Present in most primates, nails are unique features that serve many functions. They consist of compact, translucent, keratinized cells overlying the distal, dorsal digits. Like hair, nails are ectodermal appendages whose histology shares similarities and differences with the skin. Abnormal nails can result from isolated nail pathology or can be a manifestation of underlying systemic disease. In cases of clinical uncertainty, a nail biopsy may be warranted to provide a definitive diagnosis. In performing a nail biopsy, an understanding of macroscopic and microscopic nail anatomy is essential to minimize the risk of permanent nail dystrophy and to facilitate the clinicopathological correlation of nail diseases.
The nail unit includes the nail plate and supporting structures. This section explains the anatomy of the nail unit and histologic findings of each region.
The nail plate is the nail itself. It is a rigid, keratinized structure composed of around 196 rows of compact, well-differentiated keratinocytes that are called onychocytes. The curvature of the nail plate along both the transverse and longitudinal axes contributes to its strength and allows for a snug fit within the proximal nail fold (PNF) and lateral nail folds (LNFs). Histologically, the nail plate resembles a modified stratum corneum. Like the stratum corneum, the nail plate contains keratinocytes that have lost their nuclei, which contributes to the translucency of the nail plate. Contrary to the stratum corneum, the nail plate has a lower percentage of total fat and water, a more significant percentage of cysteine, and thus, many strong disulfide bonds. Additionally, onychocytes are flatter and do not desquamate like corneocytes.
The nail matrix is the origin of the nail plate. Its proximal end resides halfway between the distal interphalangeal (DIP) joint and the PNF. When its distal end extends past the PNF, an opaque half-moon shape can be seen, which is the lunula. The lunula is most prominent on the thumb and gets concealed by the PNF as one progresses to the digits on the ulnar side. The contour of the distal margin of the nail plate follows the shape of the lunula as seen in neonates and following nail avulsion. Histologically, the nail matrix has a relatively thick stratified squamous epithelium that lacks a granular layer. It has long rete ridges whose tips point distally. At the edge of the lunula, an abrupt thinning of the epidermis is visible as the epithelium transitions to the nail bed. When forming the nail plate, matrical keratinocytes flatten and lose their nuclei; this occurs in the eosinophilic keratogenous zone. Below the keratogenous zone is the prekeratogenous zone and below that lies the basal layer. Collectively, the nail unit has a lower concentration of melanocytes than skin, but relative to other nail components, the matrix contains the highest concentration of melanocytes at around 217 cells per square millimeter. Melanocytes in the matrix have a basal and suprabasal distribution, occasionally being found as isolated units in the prekeratogenous zone. This suprabasal distribution of melanocytes can cause confusion when interpreting the histology of suspicious melanoma. Additional cells in the matrix include Langerhans cells and Merkel cells.
Two sections constitute the mesenchyme below the nail matrix epithelium: a matrical dermis and a matrical hypoderm. The matrical dermis contains a thin papillary and a thick reticular layer. The matrical hypoderm is continuous with the hypodermis of the PNF. It includes clusters of adipocytes interweaving with loose connective tissue, large vessels, and nerves. The density of adipose tissue below the epithelium diminishes distally and becomes scarce at the level of the nail bed. Some have suggested the presence of an onychodermis characterized by CD10 positive fibroblasts that contribute to nail plate production through an epithelial-mesenchymal interaction. Further studies in adult nails have shown the proposed onychodermis residing deep in the dermis, therefore, challenging its ability to contribute to nail plate formation directly.
The nail bed sits underneath the nail plate, spanning between the lunula and the hyponychium. Histologically, the nail bed epithelium is composed of a monocellular basal layer, a spinous layer, and, like the nail matrix, is devoid of a stratum granulosum. Longitudinal epidermal ridging complementary with corrugations in the overlying nail plate contributes to a firm attachment between the bed and the plate. On transverse slices, rete ridges are discernable, but they are lost when cutting longitudinally. There are scant melanocytes with a basal distribution.
The nail bed dermis has one relatively uniform compartment of collagen bundles and elastic fibers. There is a rich vascular network oriented longitudinally. Glomus bodies are present in the underlying dermis, which are arteriovenous anastomoses involved in thermoregulation.
The nail folds help secure the proximal and lateral margins of the nail plate.
The hyponychium refers to the epidermis underlying the free margin of a nail. Melanocytes here have a basal distribution. The crevice that forms between the hyponychium and the nail plate harbors pathogens and can contribute to infection transmission as microbes remain despite thorough hand washing. Histologically, the hyponychium characteristically demonstrates the reappearance of the stratum granulosum, and it has an epithelial thickness that rivals the matrix.
The isthmus is the transitional zone between the nail bed and the hyponychium. As a transition zone, it expresses a keratin profile that contains a mixture of keratins expressed by the nail bed and the hyponychium. It has a discontinuous stratum granulosum and a layer of parakeratotic corneocytes firmly affixed to the nail plate. This zone helps seal the undersurface of the nail plate, preventing onycholysis.
The nail has a multitude of functions including protective, mechanical, thermoregulatory, cosmetic, and economic. Nails provide a rigid barrier for the distal digits and prevent hypertrophy that has been shown to ensue after great toe avulsions. The free edge of the nail is a useful tool for grasping small objects and scratching. The nail contributes to tactile perception by providing counter-pressure to the fingertips. The glomus bodies help regulate the body's temperature by diverting blood flow from the capillaries. Nails are a platform for aesthetics, which provides occupation for many. If nails grow too long, they can cause a functional decline as shown by diminished grip strength and typing speeds with longer nail length.
A biopsy should be conducted for specific inflammatory, infectious, or tumorous conditions. It is the most sensitive technique for diagnosing onychomycosis and is especially useful for pigmented nail lesions. An apprehension of performing nail biopsies exists due to fear of pain and scarring. Other than the fleeting pain of the anesthetic injection, the procedure should be pain-free, and biopsy of a site other than the proximal nail matrix has a minimal risk of scarring. One must attain a delicate balance between acquiring enough tissue to make an accurate diagnosis and preventing unnecessary trauma.
Before the procedure, the practitioner should set patient expectations and obtain consent. Potential risks of the procedure include bleeding, infection, permanent nail dystrophy, the possibility that the biopsy does not yield a diagnosis, post-operative pain, and temporary functional handicap of the digit. A strict sterile technique is essential to avoid osteomyelitis because most biopsies are taken down to the periosteum of the underlying phalanx . The patient should receive instruction to elevate the digit for 48 hours post-surgery to prevent edema that can create a tourniquet from the bandage compromising digital perfusion.
The two most common techniques for nail anesthesia consist of the proximal digital block and the distal digital (wing or paronychial) block. The nerves that supply the nail travel along the lateral aspects of the finger. The proximal digital block utilizes this neural anatomy by injecting a local anesthetic in the lateral aspects of the finger base, which creates a field block and requires 10 to 15 minutes to become fully effective. A tourniquet can be placed to decrease blood in the surgical field and to prevent circulatory loss of the anesthetic. Common tools used for the tourniquet include gauze, Penrose drain, foley catheter, and surgical gloves. The tourniquet should not stay in place for greater than 15 minutes. In the distal digital block, the injection site is found approximately 1 cm proximal and lateral to the angle of the PNF and LNFs; this creates a localized block, requires less anesthetic, and has a quicker effect. The preferred anesthetic is ropivacaine 1% due to its long duration, although any local anesthetic is a viable option.
Ample consideration of the biopsy site is required to yield a sample where histopathological changes will be best represented. It is essential to recall that alterations of the nail plate are often manifestations of a lesion in the nail matrix. For instance, in the case of longitudinal melanonychia, the melanin in the nail plate is deposited by matrix melanocytes; therefore, the matrix should be included in the biopsy. Nail biopsy techniques include elliptical excision, punch biopsy, shave biopsy, and longitudinal biopsy. The anatomic site guides the biopsy method.
A biopsy of the nail plate is the easiest to perform and has the lowest risk of scarring. If separation of the nail bed and nail plate is necessary or a proximal nail plate punch biopsy is needed such as with proximal subungual onychomycosis, then anesthesia is required. Otherwise, a nail plate biopsy can be clipped from the free margin as is commonly utilized for distal-lateral subungual onychomycosis. An alternative method for diagnosing distal-lateral subungual and superficial onychomycosis does not require removal of the nail plate. This modified nail plate biopsy involves a simple scraping of the subungual or superficial debris to be used for direct microscopic examination with potassium hydroxide and sent for a fungal culture. Nail plate biopsies can also be useful for psoriasis, gout, and distinguishing melanin versus blood deposits. With spectrometry, chromatography, and various other analytic techniques, nail clippings can be used to detect methamphetamine, cocaine, opioids, tetrahydrocannabinol, and many other constituents.
Contrary to the nail plate biopsy, a biopsy of the nail matrix is the most difficult to perform and carries the highest risk of permanent nail dystrophy because of its role as the origin of the nail plate. The proximal matrix produces 81% of the cells in the nail plate; therefore, if possible, the biopsy should be confined to the distal matrix to decrease scarring potential. The following clinical findings suggest nail matrix pathology and could warrant a nail matrix biopsy: melanonychia, erythronychia, leukonychia, nail matrix tumors, onychorrhexis, and nail pitting. A nail matrix biopsy can be acquired as a punch biopsy, shave biopsy, or transverse elliptical excision. The PNF may need to be retracted or reflected to expose the underlying matrix, which is possible with skin hooks or lateral release incisions at the angle of the PNF and LNFs. Nail plate avulsion may or may not be used depending on surgeon preference and clinical judgment. Some clinicians prefer to replace the avulsed nail plate following the procedure. A double-punch technique exists in which there is no retraction of the PNF. Instead, a 2 mm punch biopsy is pushed through the PNF, nail plate, and nail matrix at the expected level of the proximal nail matrix. Biopsies of lesions wider than 3 mm have a higher risk of leaving permanent dystrophy, and it has been proposed shave biopsy such lesions.
A nail bed biopsy is necessary for dyschromia, onycholysis, tumorous growths, atypical-appearing subungual warts, and to differentiate psoriasis from onychomycosis. A longitudinal elliptical excision or punch biopsy are both options. The elliptical excision necessitates avulsion of the nail plate, while the punch biopsy does not. Avulsion techniques should only be for pathologies isolated to the dermis because removal of the plate displaces part of the firmly attached nail bed epithelium compromising its histologic architecture. Partial avulsion is preferred over complete nail avulsion whenever possible. For a punch biopsy without nail plate avulsion, the nail plate can be softened by soaking the digit in warm water for a few minutes. Nail bed biopsies can create onycholysis, but they generally heal without permanent dystrophy.
Nail fold biopsies are useful for paronychia and nail fold tumors. If Hutchinson sign is present, which is an extension of pigment to the nail fold in the setting of longitudinal melanonychia, a nail fold biopsy is not sufficient because its histopathologic findings can be misleading. To protect the underlying nail bed or matrix, place a spatula or nail elevator underneath the fold. Biopsy techniques can be of the shave, punch, or excisional varieties. Nail folds heal well with secondary intention.
Another biopsy technique is the longitudinal biopsy, which incorporates all components of the nail apparatus. The incisions penetrate to the periosteum with a straight medial incision and a curved lateral one. This biopsy method provides the most information to the pathologist. It can be used for large lesions on the lateral third of the nail but is otherwise not routinely performed because of its high risk of scarring. It is suggested to choose a starting point 75% the distance from the cuticle to the DIP joint to ensure inclusion of the matrical horns (the proximal corners of the matrix.)
Detailed information should be provided to the dermatopathologist including pertinent patient history, differential diagnosis, and the exact location of the sample. Communicating the orientation of the sample is vital to optimize histopathologic assessment because, as mentioned above, the histologic features greatly vary depending on the region of the nail unit. Ink, suture, or an accompanying diagram can facilitate communication of the orientation.
Processing a nail biopsy is more challenging than a standard skin biopsy. The specimen first undergoes fixation in a 10% formalin solution for an amount of time dependent on the tissue thickness. Because of the rigid nature of the nail plate, a softening step should be performed to reduce chattering, which may impair the quality of the slides and compromise the diagnostic potential. Many softening agents are available including a combination of ethanol, methanol, acetone, glycerin, 4-hexylresorcinol, 10% to 30% potassium hydroxide, 10% potassium thioglycolate, polyoxyethylenesorbitan monopalmitate, 5% trichloroacetic acid, 4% phenol, 10% formalin, chitin-softening agent, 4 to 10% sodium hydroxide, and water. Certain common household agents have even been shown to be effective softening agents. Decalcifying agents such as nitric acid have been used, but are not recommended anymore because they may alter the morphology and disrupt molecular analysis. Children tend to have thinner nail plates; thus, softening is often not required in the pediatric population. Alternatively, plastic embedding of the nail specimen has been suggested, a process that eliminates the need for the softening step. A potential downside of this technique is that it requires specialized equipment and takes over two weeks to perform. Following softening, the specimen is embedded, the most common medium being paraffin wax. The samples are then sliced longitudinally, mounted on slides, routinely stained with hematoxylin and eosin, and evaluated using light microscopy.
In addition to hematoxylin and eosin, other stains that are useful for interpreting nail biopsies include, but are not limited to, the following:
The nail matrix is responsible for nail plate growth. The proximal nail matrix produces the dorsal nail plate, and the distal matrix produces the ventral nail plate, possibly with a minor contribution from the nail bed. Thus, in the case of melanonychia, the depth of the pigment suggests the biopsy site. If the melanin remains confined to the ventral nail plate, a distal matrix biopsy is sufficient, and a proximal matrix biopsy is required if the dorsal nail plate is involved. The distal matrix is more often involved in longitudinal melanonychia because its melanocyte population possesses more potential activity. Fingernails grow at a rate of approximately 3 mm per month, while toenails grow a bit slower at around 1 mm per month. Several factors influence the rate of growth. Lowered temperatures, female sex, non-dominant hand, first and fifth digits, certain disease states, and many medications are associated with a slower growth rate. A variety of changes can appear on the nail plate as a result of nail matrix injury. The dystrophy of the nail plate depends on the severity, duration, and location of the nail matrix insult. A mild, transient, diffuse injury to the nail matrix manifests in a transverse depression across the nail plate, known as Beau’s line. Along the same spectrum, onychomadesis, a transverse separation of the nail plate, can ensue if the diffuse insult to the matrix is more severe. In comparison, a focal, prolonged insult can produce longitudinal leukonychia.
Often underutilized, nails can offer an important clue in many clinical scenarios. Not only can they reveal a recent health history, but they can also act as a window to internal pathology. For instance, in the case of hypoxia, under-perfusion, iron-deficiency anemia, and endocarditis, the nails can display clubbing, delayed capillary refill, spooning, and splinter hemorrhages, respectively. Nail dystrophy can be the first sign of inflammatory diseases such as lichen planus and psoriasis. The nails can be a site of aggressive conditions like melanoma. A histopathologic analysis is mandatory in the setting of suspicious melanoma. A helpful mnemonic that highlights clinical features associated with subungual melanoma is the ABCDEF rule, which stands for:
In addition to pigmented lesions, nail biopsies can help differentiate other similar-appearing entities, such as subungual verruca and subungual squamous cell carcinoma, as well as psoriasis and onychomycosis. Now fully equipped with an understanding of normal nail histology, abnormal histologic findings can be explored and linked to clinical diagnoses.
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