Base for the histopathological study of nail lesions

Abstracts

BACKGROUND: In this paper a broad review of nail biopsy is presented, emphasizing its anatomy, embryology, histology and histopathology. The indication and purpose of nail biopsy as well as the techniques available are reviewed in detail. OBJECTIVE: Define the appropriate site for nail biopsy according to nail disorder, compare two different methods of nail specimen softening: soaking in 20% potassium hydroxide and in 10% aqueous solution of Tween 40 and establish a routine for the nail biopsy procedure. METHODS: Our group studied eleven patients with nail disease who presented at the Dermatology Department in Pedro Ernesto University Hospital for a definite diagnosis. Twelve biopsies were performed. RESULTS: There were no differences in the softness achieved with the two methods and none harmed the cytologic and histologic analysis. CONCLUSIONS: The authors concluded that nail biopsy is a safe and useful procedure for nail diseases when clinical and laboratory methods fail to reach a diagnosis. A complete and thorough understanding of the nail unit anatomy is necessary to perform the procedure

biopsy; nail diseases; pathology

FUNDAMENTOS: Os autores apresentam ampla revisão sobre os fundamentos da biópsia da unidade ungueal, fornecendo bases teóricas sobre anatomia, histologia e histopatologia. Abordam, detalhadamente, as indicações e os objetivos da biópsia da unidade ungueal e as várias técnicas disponíveis. OBJETIVOS: Definir os locais adequados da biópsia do aparelho ungueal para as diferentes doenças da unha, comparar duas formas de amaciamento da lâmina ungueal, uma utilizando KOH a 20%, outra, solução aquosa de Tween 40 a 10%, e estabelecer uma rotina para o processamento do material de biópsia. MÉTODOS: O grupo de estudo constou de 11 pacientes com doença ungueal, que procuraram o ambulatório de Dermatologia do HUPE/UERJ para esclarecimento diagnóstico. Foi realizado o total de 12 biópsias. RESULTADOS: Não houve diferença na dificuldade técnica, em incluir ou cortar o material, consoante ao método de amaciamento utilizado, nem prejuízo citológico ou histológico ao exame histopatológico. CONCLUSÕES: Os autores concluíram que biópsia ungueal é procedimento seguro e eficaz para esclarecer o diagnóstico das doenças ungueais quando o exame clínico ou outras técnicas falharam. Também é importante, para quem deseja realizar o procedimento, o conhecimento adequado da anatomia dessa região.

biópsia; doenças da unha; patologia

CLINICAL, LABORATORY AND THERAPEUTIC INVESTIGATION

Base for the histopathological study of nail lesions^*

Geraldo Magela Magalhães^I; Isabel Cristina Brasil Succi^II; Maria Auxiliadora Jeunon Sousa^III

^ISpecialist in Dermatology from the SBD / Ph.D. Student of Dermatology,UFRJ

^IIHead of Dermatology Service, UERJ

^IIIHead of the Dermatopathology Dept., UERJ

^{Correspondence}

SUMMARY

BACKGROUND: In this paper a broad review of nail biopsy is presented, emphasizing its anatomy, embryology, histology and histopathology. The indication and purpose of nail biopsy as well as the techniques available are reviewed in detail.

OBJECTIVE: Define the appropriate site for nail biopsy according to nail disorder, compare two different methods of nail specimen softening: soaking in 20% potassium hydroxide and in 10% aqueous solution of Tween 40 and establish a routine for the nail biopsy procedure.

MATERIAL AND METHODS: Our group studied eleven patients with nail disease who presented at the Dermatology Department in Pedro Ernesto University Hospital for a definite diagnosis. Twelve biopsies were performed.

RESULTS: There were no differences in the softness achieved with the two methods and none harmed the cytologic and histologic analysis.

CONCLUSIONS: The authors concluded that nail biopsy is a safe and useful procedure for nail diseases when clinical and laboratory methods fail to reach a diagnosis. A complete and thorough understanding of the nail unit anatomy is necessary to perform the procedure.

Keywords: biopsy; nail diseases; pathology.

INTRODUCTION

Although dermatologists do not hesitate to perform a skin biopsy to confirm diagnosis or exclude the possibility of malignancy, their behavior in relation to nail biopsy is completely different. The doubts and fears are many. What are the indications? Which is the appropriate site for biopsy in the ungual unit? Which technique is the safest, most accessible and least traumatic? Will there be a definitive sequel? How does one process the material? In this work, the authors attempted to provide answers for these questions.

Ungual Unit

Functional Anatomy

The nail (or unguis; pl. ungues) is a cutaneous annex that covers the dorsal face of the fingers and toes. The nails protect the sensitive digital extremities and serve the function of scratching, growing about 2 to 4.5mm each month.¹

Philogenetically, the nails evolved from the claws of birds and reptiles and the shells of inferior mammals - sic. They protect the distal phalange from traumatic impact; they also function as tweezers; participate in the sensory function; and are used to scratch. Also, one cannot forget their cosmetic function in humans.²

There are basically six components in the ungual unit:2

- the nail matrix, which is the root;

- the nail plate, product of the keratinization of the matrix;

- the cuticula system, including the eponychium or visible cuticle, derived from the proximal nail fold, and the hyponychium, derived from the epithelium of the nail bed;

- the support portion represented by the nail bed and phalangeal bone;

- the anchorage portion represented by the specialized mesenchyma that is proximal between the phalange and matrix and distal between the phalange and the lateral and distal digital pulp;

- the mantle composed by the nail folds: proximal, lateral and distal;

The configuration of the nail plate is defined by a delicate balance between some parameters: form, convexity, outline of the distal extremity, thickness and the sidewalls of the plate.² The first four parameters are responsible for the morphology of the plate and the latter for its position.

The blood supply of the bed and matrix is composed of a rich network of digital arteries. There are two main arterial arcades (proximal and distal) formed by anastomoses of the branches of the digital arteries, together with several arteriovenous anastomoses in the nail bed (glomus body), which are related to the thermal regulation.³

Branches of the digital nerves affect the innervation. The ungual unit of the fifth and medial half of the fourth fingers receives sensitive innervation from the ulnar nerve. The remainder is made through the median nerve.³

Histopathology

Griffin in 1977,⁴ Omura in 1985⁵ and Jerasutus in 1997⁶ provided detailed descriptions of the ungual diseases.

Knowledge of the histopathology of nail diseases is much more limited than that of cutaneous diseases, not only because fewer biopsies have been performed, but due to the different and more complex anatomical structures when compared to those of the skin.

The greatest obstacle is the possibility of losing correct orientation in the inclusion of material. Hence the importance of marking the specimens obtained, to guide their positioning for correct inclusion.

In a longitudinal section one finds the nail plate, proximal nail fold, nail matrix, nail bed and hyponychium.

The proximal nail fold presents dorsal and ventral epithelium. The dorsal surface contains sudoriparous glands, but has no pilosebaceous units. The epidermis presents the four usual layers, and the cone/papilla relationship is preserved. The ventral epithelium is cornified, as are the four layers, however it is finer, rectified and without any epidermal appendixes. The horny layer originating from this epithelium constitutes the cuticle, which functions as a seal to prevent the entry of bacteria and humidity.

The nail matrix is a thick germinative epithelium, without interposition of a granular layer, with wide cones located downward toward the proximal portion.

As their cells progress into the superior layers, the cytoplasm becomes intensely eosinophilic and their nuclei become picnotic, until disappearing entirely and the cells of the nail bed turn into onychocytes. The lunula, the visible part of the matrix, continues to show the same characteristics and forms the keratogenous zone.

Melanocytes are present in the matrix, but are few and poorly developed. They present both basal and suprabasal location, and this could be a consequence of the expression of adhesion molecules in the ungual epithelium. In the latter location, the integrins a3 and b1, which probably regulate the melanocyte-keratinocyte adhesion, are expressed in the basal and suprabasal layer.⁷ The proximal matrix is less dense, perhaps due to the protection against exposure to ultraviolet rays provided by the proximal fold. Langerhan and Merkel's cells have also been identified in this location. The matrix is divided into three zones: the proximal, intermediate and distal, of which the lunula comprises part of the latter.

The nail plate is constituted of cornified cells in a lamellar arrangement that is colored faintly by eosin and strongly by the acid resistant dyes. Their origin continues to be controversial. Zaias, in 1968,⁸ researching the formation of the nail plate in primates through auto-radiographic study, concluded that the nail plate has its origin exclusively in the matrix. Johnson (1991)⁹ stated that the ventral portion of the plate, about 20% of its thickness, is produced by the nail bed.

The epithelium of the nail bed rests below the nail plate and is distally limited by the hyponychium and proximally by the matrix. It does not present granular cells and is relatively thin. In the sagittal section it contains few parakeratotic cells. In the traverse section, it is firmly adhered to the underlying dermis by long and narrow epidermal cones, interposed to the dermal papillae.

The hyponychium becomes keratinized like the volar skin, presenting a layer of granular cells and the horny layer is thickened and more compact.

The dermis is highly vascular and supplied by digital arteries. They are numerous glomic bodies that, working as arteriovenous shunts, regulate the temperature of the fingers. Trunks and nerve endings are also numerous. The dermis rests directly on the distal phalange, without interposition of subcutaneous cellular tissue.

Nail Biopsy

General Considerations

Stone, in 1978,¹⁰ stated that nail biopsy is only justified in cases in which there is a possibility of malignanc, while in those with a diagnostic hypothesis of psoriasis or lichen planus, for instance, it would be preferable to manage the picture clinically.

Other authors^11,12 cconsider nail biopsy to be a safe diagnostic resource that is easy to perform and useful for several ungual diseases, when the clinical routine and laboratory methods fail to reach the diagnosis. It is a safe procedure that can and should be performed by a dermatologist.^11,12

The prerequisites for performing ungual biopsy are appropriate knowledge of the nail anatomy, anesthesia and hemostasia and the presence of a histopathologicaly identifiable diagnosis.¹²

The indication and methods vary according to the site and type of disease in the ungual unit.¹²

Specimens obtained from nail biopsies can present difficult orientation, processing and interpretation. Thus it is important to maintain direct contact with an experienced dermatopathologist.^5,6,12

Methods and Indications

Longitudinal Biopsy of all the Nail Elements

Initially described by Zaias in 1967¹³ it corresponds to the removal of a median longitudinal fragment. In this case, information is obtained regarding all of the elements of the nail unit: proximal nail fold, matrix, bed and plate. The author recommends that the biopsy does not exceed 3mm in width within the area of the matrix, so as to avoid a permanent scar.

In 1980, Scher¹⁴ once again provided a detailed description of this technique and suggested some modifications, broadening the field of action both in the diagnosis and therapeutics for ablation of neoplastic lesions and correction of onychrocryptosis.

The main indications include diseases of the matrix or the entire nail unit: tumors of the matrix, striated melanonychia and nail dystrophy with extensive involvement of the nail plate. Sometimes, it is also indicated for the diagnosis of psoriasis and lichen planus, when the whole ungual unit is involved.¹²

Excision and Incision Biopsy

These can be performed in the nail bed or matrix.

In the bed, the excisions should be fusiform and longitudinal when possible and limited to a width of 3mm. Some argue that suture provides better aesthetic results.¹⁵

Figura 5

In the matrix, the orientation of the largest axis of the strip should follow the crescent form of the lunula. Attention should be given to the thickness of the strip, which should not exceed 3mm.¹⁶

The indications include: tumors and vegetative lesions in the nail bed; unexplained pigmentation, onycholysis and hyperkeratosis, or when onychomycosis psoriasis are suspected. In the nail matrix, it can be used to exclude malignancy in melanonychia lesions and to diagnose tumors or dystrophy.¹²

Punch Biopsy

Scher (1978)¹¹ gave a detailed description of the technique to perform punch biopsy of the nails, demonstrating a simple method that improves the diagnostic accuracy and treatment of nail diseases, while emphasizing the importance of discarding malignant potential.

The indications include diseases of several elements of the nail unit, such as the matrix.¹²

One of the inconveniences of this technique is the fact that only one of the elements of the nail unit can be studied in each biopsy.

In 1978, Stone¹⁰ described the biopsy of the proximal nail fold using a 2mm punch in patients with chronic paronychia resistant to treatment, as well as for detecting foreign bodies as etiological agents.

Souza, in 1995,¹⁷ showed the usefulness of nail punch biopsy as the main method for diagnosis in cases in which diseases such as psoriasis and lichen planus are exclusive to the nail apparatus.

Figura 6

Other Methods

When the lesion involves the folds of the nail apparatus, other methods can be used, such as block excision, shaving or crescent shaped excision of the nail fold.¹²

Other Considerations

Elewski, in 1996,¹⁸ stressed the need for a histopathological evaluation of nails with clinical suspicion of onychomycosis in which direct mycological exams for KOH and cultures were negative.

Blecher, in 1993,¹⁹ used a high rotation motor for collecting material for the cultures. It was observed that this was a feasible option for the diagnosis of fungal infection, midway between the traditional method for collection of material from the nail for mycological exam and the biopsy.

Machler, in 1998,²⁰ used fragments of the nail plate to perform histological exam in cases with dystrophy, concluding that it is a simple and effective technique for the diagnosis of onychomycosis and psoriasis, without the inconvenience of having to identify the fungi.

Grammer-West, in 1998,²¹ described cases in which the diagnosis of psoriasis was obtained through histopathological exam of the nail plate.

Processing the Material

Processing the biopsy material involves techniques for softening the nail plate, inclusion and staining.

Alvarez, in 1967,²² described a method using inclusion in pyroxylin glycol polyethylene, in which the nail plate was softened by fixing in 5% trichloroacetic acid and 10% formalin.

Paraffin is the most often used material for inclusion of the biopsy.

Lewin (1973)²³ described several techniques for softening the nail plate. For nails of normal thickness, the methods using potassium hydroxide associated with nitric acid after fixation in formol or Tween 40 followed by formol were equally effective. For thick nails, the preferable technique is Tween 40, although the use of potassium hydroxide is also effective.

Piérard, in 1994,²⁴ used a 10% aqueous solution of Tween 40 for 24 hours with good results for the softening of their nail biopsies; followed by the usual techniques for formalization and inclusion in paraffin.

In general, the plates are stained with hematoxylin and eosin or PAS.

PATIENTS AND METHODS

The patients in this study are those who presented with lesions at the Dermatology Clinic of the University Hospital Pedro Ernesto (HUPE/UERJ), during October and November 1999, for diagnosis and therapeutic management of nail lesions.

The inclusion criteria were:

- presence of longitudinal melanonychia;

- presence of tumors in the nail unit;

- suspicion of infectious process in the nail unit in which tests for isolation of fungi were negative;

- presence of inflammatory dermatological conditions with exclusive nail involvement.

The exclusion criteria were:

- patients that refused to undergo biopsy;

- patients with psychiatric disturbances that rendered the procedure unfeasible;

- patients with a history of peripheral vascular disease;

- acute infectious process of the nail unit;

- blood dyscrasia.

The biopsies were performed in the dermatological surgery theater.

Antisepsis of the hands or feet was done with povidone iodine (PVPI) and a solution of alcohol and ether.

The local-regional anesthetic used was 2% lidocaine without adrenaline, while in the biopsies of the matrix, nerve block anesthetics was used. In the cases of biopsy of the bed, local anesthetics were used in the junction of the proximal and lateral fold, progressing to the fingertip, as described by Scher, in 1978.¹¹ A clipping was not used for biopsies of the nail bed, though this was essential for matrix biopsies.

Disposable punches of 4mm and 3mm were used for biopsies of the bed and matrix, respectively. Access to the matrix was achieved by lateral incisions in the proximal nail fold and repair. In the biopsies of the periungual elements, the authors followed the general orientations for biopsy of the skin.

Indication for biopsy of the matrix was considered to be the presence of striated melanonychia and suspicion of nail diseases originating in the matrix. Biopsies of the nail bed were performed for inflammatory diseases with involvement of the matrix and bed and were used to confirm suspicion of infection when the mycological exams were negative. Biopsies of the periungual tissue were used to diagnose tumoral lesion.

Occlusive and compressive dressings were used and these were changed after the first 48 hours and then on a daily basis.

The biopsy fragment was submitted to a preparation of 20% KOH at 56°C for 30 minutes and then to fixing of the material in formol or immersion in 10% aqueous solution of Tween 40 for 24 hours prior to fixing in formol.

The material was then forwarded to the Pathological Anatomy Department of the Medicine School of the University of the State of Rio de Janeiro, where it was embedded in paraffin, cut into 5m sections and stained with hematoxylin and eosin and with periodic-acid Schiff (PAS).

The histopathological evaluation was performed by the Dermatopathology Department of the HUPE-UERJ.

RESULTS

A total of 12 nail biopsies were performed in 11 patients, of which two were incision and 10 punch biopsies, the results are summarized in table 1.

DISCUSSION

While performing the biopsies, the authors encountered several difficulties. In 100% of the cases in which the nails presented onycholysis and thickening of the nail plate, there was a separation between nail plate and nail bed during the procedure, leading to the need for nail plate and nail bed to be mounted separately.

Regarding biopsies of the bed, in 1995¹⁷ Souza used a 3 or 4mm punch; Rich,¹² (1992) described a technique using two punches; recommending the use of a 4mm punch for sampling the nail plate followed by a 3mm punch for removal of a fragment of the nail bed; in the cases reported in the present work, there was great technical difficulty in the use of disposable punches, because the nail plate loosened during the biopsy and became adhered to the internal part of the punch, which did not happen when non disposable punches were used.

In biopsies of the matrix, the choice of a 3mm punch or transversal strip, is recommended and accompanying the curvature of the lunula while avoiding the proximal matrix, whenever possible.^15,16

Given that in this type of biopsy, exposure of the matrix for preparing a specimen of the proximal fold is indispensable, the authors opted for a disposable 3mm punch, with a view to preventing definitive sequels in the nail.

Regarding anesthesia, a nerve block is considered fundamental as the anesthetic procedure in the cases of biopsy of the matrix, since a surgical field free from blood is necessary to facilitate identification of the site in the matrix that presents alterations and thereby guiding the biopsy. When performing a biopsy in the nail bed, local anesthesia was applied and there was good tolerance on the part of the patients, as well as effectiveness of the blockade. It was not necessary to use clipping since the bleeding was minimal and it did not affect the procedure.

Another decisive factor for a precise histological evaluation of the biopsy is the orientation of the material obtained. This usually involves small fragments and an incorrect orientation can invalidate the results. To indicate the orientation one can use colors or mark the specimen with a small lateral incision to define whether the inclusion was transversal or longitudinal. The immediate cleaving of the material as a means to guide the inclusion is not advisable as there is a risk that this might alter the histopathological exam.

In 1997,⁶ Jerasutus demonstrated in detail the histopathological alterations to the nail bed following a transverse inclusion: the epidermal cones are narrow and long, inserted by dermal papillae, forming regular longitudinal folds along the nail bed. Therefore a longitudinal inclusion is recommended.

The patients were followed-up by the authors and no sequela were observed among those submitted to biopsy of the nail bed. The two patients submitted to biopsy of the matrix were followed-up until normal and complete growth of the nail bed, without furrows or onycholysis, which was considered a final result without sequela. However, the authors' patient sample was small and consequently may not be representative of the real incidence of complications.

Regarding the processing, the biopsies of the matrix and periungual tissue were fixed directly in formol, which did not alter the inclusion or histopathological evaluation.

Biopsies of the bed were submitted to one of two methods of softening, using either 20% KOH, heated to 56°C for 15 minutes, or 10% aqueous solution of Tween 40. Regarding the sectioning of the material in the microtome, the difficulty was linked to the greater or lesser degree of thickening of the nail plate, as was already expected and overcome by the use of new blades in the microtome after every four different specimens processed.

In the histopathological evaluation, no cytological or histological damage was observed in the samples. Both techniques are easily reproduced and have a low cost.

It was not possible to perform statistical analysis of the group given its limited number of samples and lack of homogeneity. The blades in the microtome were frequently changed to facilitate the sectioning, which also interfered in the evaluation of the effectiveness of the softening techniques.

As for the results obtained from the histopathological exam:

- the clinically suspected cases of onychomycosis that presented negative mycological exams were not confirmed by histopathology. The number of cases was small (n = 3), which did not allow the authors to reach any conclusions. However costly procedures with side effects were avoided. It was suspected that these were cases of traumatic onychopathy; which frequently affects the hallux, the site of the biopsies focused here and that can present clinically as onycholysis or reactive hyperkeratosis;25

- in one of the clinical cases, subungual wart was suspected and the histopathology revealed no histological characteristics of infection by HPV, although the authors were surprised by hyphae in the staining by PAS. Unfortunately, no previous mycological exam had been done. This fact suggests how important it is to perform staining for the isolation of funghi in nail biopsies, above all in those cases with ungual hyperkeratosis;

- in the case of striated melanonychia, it is important to exclude, with certainty, the possibility of malignancy, which was equally valid for the case of periungual tumor that was also biopsied;

- in the case of the child with trachyonychia, the histological findings in the biopsy of the bed were only secondary to the primary alterations occurring in the matrix. Hence it would have been better to perform a biopsy in the matrix;

- in the case with clinical characteristics of psoriasis, biopsy of the nail bed was sufficient to confirm the diagnosis.

- in the case of lichen planus, since the disease affects both the nail matrix and bed, biopsy of the bed was preferred. However, when the lesion is in the very initial stage and restricted to the matrix, as in one of the biopsy cases, a biopsy of the matrix is essential.

In the cases of suspected onychomycosis, in which the mycological exams were negative or whenever it was necessary to exclude psoriasis, the histopathological exam of nail plate fragments, midway should not be neglected. Suarez (1991)²⁵ demonstrated its usefulness for diagnosing onychomycosis in the cases of negative mycological exam for fungi. Machler in 1998,²⁰ and Grammer-West 1998,²¹ working with cases of onychodystrophy, endorsed its usefulness for the diagnosis of onychomycosis and psoriasis.

CONCLUSIONS

Nail biopsy should only be carried out by dermatologists who have a broad knowledge of the anatomy of the nail unit, which is indispensable in all aspects ranging from choice of the site to orientation of the inclusion and interpretation of the results.

The appropriate sites for biopsy in the ungual apparatus for the various diseases of the nail are:

- striated melanonychia or tumors in the matrix: matrix;

- ungual dystrophy involving the entire extension of the nail plate: matrix;

- mass or tumor in the nail bed: bed;

- doubtful or non documented psoriatic or fungous nail: bed;

- unexplained onycholysis: bed;

- subungual hyperkeratosis: bed;

- vegetative lesion and unexplained subungual pigmentation: bed;

- chronic paronychia, tumors or masses of the ungual fold causing ungual dystrophy: ungual fold.

In the nail biopsies there was no difference in the technical difficulty of including or sectioning the material relative to the softening the method used.

There was no cytological or histological damage to the biopsies with either of the two techniques used for softening the nail plate.

Nail biopsy is safe a procedure that can help in the diagnosis of ungual diseases.

A routine for nail biopsy is suggested below:

1) site: use the nail alteration presented as a guideline;

2) anesthesia: regional for biopsies of the matrix and local for the nail bed;

3) clipping should be used for biopsy of the matrix and is dispensable for those of the nail bed;

4) punch: use a disposable 3mm punch for biopsies of the matrix and a non disposable 4mm punch for biopsies of the nail bed;

5) mark the biopsy specimen with colors or trim one of its sides, in order to guide the inclusion: transversal or longitudinal;

6) fixing: directly in formol for biopsies of the matrix and the use softening techniques is advised, either 10% Tween 40 in aqueous solution or 20% KOH for 15 minutes at 56°C for biopsies of the nail bed.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Ivan Curtiss Silviano Brandão for revising the english version of this paper.

REFERENCES

Received in March, 30^th of 2001.

Approved by the Consultive Council and accepted for publication in August, 23^th of 2002.

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• 17. Souza MCF, Corredor JG, Duque H, Mendonça IRM, Azulay RD. O valor da biópsia da unha para diagnóstico histopatológico da psoríase e do líquen plano ungueal. Dermatológica 1995:29-31.
• 18. Elewski BE. Diagnostic techniques for confirming onychomycosis. J Am Acad Dermatol. 1996;35(3):S6-S9.
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• 21. Grammer-West NY, Corvette DM, Giandoni MB, Fitzpatrick JE. Clinical Pearl: Nail plate biopsy for the diagnosis of psoriatic nails. J Am Acad Dermatol. 1998;38(2):260-262.
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*

Work done at the Dermatology Service of the UERJ - Hospital Pedro Ernesto, as final monograph of course.

Publication Dates

• Publication in this collection
  01 Nov 2005
• Date of issue
  Feb 2003

History

• Accepted
  23 Aug 2002
• Received
  30 Mar 2001