Key points

Introduction

Nail clipping is one of the simplest diagnostic techniques performed in medicine, but it is often underused in the diagnosis of disease. Compared with many other tests in medicine, both patients and physicians have an instant familiarity with the procedure, because nail clippings are performed as part of routine grooming procedures. The benefits of performing nail clipping for diagnostic purposes are numerous, including minimal risk to the patient, increased diagnostic information about a nail disorder, rapid completion in the office, and in some cases the preparation of permanent glass slides that can be referred to in the future for further diagnostic study.

Dermatoses affecting the nail unit can be difficult to diagnose. Infectious, inflammatory, and neoplastic disorders of the nail unit can mimic each other on clinical examination. Histopathology is helpful, and often necessary, to establish a specific diagnosis and to distinguish such similar-appearing entities. However, the need for tissue diagnosis can lead to unease, because clinicians are often reluctant to obtain a soft tissue biopsy specimen from the nail unit. Barriers to obtaining a soft tissue specimen from the nail unit include concern for a subsequent permanent nail unit dystrophy, lack of prior training in nail unit surgery, and the perceived technical difficulty of a nail unit biopsy.

As opposed to obtaining a soft tissue specimen from the nail unit, obtaining a nail clipping does not require any specialized training, and can be used to evaluate a wide range of nail disorders. This article describes a variety of uses of nail clippings to help diagnose dermatoses of the nail unit, as well as best practices in order to obtain the specimen, and correlate the findings within the specific clinical context. This article covers the use of nail clippings for the diagnosis of onychomycosis, nail unit psoriasis, subungual hematoma, and onychomatricoma (OM), and for forensics, surgical planning for nail unit biopsy of melanonychia, as well as distinguishing nail cosmetics from other dermatoses.

Introduction

Nail clipping is one of the simplest diagnostic techniques performed in medicine, but it is often underused in the diagnosis of disease. Compared with many other tests in medicine, both patients and physicians have an instant familiarity with the procedure, because nail clippings are performed as part of routine grooming procedures. The benefits of performing nail clipping for diagnostic purposes are numerous, including minimal risk to the patient, increased diagnostic information about a nail disorder, rapid completion in the office, and in some cases the preparation of permanent glass slides that can be referred to in the future for further diagnostic study.

Dermatoses affecting the nail unit can be difficult to diagnose. Infectious, inflammatory, and neoplastic disorders of the nail unit can mimic each other on clinical examination. Histopathology is helpful, and often necessary, to establish a specific diagnosis and to distinguish such similar-appearing entities. However, the need for tissue diagnosis can lead to unease, because clinicians are often reluctant to obtain a soft tissue biopsy specimen from the nail unit. Barriers to obtaining a soft tissue specimen from the nail unit include concern for a subsequent permanent nail unit dystrophy, lack of prior training in nail unit surgery, and the perceived technical difficulty of a nail unit biopsy.

As opposed to obtaining a soft tissue specimen from the nail unit, obtaining a nail clipping does not require any specialized training, and can be used to evaluate a wide range of nail disorders. This article describes a variety of uses of nail clippings to help diagnose dermatoses of the nail unit, as well as best practices in order to obtain the specimen, and correlate the findings within the specific clinical context. This article covers the use of nail clippings for the diagnosis of onychomycosis, nail unit psoriasis, subungual hematoma, and onychomatricoma (OM), and for forensics, surgical planning for nail unit biopsy of melanonychia, as well as distinguishing nail cosmetics from other dermatoses.

Best practices for obtaining a nail clipping for histopathologic examination

In order to maximize diagnostic yield from a nail clipping, it is important to obtain a sample that is at least 4 mm in length. A common error is collecting a sample that is too small. If a very small sample is submitted, the nail plate tissue that is examined may not be representative of the overall pathologic process in the nail unit. In addition, a tiny sample may be damaged during routine laboratory processing procedures, and not survive the process. If a patient’s nail is not long enough to obtain a sufficient sample, the patient should allow the nail to grow, and return to have a nail clipping at a later date. It may be useful to have the office staff explain to patients who are being evaluated for a nail problem, either verbally or in preprepared paperwork that is mailed to patients before their visits, that they should allow their nails to grow to a sufficient length and, in particular, should not clip their own nails before evaluation.

If the clinician is interested in performing a fungal culture in addition to the nail clipping, the target nail should be cleansed either with an alcohol swab or soap and water before clipping the nail, but this step is not required for histologic assessment alone. Cleansing the nail helps to remove the presence of any contaminating bacteria. There are a variety of nail instruments that can be used to obtain the nail clipping. A dual-action nail nipper is particularly helpful because of its hinged shape, and can create a large force at the point of clipping, making the clipping easier, especially of thick nails ( Fig. 1 A). A heavy-duty nail nipper may be used as well. Routine nail clippers, as are used by laypersons at home, are not recommended to obtain nail clippings in the medical office setting because of their inferiority compared with the aforementioned instruments.

Optimal sampling of a nail clipping. ( A ) The nail is clipped back with a double-action nail nipper. ( B ) Subungual debris is removed with a small curette and placed on a Dermapack.

When performing the nail clipping, the nail should be clipped as far back as possible without causing pain or bleeding. This method ensures that a sufficient sample is obtained and, in the case of onychomycosis, that the active area of infection is included. In the case of onychomycosis, if only a small distal sample of the nail plate is obtained, it may provide a false-negative result because of a sampling error. If onycholysis is present, the nail should be clipped back to the most proximal attachment of the nail plate to the nail bed. It is helpful to pathologists evaluating specimens to be provided with sufficient clinical context, as well as any special requests for processing or interpretation of the nail clipping specimen.

After the nail is clipped, and if a fungal culture is desired, a small curette can be used to gently dislodge any subungual material from the nail bed, which is sent for culture (see Fig. 1 B). The clipped nail plate can be put into a formalin-filled bottle and sent to the pathology laboratory.

Best practices for preparing a nail clipping for histopathologic examination

Because of the unique anatomy and physical characteristics of the nail unit, the pathology processing techniques required for nail clipping specimens are more complex than those for routine cutaneous specimens. The nail plate is rigid and often requires adequate softening to create high-quality sections, which in turn allow accurate diagnoses. There are numerous options for softening nail plate specimens, including 4% phenol, 5% trichloroacetic acid in 10% formalin, chitin-softening agent, cedar oil, and 10% Tween 40, as well as sodium hydroxide (NaOH). André and colleagues use Mollifex Gurr, dipping the paraffin blocks in it for 2 to 12 hours, depending on nail plate thickness.

A study by Lewin and colleagues compared different methods of softening nail specimens for histologic sectioning. For nails of normal thickness, several methods were equally effective; namely, soaking in potassium hydroxide (KOH) or nitric acid after fixation in formaldehyde solution, or Tween 40 treatment of normally processed paraffin blocks. For thick nails, treatment with Tween 40 was preferred, although KOH was adequate.

Orchard and colleagues performed an evaluation of several methods using softening agents to facilitate the preparation of reproducible, high-quality, formalin-fixed, paraffin-embedded sections of nail tissue. They collected nail plate specimens from 3 normal volunteers. The clippings were fixed in 10% neutral buffered formalin for 12 hours. Next the investigators applied the softening agents, including Veet (Reckitt Benckiser), Easy fabric conditioner (powder and liquid products), Fairy Liquid (Procter and Gamble), 30% KOH, and distilled water for 10 minutes each following paraffin wax embedding. The sections were cut at a thickness of 4 μm. The block was then faced off and treated with a different softener for evaluation. The investigators then evaluated sections taken after each softener was used and assessed the quality and number of sections before specimen damage became apparent. The investigators found that Veet and Easy fabric conditioner were the best softening agents, closely followed by Fairy Liquid. These agents were superior to distilled water and 30% KOH, which provided fewer high-quality sections after application for 10 minutes.

Nazarian and colleagues detailed a protocol using pretreatment with NaOH in the evaluation of 45 samples from fingernails and toenails of patients with clinically suspected onychomycosis. The specimens were soaked in 20% NaOH for 2 to 3 hours, then rinsed for 5 minutes under tap water before routine processing, paraffin embedding, and routine hematoxylin-eosin (H&E) periodic acid-Schiff (PAS) staining. This softening method provided higher-quality sections compared with no softening treatment. However, this study did not compare the quality of the specimens with that obtained using other softening agents. In addition, the investigators noted that NaOH treatment led to some areas of the nail plate having fainter PAS staining of fungal elements. However, this did not result in a false-negative diagnosis in any case. The investigators also suggested that NaOH may damage melanin and hemosiderin pigments, and therefore caution should be used when evaluating cases with clinical history of melanonychia.

Chang and colleagues advocate using plastic embedding of nail plate specimens in 2-hydroxyethyl methacrylate as an alternative to softening agents, which allows thinner, uniform sections that adhere well to glass slides, and decreases the chance of dislodging the nail plate from the embedding media. However, the investigators note that this technique is technically difficult, costly, time consuming, and requires a motorized microtome and glass knives.

In our laboratory, we use 10% NaOH for pretreatment of nail plates. After a nail plate specimen is received in 10% formalin, the formalin is removed and replaced with 10% NaOH. The time of incubation is based on the size of the specimen. The specimens are monitored closely throughout the timing process to prevent degradation. Small specimens are incubated for approximately 45 minutes, whereas bigger specimens may be incubated for 2 to 3 hours. Once softening is completed, the nail plates are washed in running water for 10 minutes then returned for processing and embedding.

In order to help the nail specimens adhere to the glass slides, we coat the slides with a light film of adhesive solution and then let dry. This technique allows the nail to remain attached to the glass slides through H&E, special stain, and rigorous immunoperoxidase staining processes, if needed. Otherwise, the tissue may fall off the slide and be lost. Once the tissue block is prepared, it is faced off and then allowed to sit in a tray containing 4% ammonium hydroxide for about 30 to 45 minutes before cutting to ensure easy passage of the knife through the nail clipping specimen. This step further ensures that the clippings adhere to the slide, because the adhesive solution alone may not suffice. Once the nail clipping sections are on the slide, the slides are placed in a 60° to 65°C oven for approximately 1 hour to allow the sections to dry sufficiently before proceeding with staining.

When assessing a nail clipping for onychomycosis in particular, it is important to keep in mind that hyphae, when present, are most commonly localized in areas of subungual hyperkeratosis and the ventral aspect of the nail plate. Given this phenomenon, Chang and colleagues described a novel method of isolating subungual hyperkeratosis from the nail plate for histologic evaluation during the grossing stage of specimen preparation, before PAS staining for onychomycosis. After nail clippings were submitted to the laboratory, the subungual hyperkeratosis was trimmed from the base of the nail plate using standard grossing instruments. This subungual material was then processed directly without the need for additional steps of softening. The investigators report that this technique provided the diagnosis, such that fungal elements were identified in the samples, in 64 of 66 (97%) cases of onychomycosis. This method spared the added technical difficulty and cost of nail plate softening and processing. In 3% of the specimens in which the subungual hyperkeratosis was PAS negative, the nail plate was positive. Therefore, the investigators recommend that, in cases in which the PAS stain of the subungual debris is negative, sections of the nail plate should be examined to avoid false-negative results when using this technique.

Recently, there has been some debate regarding the fungal stain of choice for identifying hyphae for the diagnosis of onychomycosis. One study by D’Hue and colleagues showed the superiority of Gomori methenamine silver (GMS) compared with PAS in detecting the presence of hyphae in nail plate biopsies. The investigators performed GMS staining on 20 PAS-positive and 51 PAS-negative nail clippings with a clinical diagnosis of onychomycosis. All 20 of the PAS-positive cases were GMS positive, and 5 of the PAS-negative cases were GMS positive. In addition, GMS stains were easier and less time consuming to interpret compared with PAS.

A similar study was performed by Reza Kermanshahi and Rhatigan. These investigators compared PAS and GMS stains on sections from 30 cases of clinically suspected onychomycosis using sections cut at similar depths of the tissue blocks. The PAS stain was positive in 22 of 30 specimens, and GMS was positive in 19 of 30. On samples that were originally negative with GMS but positive with PAS, cutting deeper into the block revealed GMS positivity. The investigators therefore suggested that the two stains are equivalent for detecting the presence of hyphae. They also explain that although the PAS stain is simple to perform, the GMS stain can be technically difficult and requires experienced histotechnologists.

A study of efficacy of histopathologic PAS staining of nail clippings by Mayer and colleagues revealed that parakeratosis and globules of plasma were statistically significantly more common in the nail samples with fungal elements than in those without on examination of H&E-stained specimens. The investigators concluded that this finding may indicate an ongoing inflammatory process associated with onychomycosis.

Another study, by Barak and colleagues, concluded that there was no difference in the sensitivity between PAS versus GMS. To evaluate the efficacy of the GMS stain, the investigators identified 326 PAS-negative nail plates submitted for evaluation of possible onychomycosis, which were then stained with GMS. Staining of additional levels with GMS highlighted fungal hyphae in 14 (4.3%) of the 326 PAS-negative nail plate specimens, whereas 312 specimens remained negative.

As a control, the investigators identified 190 additional nail plates negative for PAS on the first level and evaluated a deeper section by a second PAS stain. In this group, 8 (4.2%) revealed fungal hyphae by this methodology. Although the rate of positive staining was not statistically different (4.2% vs 4.3%; P = .57), PAS was 2.6-fold less expensive.

Taken together, the investigators conclude that the PAS stain is the optimal method for diagnosing onychomycosis.

A recent study by Idriss and colleagues showed fungal fluorescence of routinely stained sections as a useful diagnostic tool in the diagnosis of onychomycosis. Forty-six of 48 routinely stained PAS-positive onychomycosis nail plate specimens showed positive fluorescence, whereas 20 of 23 PAS-negative control specimens remained negative on fluorescent examination. The sensitivity and specificity of the method were 96% and 90%, respectively. The investigators commented that fungal fluorescence has several distinct advantages, such as no delay associated with a special stain, cheaper cost compared with PAS, and that it can be performed when tissue is exhausted and not available for special stains.

Onychomycosis

Onychomycosis is the most common disease of the nail unit in adults, presenting with yellow patches and streaks, onycholysis, and subungual hyperkeratosis ( Fig. 2 A). Approximately 75% of onychomycosis is caused by dermatophytes, including Trichophyton rubrum , Trichophyton mentagrophytes , and Epidermophyton floccosum ; however, 5% to 15% can be caused by nondermatophyte fungi, such as Aspergillus , Scopulariopsis brevicaulis , Scytalidium dimidiatum , and Fusarium . Many practitioners empirically treat nail dystrophy as onychomycosis, believing a clinical examination is sufficient for diagnosis and treatment. However, this practice may lead to expensive, ineffective treatment, and drug-related risk exposure without definitive diagnosis of onychomycosis. The American Academy of Dermatology’s contribution to the Choosing Wisely campaign specifically highlights clinical similarities between onychomycosis and other nail dermatoses, and urges clinicians not to prescribe oral antifungal therapy for suspected onychomycosis without confirmation of the diagnosis, in order not to subject patients to the risk of unnecessary drug side effects and to avoid inappropriate treatment of the underlying disorder.

Clinical and histologic features of onychomycosis. ( A ) The great toenail shows xanthonychia and onycholysis. ( B ) At low power, the nail plate shows thickening and has subungual debris (H&E, 40×). ( C ) At medium power, the nail plate shows parakeratosis, bacteria, and neutrophils at the ventral aspect of the nail plate (H&E, 200×). ( D ) High-power view of the nail plate shows many hyphal elements within the nail plate (PAS, 200×).

Although there are several methods for diagnosis of onychomycosis, such as fungal culture and direct microscopy with KOH, nail clipping for histology with PAS can be particularly helpful. In a recent study by Wilsmann-Theis and colleagues that included 631 samples of onychomycosis shown by at least 1 method, PAS staining of a nail clipping had a higher sensitivity (82%) compared with culture (53%) and direct microscopy (48%). The combination of fungal culture and nail clipping for PAS yielded a sensitivity of 96%, which was higher than combinations of other methods explored in the study.

The advantage of mycologic culture is that it does not only allow the identification of specific fungal species. However, this test is highly operator dependent and therefore less sensitive, at only 35% to 53%. In addition, cultures require long incubation times and can take 4 weeks to return a diagnosis, whereas a nail clipping has a faster turnaround of 2 to 7 days, depending on the laboratory and the complexity of the specimen.

Histopathologic examination of a nail clipping is the least likely modality to be affected by sampling methods or operator experience. Although it is possible to examine the morphology of pathogens with this method, thereby narrowing the differential diagnosis, histologic evaluation of nail clippings cannot precisely identify pathogens and their susceptibilities, or whether the organisms were viable at the time of sampling. It is the most sensitive test to assess for residual infection after therapy with oral antifungal medication. In addition, another disadvantage of obtaining nail clipping for histology is cost, because this method is more expensive than culture and/or direct microscopy.

A nail clipping is also helpful in identifying cases of onychomycosis caused by nondermatophyte fungal infection or coinfection with dermatophyte and nondermatophyte fungi. Several nondermatophyte fungi are resistant to standard antifungal therapies, and therefore culture is necessary to guide therapy. In these cases, the nondermatophyte hyphae may be identified in the nail plate on histologic examination, whereas mycologic culture may be negative. Culture plates use Sabouraud dextrose agar and may contain antibiotics such as cycloheximide that are used to suppress contaminants such as nondermatophyte fungi. Therefore, these hyphae are visible with a PAS examination of the nail plate, but may not grow in culture. The identification of fungal elements within the parenchyma of the nail plate on histologic evaluation in combination with a negative fungal culture from the same nail can therefore raise the possibility of a nondermatophyte onychomycosis.

In addition, nondermatophyte fungi can mask the presence of dermatophytes. Stefanato and Verdolini reported a case of onychomycosis caused by S brevicaulis disguising a dermatophyte infection. Both fungal forms were evident on histologic sections of subungual hyperkeratosis. Culture was only positive for S brevicaulis and repeatedly negative for dermatophyte fungi. On histologic examination, the two fungi maintained separate boundaries, causing territorial demarcation between the infected regions.

Another important histologic phenomenon that may be gleaned from histologic examination of a nail clipping of onychomycosis is the presence of a dermatophytoma. Clinically, a dermatophytoma can present as a round yellow or white patch in the nail plate. A study by Martinez-Herrera and colleagues reported 7 cases of dermatophytoma that clinically presented with white or yellow nail bands with hyperkeratosis, with 3 patients having total dystrophic onychomycosis, 3 with distal subungual onychomycosis and 1 with superficial white onychomycosis. Microscopic examination of a dermatophytoma shows a dense mass of dermatophyte hyphae. Approximately 18% of patients with onychomycosis present with clinical findings suggesting a dermatophytoma. This presentation is associated with a failure to achieve a mycologic cure following oral antifungal treatment, and treatment with chemical or physical debridement in combination with oral antifungal therapy is recommended. Burkhart and colleagues introduced the concept of biofilm in dermatophyte infections, describing populations or communities of micro-organisms that adhere to surfaces and then aggregate, forming a mass or fungal ball because of an extracellular polysaccharide. The biofilm may account for the observed poor therapeutic response and recurrence in the treatment of onychomycosis. Recognizing dermatophytoma on histology and reporting it to the clinician can therefore help guide management and lead to a greater chance of mycologic cure.

The characteristic histologic features in evaluation of onychomycosis are subungual hyperkeratosis, neutrophilic infiltrate, parakeratosis, hemorrhage, and serum crusts (see Fig. 2 B, C). Most commonly the fungi are located in the ventral aspect of the nail plate. The fungal hyphae on routinely stained sections are difficult to visualize, and are best visualized with PAS or GMS staining. The use of the PAS or GMS stain is essential because there are many overlapping features of onychomycosis and nail unit psoriasis, and it is the presence of hyphae that is the defining feature of onychomycosis (see Fig. 2 D).