominant mode of inheritance with the abnormal gene coding for an enzyme required to transport calcium into cells. Although the exact mechanism by which the lack of this gene causes disease is still under investigation, it appears that it disrupts desmosomal connections. The disease typically presents as warty, brown papules and plaques involving the seborrheic and flexural areas, hands, mouth, and nails.[51] In contrast to HHD, acantholysis in Darier disease is usually more discrete and localized mostly in the perifollicular epidermis. Although diagnosis may require a skin biopsy, a smear may aid in the detection of dyskeratosis and in differentiating this disease from others. If a biopsy is indeed needed, prominent follicular dyskeratosis may be detected in the form of numerous corps ronds and grains. Varying degrees of papillomatosis may also be present.[52] 3. Conclusion We function in an interesting time in medicine. As physicians, we are expected to make diagnoses not only rapidly, but precisely. Our patients want reliable results. Insurance companies and governments are consumed with the rising cost of healthcare. These are but some of the many factors that influence a physician making decisions about the diagnosis and management of patients. In addition, we are bombarded with new technologies, such as confocal in vivo microscopy, that may allow diagnoses to be made with much less patient invasion. It may be wise to remember the Tzanck smear in these times a useful, rapid, inexpensive, minimally invasive, office-based test with applications that extend far beyond herpes virus infections. Its precision, however, depends on the expertise of the investigator.[53] We should strive not only to utilize and master this test, but also to teach and instil its use in our training of physicians and medical students. We must also realize its limitations. Although the direct smear can be a valuable addition to our armamentarium, helping the physician establish the correct diagnosis from broad differential categories, it is worth mentioning the restrictions posed on the physician. The Clinical Laboratory Improvement Act (CLIA) of 1988 requires certification and routine inspection of non-exempt office laboratories. Since 1993, the CLIA has recognized cytodiagnosis with the Tzanck smear to be a test of moderate complexity, requiring the procedure to be performed (and billed) in certified
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Hailey-Hailey disease (HHD) is an autosomal dominant genodermatosis in which epidermal keratinocytes subjected to friction dissociate and gradually lose their cell-cell connections.[48] Because of its similarity to pemphigus, HHD is also called familial benign chronic pemphigus. The skin lesions resemble large verrucous granulations in which the center is composed of a moist granular tissue bordered by vesicles and pustules. These lesions tend to coalesce into patches in intertriginous areas exposed to chronic friction and humidity.[49]
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laboratories. However, although regulations impose restrictions on performing a Tzanck smear in the office, the benefits of this quick and inexpensive diagnostic test outweigh the expense required for special certification from the CLIA. The Tzanck smear should not be considered a substitute for more diagnostic tests (biopsies, etc.), but rather an adjunct to the physical examination. The ability to achieve important clinical information from the analysis of cytology should not be underestimated. Acknowledgments
No sources of funding were used to assist in the preparation of this article. The authors have no conflicts of interest that are directly relevant to the content of this article.

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Correspondence: Dr Brent Kelly, Assistant Professor of Dermatology and Dermatopathology, Department of Dermatology, University of Texas, Medical Branch, 301 University Boulevard, Galveston, TX 77555-0783, USA. E-mail: bckelly@utmb.edu

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Am J Clin Dermatol 2009; 10 (3)