Histology for Pathologists

Editors: Mills, Stacey E.

Title: Histology for Pathologists, 3rd Edition

Copyright 2007 Lippincott Williams & Wilkins

> Table of Contents > I - Cutaneous Tissue > 2 - Nail

2

Nail

Julian Conejo-Mir

Luis Requena

Introduction

Fingernails are an important epithelial mini-organ system of the hand, with a complex anatomical structure well described in the last decades. They are important in certain animals for the apprehension and capture of prey. In primates and humans, the nails have two different functions: as a protective plate and to enhance sensation of the fingertip. The protection function of the fingernail is commonly known, but the sensation function is equally important. The fingertip has many nerve endings in it allowing us to receive volumes of information about objects we touch. The nail acts as a counterforce to the fingertip providing even more sensory input when an object is touched.

Although most pathological specimens from the nail show well-known changes such as psoriasis, lichen planus, and the characteristic malignant tumors, a broad spectrum of other changes may be found. For the pathologist, knowledge of the normal histology and its more common variations is important in establishing a correct diagnosis. Unfortunately, much of the literature on the nail can be troublesome and confusing because a great profusion of names and concepts exists and has changed over the years; also, many newer concepts of the embryology, physiology, genetics, immunohistochemistry, and nail growth mechanism find their way slowly into textbooks. This chapter emphasizes those observations and theories related to clinical pathology.

History

Historical interest can be traced to the works of Galen in the second century BC when he noticed the nail's resemblance to hair structure. However, the real study of the nail begins at the end of the nineteenth century, mostly by Germans like Zander (1), Kolliker (2), and Unna (3). The first studies dealt with embryology and anatomy and their comparison to birds and primates (1,4,5). After the initial spark of interest, the nail literature was enriched by many authors,

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During the 1950s, Lewis (8) challenged this view and published his idea of the nail unit, consisting of a dorsal, intermediate, and ventral nail, with differentiation based on the use of a silver-proteinate stain. In 1963, Zaias (9) extended the concept of the nail unit, including the proximal nail fold, the matrix, the nail bed, and the hyponychium, all of which contribute to the formation of the nail.

During the last 25 years, most studies on the nail have fundamentally tried to explain its biochemistry and physiology, with emphasis on analyzing nail growth; also considered were ultrastructure and, most recently, the immunohistochemistry of the nail. At the same time, new conditions, particularly tumors, unknown until a few years ago, have been described from the point of ungual structures, especially keratoacanthomas, merkelomas (Merkel cell tumors), and subungual Bowen's disease. In the last five years, however, publications have been very scarce following the great proliferation of papers in previous years.

Difficult biopsy access, as well as the complicated orientation and specimen handling (with the resulting difficulties of interpretation), are the main reasons why there are few histologic and histopathologic studies on the nail.

Embryology

Whereas the embryonic development of the fetal skin has been divided into eight stages, using scanning electron microscopy (10), the embryonic development of the nail shows only five stages (11,12): (a) plate phase; (b) fibrillar phase; (c) granular phase; (d) squamous phase; and (e) definitive nail phase, or end phase (Table 2.1).

The earliest recognizable fingers are seen in 42- to 45-day-old embryos (16 mm crown-rump), while the toes lag somewhat and are seen at 52 to 54 days of age (18.5 mm) (13). Studies using optical microscopy showed that the ungual morphogenesis begins at the embryonal age of 10 weeks, with a smooth, shiny quadrangular surface delineated by continuous shallow grooves. This surface of the phalanx is the primary nail base of Zander (1) or primary nail field of Zaias (9), delimited proximally by a transversal groove (the proximal nail groove).

Studies we performed using scanning electron microscopy showed that the formation of the nail begins very early, at the embryonal age of seven weeks, with an accumulation of strongly active cells, abundant mitosis, and cellular damage, followed by necrosis, with the presence of T lymphocytes in the primary nail base (Figure 2.1). This phenomenon, named apoptosis, occurs in all the epidermal accumulated cells following a transversal band in the dorsal area of the distal third of the fingers. Apoptosis of these epidermal cells is the most important step in the nail's development because it permits an immediate epidermal invagination identical to the one in the hair follicle except for one difference: in the hair follicle, the process starts at the age of 2.5 to 3 months. We observed apoptosis in nail development in this first phase only. Yet, the two processes are so identical that sometimes the layers of the nail have been compared with those of the hair follicle. The result is the formation of a transversal groove, which subsequently becomes the proximal nail fold.

An interesting feature of the first stages is the excessive size of the primitive nail plate (2.5 to 3 months), nearly occupying the total distal third part of the finger. This plate stays attached to its surroundings through some periungual-fixing filaments (Figure 2.2). At the age of 11 weeks, all the proximal and lateral nail folds are already formed. The transversal distal fold, corresponding to the hyponychium, is completely keratinized at the age of 3.5 months (Figure 2.3). Afterward, the epidermal cells of the nail field suffer a process of keratin formation that is different from the rest of the embryo. The result is a keratinized structure, covering the whole nail bed from the age of 14 weeks on and sometimes confused by some authors with a false nail (Figure 2.4) (8,9). The production of the true nail plate starts from the matrix cells, located in the proximal nail groove and the most proximal portion of the nail bed. Its presence in the

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The nail unit at this stage shows grooves form by invaginations of primitive ectoderm in regions that will become nail folds. These grooves delimit rectangular areas at distal aspects of dorsa of fingers and toes where nail plates will be situated subsequently. These areas are covered by primitive epithelium that, approximately at the fourteenth week of intrauterine-life (Figure 2.5), appears composed of a basal layer of primitive germinative cells, three or four layers of primitive keratinocytes with clear or pale cytoplasm, and a thin and eosinophilic acellular layer at the surface. This primitive epithelium covering the dorsum of a distal phalanx develops two clusters of epithelial cells at their proximal and distal ends. The proximal bud of primitive epithelial basaloid cells proliferates backward and downward, descending at an angle into the subjacent primitive mesenchymal tissue. The superficial part of this wedge of epithelial cells will become the proximal nail fold, and the deeper part will eventuate in the dorsal and intermediate nail matrices. At the junction between the superficial and deeper parts, there is a crease of cornified cells that will be the cuticle of the fully developed nail. At this stage of development, the primitive mesenchymal tissue underlying the future nail is a highly cellular tissue with abundant ground substance. At this time, the future distal phalanx is represented by primitive cartilaginous tissue with the earliest evidences of focal calcification. Distally, the primitive epithelium forms another cluster of cells with a distal ridge that will become the hyponychium.

From the fifth month on, the definitive nail plate starts to grow in a distal sense until it reaches the hyponychium at the time of birth. The growth mechanism of the definitive nail is discussed later in the section entitled Nail Growth.

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Genetics and Nail Keratins

Epithelial cells possess three cytoskeletal systems: actin microfilaments, microtubules, and keratin intermediate filaments. The protective structural role of keratins was clearly revealed in the early 1990s, when mutations in human keratin genes were discovered in a variety of human genetic diseases characterized by fragility and/or overgrowth (hyperkeratosis) of specific epithelial tissues (15). It is not precisely known how mutations in these keratins lead to hyperkeratosis of the nail, but fragility of the underlying nail bed keratinocytes presumably leads to release of cytokines and other inflammatory factors that act upon the proliferative cells of the nail matrix and produce overgrowth of the nail (16).

Keratins are a large family of intermediate filament proteins encoded by more than 50 distinct genes in humans (17). About half of these are the epithelial keratins that are found in soft epithelial tissues of the human body. The rest are the trichocyte or high-sulphur hard keratins of which hair and nail is composed. Both epithelial keratins and hard keratins can be further subdivided into type I and type II proteins on the basis of their size, charge, and amino acid sequence characteristics (18). Recent work has shown that the human hair keratin family consists of nine type I and six type II members, whose genes are organized as distinct clusters within the type I and type II epithelial keratin gene domains on chromosomes 17q21.2 and 12q13.3, respectively (16). The functional type I (K9 K23; Ha1 Ha8) and type II keratin genes (K1 K8; Hb1 Hb6) are each clustered on distinct chromosomes in the human and mouse genomes. The pairwise and differentiation-related regulation of most type I and type II keratin genes provides a unique handle to track differentiation within epithelial tissue (19). A family-wide, crucial function of keratin filaments is to endow epithelial cells with the ability to withstand mechanical and other forms of stress.

Accordingly, mutations in keratin genes are responsible for a number of genetically based fragility disorders involving specific cell types in skin and other epithelia (20,21). Among type I keratin genes, K17 stands out in multiple ways. In mouse embryonic skin, it is first expressed in ectodermal cells committing to a nonepidermal cell fate (i.e., all appendages and periderm) in response to mesenchymal induction (22). Concomitant with skin maturation, mK17 expression becomes restricted to specific cell layers and compartments within all major types of epithelial appendages. Both hK17 and mK17 can be coregulated with distinct type II keratin genes (e.g., K5, K6a, K6b, K6hf) in mature epithelial settings (23). In addition to its constitutive expression in epithelial appendages, K17 expression is induced in mature interfollicular epidermis subjected to various types of acute challenges (e.g., injury, ultraviolet light exposure, inflammation) or during diseases (e.g., psoriasis, basal cell carcinoma). Mutations affecting a particular segment of hK17's coding sequence can cause distinct disorders of the skin, related to ectodermal dysplasias (15).

Several genodermatoses, such as Darier's disease, X-linked dyskeratosis congenita (DC), and pachyonychia congenita (PC), are associated with characteristic nail changes (24). A causative gene for Darier's disease, which resides at 12q23-q24.1, encodes the sarco/endoplasmic reticulum Ca²⁺-ATPase type 2 isoform. Mutations in XAP101 gene, which maps to Xq28, are responsible for DC. Thus, mutations in DKC1 gene encoding dyskerin have been also found (25). Pachyonychia congenita is a group of autosomal dominant ectodermal dysplasias, whose most obvious phenotype is hypertrophic nail dystrophy leading to a phenotype of grossly thickened nail. There are two main types of PC: the Jadassohn Lewandowsky form (type 1) and the Jackson Lawler form (type 2). The epithelia affected in pachyonychia congenita type 1 express the keratin pair K6a and K16, and the condition is caused by dominant-negative mutations in these genes (26). The pachyonychia congenita type 2 variant is caused by mutations in keratins K6b and K17 (27). All the tissues affected in pachyonychia congenita type 2 express K6b and K17 (28,29,30).

Gross Anatomy

It was first noted early in the twentieth century that the nail unit was comparable in several respects to a hair follicle sectioned longitudinally and laid on its side. The epithelial components of the hair follicle and nail apparatus are differentiated epidermal structures that may be involved jointly in several ways as congenital and hereditary anomalies and acquired conditions such as alopecia areata, lichen planus, iatrogenic causes, and fungal infection (31).

Various types of differently keratinizing epidermis make up the nail. What commonly is termed the nail plate is the horny end product of the most important epidermal component, the matrix. Usually, this nail plate is slightly convex or flat, rectangular, and of varying size between approximately 1 1 and 2 3 cm, depending on the finger (Figures 2.6,2.7,2.8). In the hand, this is usually 25 to 50% of the dorsal surface of the fingertips, whereas in the big toe it occupies about 75%. The nail is translucent and becomes rosy from the underlying vascular network. However, change of colors (erythronychia, melanonychia) can be observed in the nail plate and may indicate inflammatory diseases (lichen planus, lupus erythematosus); benign or malignant neoplasms, main subungual melanoma; and scarring of the dermis or epidermis (32). The white appearance of nails in leukonychia seems to be a result of an abnormal keratinization of cells originating from the proximal nail matrix, which leads to the presence of abundant intracellular vacuoles and to a lesser compactness of keratins. Genes mapping within this chromosomal region include the genes coding for type II (basic) cytokeratins and

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In the proximal portion of the nail, there is an arch called the lunula. The thickness of the nail plate is 0.5 mm in women and 0.6 mm in men (34). The nail plate is delimited by three folds: two lateral and one proximal (Figure 2.9). If the nail plate is avulsed, the grooves become visible where the nail plate rested. These potential spaces are only real spaces in abnormal conditions of the nail, as in paronychia. In the lateral nail grooves, the epidermal lining does not contribute to the formation of the nail plate except in the most proximal portions, where it becomes continuous with the epidermis of the proximal groove or matrix.

The proximal nail fold is the most important one, since, as we shall note later, its contribution to the formation of the nail plate is fundamental (35). This fold shows two portions: a dorsal portion, lodging the matrix, and a ventral portion. Twenty-five percent of the total surface of the nail plate is located under the ventral portion of the proximal nail fold. The terminal tendon of the digital extensor is closely related with this area, and the thin nature and proximity with the nail matrix must be kept in mind during surgery (36).

A white crescent-shaped lunula can project from under the proximal nail fold. It is usual on the thumbs and common on other fingers and on large toenails. The lunula

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At the point of separation of the nail plate and the nail bed, the subungual epidermis may be modified as the solehorn (38). In humans, this structure may only be vestigial, its original significance only being evident from comparative anatomical studies. However, in certain diseases, it could be the seat of distal subungual hyperkeratosis or parakeratosis; for example, in pachyonychia congenita and pityriasis rubra pilaris (39). The distal limit of the ungual layer is the hyponychium, which determines the formation of the distal fold, a keratinized structure that continues until the fingertips. A subungual extension of the hyponichium and obliteration of the distal groove is named the pterygium inversum unguis (40). This term was coined because of the similarity between the behavior of the hyponychium and the eponychium in classic cases of pterygium unguis.

On close examination, two further distal zones can often be identified: the distal yellow-white margin and, immediately proximal to this, the onychodermal band (41). This band is a barely perceptible narrow transverse band, 0.5 to 1.5 mm wide, that is more prominent in acrocyanosis. The exact anatomical basis for the onychodermal band is not known, but it appears to have a different blood supply from the main body of the nail bed (42). It is possible to explore it through a strong compression of the distal zone of the finger, leaving behind a white band. The band's color can occasionally be modified by diseases (39,43).

Several studies have been published about the exploration of the nail apparatus. Although ultrasound transmission can be useful for studying the nail plate thickness (44), magnetic resonance imaging (MRI) permits the detection of subungual lesions smaller than 1 mm in diameter (45).

Microscopic Anatomy

The Nail Plate

Microscopically, the nail plate consists of closely packed, adherent, interdigitating corneocytes lacking nuclei or organelles (Figures 2.10,2.11). Many intercellular links, including tight, intermediate, and desmosomal junctions, are present (46). The nail plate is made-up of three layers: a thin dorsal layer, a thick intermediate layer, and the ventral

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Hamilton et al. (49) believed that the progressive increase in the thickness of the nail plate with age is attributable to the increasing size of the cells in the plate, consecutive to the frictional loss of nail; however, Johnson and Shuster (50) studied 20 normal great toenails to find the determinant of final nail thickness and length at its point of detachment at the onychodermal band. They confirmed that the increase of nail thickness with age is independent of the frictional traumatisms on the plate. Cutting tests showed that fracture of the nail plate occurred because the energy to cut nails transversely, at approximately 3 kJ/m², was about half that needed (approx. 6 kJ/m²) to cut them longitudinally (51).

Corneocytes of the human nail plate have been studied by Germann et al. (52). Corneocytes of the dorsal nail plates of normal nails are irregular and polyhedral, nonnucleated, and with distinctly irregular networks. These horny cells from nail plates increase in size with age: babies have small cells, adults have significantly larger cells, and aged subjects have significantly larger cells than the younger adults. These authors also commented that the faster-than-normal growing nail plates yield smaller cells; for example, corneocytes from psoriatic patients are smaller than normal, whereas corneocytes from slow-growing nails, such as from persons having lichen planus or dyskeratosis congenita, are larger than normal.

Frequent gap junctions were observed near the area where lamellar granules were discharging their contents, and it was suggested that a certain substance might be able to pass through the nail plate using such intercellular channels. Perhaps such channels explain the greater permeability of the nail plate to polar molecules compared with the permeability of the skin (53). The water content of human nail plates has been determined using a portable near-infrared spectrometer with an InGaAs photodiode array detector and PLS regression by Egawa et al. (54).

Transonychial water loss (TOWL) in vivo has been studied by Nuutinen et al. (55). These authors have demonstrated that TOWL values decrease with age and patients with eczema, psoriasis, and onychomycosis have significantly lowered TOWL values compared with healthy subjects.

The biochemical composition of the nail plate has been widely studied. Calcium, found as the phosphate in hydroxyapatite crystals, is an important component of the nail plate; it is intracelluarly bound to phospholipids, particularly in the dorsal and ventral nail plate (56). Calcium concentration is approximately 0.1% of the weight, 10 times greater than in hair (57). However, some authors believe that the proportion of calcium in the nail contributes little to the hardness of the nail plate in human beings (39,58). Also, it is possible that calcium is not an intrinsic part of the nail but is incorporated from extrinsic sources, such as soaps; the nail is relatively porous, and calcium could enter as ionic calcium or bound to fatty acids. Other metals such as copper, manganese, zinc, and iron are also found in small quantities in the nail plate, although their function is still unknown (58,59). Lipids are also an important component of the nail plate. Helmdach et al. (59) have demonstrated that nail plate lipid composition varies with age and sex: the lipid composition of the fertile years shows distinct profiles compared to that of childhood and old age, suggesting an influence of sex hormones on nail lipogenesis.

The existence of sulfhydryl and disulfide groups has been demonstrated in the nail plate. During early embryonic life, there is a very high concentration of the sulfhydryl groups (9), which decreases as the delivery date approaches and stabilizes at about the age of 3 years (14). These sulfurous radicals are formed at the expense of amino acids, such as cystine. Quantification of cysteine and cystine can be performed by hydrolysis (60). Total sulfur concentration is similar in the dorsal and intermediate plates. Compared with hair, the nail plate also contains glutamic acid, serine, and less tyrosine (57,58,59,60).

In certain diseases, the quantity of various organic and metallic components of the nail plate can be increased. A brief listing is presented for reference: total nonprotein nitrogen, urea nitrogen, ammonia nitrogen, and uric acid in gout (61); creatinine in chronic renal failure (62); sodium in cystic fibrosis (63,64); calcium in older subjects (48,57);

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An analysis of the keratin of the nail plate revealed the following (68): (a) -fibrillar, low-sulfur protein; (b) globular, high-sulfur matrix protein; and (c) high-glycine tyrosine-rich matrix protein. All these fractions are also present in hair. The hardness of nail is due to the high-sulfur matrix protein, contrasting with the relatively soft keratin of the epidermis.

Proximal Nail Fold

The proximal nail fold is an invaginating, wedge-shaped fold of the skin on the dorsum of the distal digit, and the nail plate arises from under this fold (Figure 2.12). The proximal nail fold consists of two layers of epidermis: the ventral portion overlying the newly formed nail plate and the dorsal portion that forms the dorsum of the finger epidermis. The keratinization process in both portions does not differ from that of the epidermis elsewhere, possessing a granular layer that is absent in all parts of the nail matrix.

The dorsal portion of the proximal nail fold consists of a continuation of the epidermis and dermis of the dorsal digit with sweat glands but no follicles or sebaceous glands. At the distal tip, a thick corneal layer called the cuticle shows on the dorsal surface of the nail plate (Figure 2.13). Its function is the protection of the nail base, particularly the germinative matrix. Loss of the cuticle often allows acute and chronic inflammatory and infective processes to involve the nail matrix, leading to secondary nail plate dystrophies.

The ventral portion is thick-skinned, has no appendages, and is closely attached on the dorsal surface of the nail plate. The epithelium of the ventral surface of the proximal nail fold has been called the eponychium (9,37,39). Diseases that affect the ventral portion of the proximal nail fold can affect the newly formed nail plate. For this reason, some authors think that the proximal nail fold contributes to form the superficial layer of the nail plate. In particular, the apparition of pits and grooves (Beau's line) on the nail is due to parakeratotic and growth detention phenomena, respectively, in the ventral portion of the proximal nail fold.

Matrix

The ventral surface of the proximal nail fold forms the roof of the proximal nail groove; the nail matrix forms its floor, and the nail plate lies between the two. The matrix is divided into three parts (8,9,39): dorsal, intermediate, and ventral. Of these, the dorsal and, above all, the intermediate portions play an important role in nail plate formation. In particular, the true matrix is the intermediate portion. For this reason, when we discuss the histology of the matrix, we are fundamentally referring to the intermediate portion. The ventral portion corresponds to the nail bed; the controversy about its participation in the formation of the definitive nail plate is discussed in the section entitled Nail Growth.

The main body of the matrix is composed of epithelial cells, with melanocytes, Merkel cells, and Langerhans cells scattered among the epithelial cells.

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Epithelial Cells

The matrix is an easily identified thick squamous epithelium, situated immediately below the ventral portion of the proximal nail fold (Figure 2.14). Its main feature is its thickness, with between 8 and 15 mamelons (protuberances) (Figure 2.15). Its undulation can be seen for only a few millimeters, flattening itself in the area corresponding to the nail bed. As in the epidermis of the skin, the matrix possesses a very active germinative basal layer of immature basaloid cells, producing keratinocytes which differentiate, harden, die, and contribute to the nail plate (Figure 2.16). The nail plate is formed by a process that involves flattening of the basal cells of the matrix, fragmentation of nuclei, and condensation of the cell cytoplasm to form horny flat cells. An important histologic feature is the lack of granular layer. Acanthosis and papillomatosis are only seen in the nail unit in the matrix and, distally, in the hyponychium (Table 2.2).

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Melanocytes

The nail matrix possesses melanocytes, just as the hair matrix does. The matrix of Caucasian patients contains sparse, poorly developed melanocytes (Figures 2.17, 2.18, 2.19) (69). It is difficult to observe melanocytes in the proximal matrix zone using light microscopy, but their number is progressively increased distally. Nevertheless, the number of melanocytes is always less than in normal skin (69,70,71,72,73).

There are distinct differences in the distribution of melanocytes in adult skin and nail matrix. Immunostaining of nail matrix melanocytes revealed that they are not singly interspersed between the keratinocytes of the basal layer, but they are frequently arranged in small clusters among the suprabasal layers of the nail matrix (73,74). A similar pattern of distribution of melanocytes has been described in fetal skin and in fetal skin equivalents, in which the melanocytes are grouped and localized both basally and suprabasally. The suprabasal location of nail matrix melanocytes may be a consequence of differences in the distribution of adhesion molecules in the nail epithelium (73,74).

Higashi and Saito (75) demonstrated that the number of melanocytes and the intensity of the dopa reaction in them were much greater in the distal than the proximal matrix. The melanocyte count in normal epidermis was reported to be 400 per 2.784 mm² (74,75), while the range was 208 to 576 in the distal areas of the intermediate nail matrix (75).

Ultraviolet rays and trauma are factors that could influence a more extensive distribution in the distal zone (73,74). In some races such as Japanese, the matrix contains several hundred well-developed melanocytes per millimeter (75). Also, it seems that melanocytes of the nail matrix in Oriental races have larger dendritic processes than Caucasians.

Pigment, therefore, arrives in the nail plate as in the keratinized cells of the stratum corneum and hair cortex (76). Nail pigmentation is most evident in African Americans, in whom it is commonly seen as longitudinal linear streaks, although this anomalous distribution of pigmentation can also be seen in pathological states such as subungual pigmented nevi and melanomas in the matrix zone (77). Abundant melanosomas of these subjects have a protective effect against ultraviolet radiation, since variations in racial pigmentation are due to the number and size of melanosomes produced (78).

The location of melanocytes in the matrix is directly related to the location of pigmented bands, most of which originate in the distal matrix and do not cross the lunula (73). Longitudinal melanonychia in AIDS patients is a common finding. Although this pigmentary nail change

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Langerhans and Merkel Cells

Langerhans and Merkel cells have also been identified in the matrix (70), although their signification is unknown. Studies of the Langerhans cells in the nail matrix are almost absent. Nevertheless, interesting studies about the Merkel cells have been recently published. Moll and Moll (81) studied the Merkel cells (MCs) in ontogenesis of human nails, using immunohistochemical stains with cytokeratins 18 and 20 in human fetuses of 9 to 22 weeks of life. These authors have concluded the number of MCs are detected very early (nine weeks) in the matrix primordium. However, MCs were found to decrease in number with aging of the fetuses: MCs were only seen in the proximal nail fold at 12 to 15 weeks and were essentially absent from the epithelium of the ventral matrix and nail bed in the adult.

Lunula

The intermediate matrix continues forward with a visible, white half-moon-shaped area called the lunula. The lunula is shown to be linked to a well-defined area in the underlying dermis with a specific histology and microvascularization. Although always present, it cannot be seen in some fingers but is most visible in the thumbs. The typical white color is related to some histologic features of this area. Lewin (34) confirmed that the opacity of the proximal nail plate, the relative avascularity of the subepidermal layer, and the loose texture of the dermal collagen are responsable for its color. Samman (82,83) thought that it was a combination of incomplete keratinization in the nail plate and loose connective tissue in the underlying tissue. Zaias (84) believed that the nail plate would be thinner in the lunula because it coincides with the keratogenous zone, the zone of cytoplasmic condensation in the matrix just before cells form the nail plate. The length of the subnail matrix area distal to the free edge of the proximal nail fold is highly correlated with the length of the lunula (85).

Other special histologic features of this zone of the matrix, including a different chemical composition of the nail plate and a different distribution of the dermal fibers, have been related to the typical white color of the lunula (86,87), although not one of these factors has been confirmed. We do not even know the exact function of the lunula.

Nail Bed

The nail bed begins where the intermediate matrix ends, and some authors prefer to designate the ventral matrix as the site (39,86). A histologic appreciation of the end of the intermediate matrix and the beginning of the nail bed is very easy. The nail bed epidermal layer is usually a flat epithelium no more than three- or four-cells thick and without melanocytes (Figures 2.20,2.21,2.22). The transition zone from living keratinocytes to dead ventral nail plate cells is abrupt, occurring in the space of one horizontal cell layer in a manner very similar to what occurs in the Henle layer of the internal root sheath of the hair follicle (87).

During its early development, the nail bed exhibits a keratinization process differing from the adult's, with a prominent granular layer at 17 to 20 weeks of development. However, after birth, the nail bed, like the matrix, keratinizes without a granular layer. lt is less active than the matrix, with a longer turnover time than the matrix and skin (88). A thin parakeratotic keratin is produced, apparently dragged forward by the nail plate growing over it rather than becoming incorporated into the nail.

In the nail bed, the dermis fits into the longitudinal and parallel nail bed ridges in tongue-and-groove fashion. The fine capillaries of the nail bed run in these parallel dermal ridges, and disruption of these accounts for the splinter hemorrhages commonly seen in normal and disease states (42). There is no fat tissue in the nail bed, although scattered dermal fat cells may be visible microscopically.

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The nail bed epidermis moves distally toward the hyponychium. The cells that appear to be the germinative population lie near the lunula, so close together that they may be confused as belonging to one population. The distal movement from this position may also help explain why during development the nail bed epidermis seems to lose keratohyalin granule layers from a proximal-to-distal direction concomitantly with the formation of the primitive nail plate (89).

In some pathologic states, the nail bed shows a granular layer in which the activity in the nail bed is greatly increased, such as occurs in onychogryphosis, pachyonychia congenita, and psoriasis (90); in these cases, the horny cells produced push the nail plate upward and give it a claw-like appearance.

Histochemical studies of the nail bed prove the presence of bound phospholipids in the nail bed epidermis (Table 2.3). Bound cysteine can be detected in the transition zones: acid phosphatase and nonspecific esterase are absent in the dorsal and intermediate zones (39,56).

Immunohistochemical studies have demonstrated that nail bed expressed all the target antigens found in the normal nonappendageal basement membrane. In particular, there was normal expression of the epidermal-associated antigens, the 230- and 180-kDa bullous pemphigoid antigens, and the 6 4 integrin. There was also normal expression of the lamina lucida antigens LH39, GB3, and laminin. Sinclair et al. (91) pointed out that the dermal-associated components, namely the 285-kDa linear immunoglobulin A (IgA) antigen, the extracellular matrix glycoproteins chondroitin sulphate, type VII collagen and its closely associated proteins, and the poorly characterized antigen for LH24 and LH39 were all normally expressed. All the former molecules were also found in the proximal nail fold, nail matrix, and hyponychium.

The presence of antimicrobial peptides in nails (mainly cathelicidin LL-37, demonstrated by immunostaining and which has activity against relevant nail pathogens) may account for the ability of the nail unit to resist infection

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Hyponychium

The most distal portion of the nail bed is the hyponychium, representing the union between the nail bed and the fingertips (Figure 2.23); its histologic characteristics are rather peculiar. This transition zone presents a notable change of appearance after a few millimeters because the epithelium undergoes keratinization similar to that of the epidermis (Figure 2.24). The result is marked acanthosis and hyperplasia with the crests oriented almost horizontally; this is associated with normal appendages (Figure 2.25). An area of abundant keratohyalin granules is present, and the horny layer produced tends to accumulate under the free edge of the nail plate, producing a keratin horn similar to the cuticle. The hyponychium is the first site of keratinization in the nail unit (8,9,11,12,13) and of all epidermis in the embryo (93). The function of this anatomical formation is to render the nail bed impermeable to protect it from external agents (94). If this structure fails, dermatophyte invasions will be frequent, producing onychomycosis (95).

Terry (41) describes an intermediate zone between the nail bed and the hyponychium, which he called the onychodermal band. Terry speculated that this area, normally from 0.5 to 1.5 mm wide, had a blood supply different from the remainder of the nail bed, a fact later confirmed by other authors (42). For this reason, the color is paler than the pink nail bed and has a slightly amber tinge with a translucent quality. The onychodermal band occasionally changes its color, especially in cirrhosis and other chronic diseases (39,43).

Lateral Nail Folds

The lateral nail folds have a structure similar to the adjacent skin but are normally devoid of dermatoglyphic markings and folliculo sebaceous units. Acanthosis and hyperplasia of the epithelium are present, similar to that of the hyponychium. Keratinization within the nail folds proceeds by keratohyalin formation in the granular layer (Figure 2.26). The epidermal lining of these grooves does not contribute to the formation of the nail plate except in the most proximal portions of the grooves, where it becomes continuous with the epidermis of the proximal nail fold or matrix.

When the lateral border of the nail plate pathologically breaks this fold, abundant granulation tissue forms, constituting the onychocryptosis, a frequent pathologic alteration of the great toenail.

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Immunohistochemistry of the Nail Unit

Nail Plate

The cornified envelope of the epidermis is formed by several precursor proteins, including involucrin, keratolinin, loricrin, pancornulin, sciellin, 195-kDa protein, keratin, and filiagrin. Baden and Kvedar (96) used monoclonal antibodies to demonstrate that pancornulin is present in the nail fold and proximal matrix, and sciellin is detectable in the nail fold, matrix, and bed. Similarly, in studies of human nails (which contain hard keratins), the use of immunofluorescence, immunoblotting, and PCR (polymerase chain reaction) have shown that trichohyalin, a 200-kDa protein of the inner root sheath and medulla, was present in the ventral matrix but not in the nail bed; a few scattered cells stained for trichohyalin were observed within the nail plate (97).

Heid et al. (98) studied the keratin expression patterns observed in the human fetal nail matrix and revealed that the nail develops from both skin- and hair-type differentiating cells. Kitahara and Ogawa (99) demonstrated that AE13 antibody reacted with the dorsal nail matrix. Because AE13 antibody recognized hard keratins that are characteristic of differentiation in hair, these results show that adult nail develops in such a way that hair-type differentiation is confined to the ventral nail matrix, supporting the Heid et al. study's results (100,101).

Keratinocytes

Expression of keratins in the different compartments within the nail unit have been demonstrated in some recent articles (102,103,104,105,106,107,108,109,110,111,112). The characteristics of the different keratins found at different sites could be relevant to our understanding of the biology of the normal nail and changes seen in several diseases.

Analysis of human nail plate by gel electrophoresis demonstrates a range of keratins of two characteristic types, as we commented formerly (see Genetics and Nail Keratins). Soft, or epithelial, keratins represent the major structural intermediate filament isolated from human skin but constitute only 10 to 20% of the keratin found in nail (99). Hard keratins, characteristic of hair and nail differentiation, exist in the same acid-base heterodimer configuration as soft epithelial keratins but have additional resistance. Molecular classification of hard keratin proteins on gel electrophoresis describes eight major (Ha1 4 and Hb1 4) and two minor (Hax and Hbx) proteins, all of which are probably present in human nail (103). This family has been extended by genomic analysis into at least seven type I Ha keratins and six type II Hb keratins. Further keratins and their isoforms are likely to be discovered. In addition to the hard keratins, epithelial keratins isolated from the nail plate include K1, K10, K5, K14, K6, K16, K17and K19 from fetal nail (102,103) (Figure 2.27).

Ha1 is one of the major hard keratins found in nail, where hard keratin represents 80 to 90% of nail keratin. Keratins K5, K14, K1, and K10 were also detected in the nail. Since Berker et al. (102) have found a low expression of the differentiation-specific keratins K1 and K10 in the keratogenous zone of the ventral matrix, this feature was not demonstrated by other authors (104).

Keratins K6, K16, and K17 are normally found in hyperproliferative epidermis, such as in psoriasis or in wound healing (105). Studies of proliferative compartments in the nail unit suggest that the nail bed is not a major contributor to the nail plate. It may be that the ventral aspect of the proximal nail fold and not the nail bed is the source of nail plate K6 and K16, and the matrix and not the nail bed provides K17. Keratin K14 is synthesized in the basal layer, and K14 protein was detected throughout the epithelium, as has been noted in other tissues. However, the marker of basal keratin conformation, LH6, was also seen throughout the nail bed. This is unusual and may reflect the absence of the expression of the suprabasal keratins K1 and K10, which are thought to obscure the epitopes detected by LH6 in normal stratified epithelium. This persistence of LH6 antigen is also seen in the outer root sheath of the hair follicle; because this is also the site of expression of K16, K6, and K17 (106), the data support the analogy drawn between the nail bed and outer root sheath (107). However, expression of K1 and K10 is found to a degree in the upper outer root sheath superficial to the level of the sebaceous gland.

The absence of K1 and K10 from the nail bed correlates with a reduction in terminal differentiation. Lack of cornification is also seen in mucosal epithelium in combination with the presence of K16 and K17. However, mucosal differentiation is defined by the presence of K4 and K13, which was absent in the nail bed (108). The exact significance of these keratins remains to be established.

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Additional molecular studies have demonstrated the homology between hair and nail keratins (109). Retinoic acid inducible gene-1 was originally identified as an orphan G-protein coupled receptor (oGPCR) induced by retinoic acid. Three highly homologous oGPCR (GPCR5B, GPCR5C, and GPCR5D) have since been classified into the RAIG1 family. Inoue et al. (109) studied the unique tissue distribution of GPCR5D and its mechanism of expression by in situ hybridization in hair shafts, nail, and filiform papillae of the tongue. They found that GPCR5D is expressed in differentiating cells that produce hard keratin, including cortical cells of the hair shaft, the keratogenous zone of the nail, and in a central region of the filiform papillae of the tongue. GPCR5D transcript is expressed in hair follicles during mid- and late anagen, and catagen but not at telogen and early anagen phases. The differentiation-inducer, all-trans retinoic acid, induces GPCR5D expression in cultured hair bulb cells. Because the tissue distribution of GPCR5D indicates a relationship with hard keratins that constitute the major structural proteins of hard epithelial tissues, they investigated the effect of GPCR5D on acid hard keratins.

Analyses of cultured cells showed that transient overexpression resulted in suppression of Ha3 and stimulation of Ha4 hair keratin gene expression. The expression was maintained in the hair follicles of whn-deficient (nude) mice, suggesting that this gene is regulated by a signal pathway different from that of hair keratin synthesis. These data provide a framework for understanding the molecular mechanisms of GPCR5D function in hard keratinization.

For the expression of hard keratin by matrical keratinocytes is primordial the influence of matrical fibroblasts. Okazaki et al. (110) demonstrated that, even in non nail-matrical keratinocytes, expression of hard keratin could be induced by nail-matrical fibroblasts. These investigators constructed three different skin equivalents: (a) ventral keratinocytes (from the ventral side of the digit) were cocultured with ventral fibroblasts (group A); (b) ventral keratinocytes were cocultured with nail-matrical fibroblasts (group B); and (c) nail-matrical keratinocytes were cocultured with ventral fibroblasts (group C). Immunohistochemical examinations with antihard keratin antibody (HKN-7) revealed hard keratin expression in groups B and C. The HKN-7-positive cells were distributed continuously in the entire epithelial strata or in the suprabasal layer in group B, whereas HKN-7-positive cells were distributed spottily in group C. This study indicated extrinsic hard keratin induction in non nail-matrical keratinocytes by nail-matrical fibroblasts and suggests that non nail-matrical epidermal grafts may be effective in the treatment of de-epithelialized nail injuries. In addition, it is possible that lost nails could be reconstructed with grafts of tissue-engineered nail equivalent.

Human carcinoembryonic antigen (CEA) and CEA-related molecules play an important role in adhesion of the nail plate to the nail bed. Egawa et al. (111) demonstrated that a CEA family antigen with NCA (CD66c)-like immunoreactivity was strongly expressed in the nail. A strong expression of the NCA-like antigen was only seen in the keratinocytes distributed in the upper epithelial cell layers of the major central portions of the nail bed, to which the nail plate is firmly bonded; expression was stronger at the more distal portion of the nail bed and was absent in the nail matrix, the hyponychium, and the lateral folds. The results are interesting because the nail plate is firmly bonded to the nail bed, less so proximal to the matrix margin, and it has been shown that the bed epithelium travels at the same speed as the nail plate, indicating that the bed epithelium has a proximal site of origin and a distal end.

Plasminogen activator inhibitor type 2 (PAI-2) was detected in the differentiating cells of the matrix and nail bed (112). These authors, using transfected cell lines that express high levels of PAI-2, have suggested that this inhibitor may confer protection against programmed cell death. This consistent, selective distribution of PAI-2 in the postmitotic, maturing cells prior to terminal keratinization and death suggests that (a) PAI-2 may be considered as a differentiation marker for many epithelial cell types, and

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Merkel Cells

Lacour et al. (113), in a double indirect immunofluorescence and immunoelectron microscopic study with the monoclonal antibody Troma-1, have only found MCs in the proximal nail fold of the adults, with a concentration greater than 50 MCs/mm².

Immunohistochemically, keratins K8 and K18 have been used as markers of MCs (81). Merkel cells have neuroendocrine characteristics and are of uncertain function, although their prominence in the nail unit in the early stages of fetal development has been noted, and a role in ontogenesis has been proposed. The number and location of cells demonstrating K8 and K18, which included the rete ridges of the digit pulp, suggested that these cells were MCs rather than they contributing directly to nail plate formation.

Melanocytes

Tosti et al. (114) have recently studied the melanocyte characterization of the normal nail matrix using immunohistochemistry techniques. These authors found nail matrix melanocytes reacted with the antibodies anti-PEP-1, anti-PEP-8, and anti-TMP-1, which recognize the tyrosinase-related protein-1, the tyrosinase-related protein-2 (DOPA-chrome tautomerase), and the tyrosinase-related protein encoded by pMT4 (Table 2.4). This confirms that, even if normally quiescent, nail matrix melanocytes possess the key enzymes responsible for the formation of melanin pigment (115).

Expression of integrins in the nail matrix have been studied by Cameli et al. (116). These authors found that 2 1 and 3 1 expression differ in nail matrix epithelium. In the nail matrix, these integrins are not only expressed on the basal layer, but also on the fourth to fifth suprabasal layers, with suprabasal expression gradually decreasing from the distal to proximal matrix (Table 2.5). As in the normal human epidermis, 1, 4, and 5 integrins subunits are not expressed in the nail matrix; in the same way, ICAM-1, the ligand of LFA1, was negative in the matrix cells. The expression of 1 subunits in the suprabasal layers of the nail matrix indicates a very strong cohesion between nail matrix cells, thus probably revealing them to be an essential prerequisite for the development of a compact nail plate. Cultures of nail matrix cells may represent a useful model to study the biologic properties of nail structure (117).

Ultrastructural Anatomy

Very few studies of the normal ultrastructural morphology of the nail exist (10,11,12,13,46,47,69,70,71,72,76,89) because of varied difficulties (70): (a) achieving proper fixation and

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The proximal end of the human toenail is composed of several layers of epithelial cells. Hashimoto and coworkers (69,70,71,72) make the distinction between a proximal dorsal, apical, and ventral matrix, although noting that there are few differences between them. They found that the cells composing the proximal matrix were: (a) relatively small, elongated basal cells attached to the basal lamina; (b) relatively large, round, or polygonal squamous cells filling the more central portion of the matrix; (c) melanocytes; (d) Langerhans cells; and (e) Merkel cells.

Moreover, there exists a system of attachment to the dermis, showing the surface of the basal cell with frequent fingerlike elongations that interdigitate with the papillary dermis (Figure 2.28). This results in the formation of numerous micropapillae, with bundles of very fine fibrils (11 to 12 m). The subjacent dermis of the matrix zone shows poor vascularity and scarce collagen fibers, with abundant basic matrix.

The basal cells are very active, with frequent mitotic figures. They showed an elongated nucleus and cytoplasm with numerous, slender projections (villi) intricately interdigitated with neighboring cells. Tonofibrils were also seen as a perinuclear ring with an interposition of the nuclear clear zone in which the majority of mitochondria, transferred melanosomes, and occasional centrioles are located. The suprabasal matrix cells are also round, with frequent mitotic figures. Generally, the long axes of these cells were oriented axiodistally, suggesting the direction of their migration. Large intercellular spaces were often seen between these suprabasal cells. The extensive interdigitation of the peripheral villi as seen in the basal cells disappeared, and multiple desmosomal junctions alone connected these cells (Figures 2.29 and 2.30).

Abundant desmosomes can be seen in the intermediate layers with high condensations of intermediate fibrils. The aspect of the intermediate layer of the nail matrix is similar to the upper layers of the normal epidermis (Figure 2.31). The cells have lost their organelles, and their cytoplasm is nearly filled with tonofibrils. For this reason, the keratinization process is very abrupt, passing from three to four cellular lines to completely keratinized corneocytes.

Other Tissues of the Nail Unit

Dermis

The dermal component of the nail structures is a very specialized tissue, unique in that it is limited by the underlying phalanx and closely associated with its vasculature and nerve supply. There is no subcutaneous tissue, as previously noted.

Dermis, epithelium, and nail plate in the nail bed present special histologic features due to the great traction that is supported. The dermis is very thick with a dense collagen layer. These fibers are vertically situated in the proximal zone of the nail bed (Figure 2.32) and inclined at an angle of 45 degrees in the zone adjacent to the hyponychium (Figure 2.33).

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There are few studies about the nerve supply of the nail. The matrix and nail bed present sparse nerve endings and few Vater-Pacini (118) and Meissner corpuscles (119). Intraepithelial nerve fibers were described at the beginning

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The hyponychium is the area with greater abundance of nerve endings of the nail and with abundant Meissner and Merkel-Ranvier corpuscles, as in the lateral nail folds (120). This histologic feature gives the hyponychium an important role in the fine sensibility of the finger.

Bone

The nail apparatus includes the subjacent bone. Although the bone has been ignored during the last years, a recent study of postamputational repair following digit-tip amputation revealed an unexpected correlation between nail regrowth and bone regrowth. In this way, Zhao and Neufeld (123) have studied this relationship, observing that in the absence of nail, bone did not regrow at distal levels, and, conversely, when the nail was surgically retained, bone regrew from proximal levels.

Blood Supply

The nail has a rich vascularization that deserves separate mention. The arterial blood supply of the nail bed and matrix is derived from paired digital arteries. The most important studies have been published by Flint in 1955 (124), Ryan in 1973 (125), and Smith et al. in 1991 (126), concluding that the main supply passes into the pulp space of the distal phalanx before reaching the dorsum of the digit. An accessory supply arises further back on the digit and does not enter the pulp space. The digital arterial system manifests three characteristic anatomic features: arched anastomotic arteries in the deep dermis; more superficial terminal arteries branching to supply the rete (127), and the great tortuosity of the arterial architecture subjacent to the nail apparatus (Figure 2.36). The arteries possess inner longitudinal and outer circular coats of smooth muscle (Figure 2.37). Oxygen-sensitive microelectrodes (tip diameter of 5 m) to measure the distribution of P_O2 in dermal papillae of the fingernail folds of healthy human subjects have been developed by Wang et al. (128).

The vasculature in the nail bed is unique in that it must supply a vascular structure between two hard surfaces, the nail plate and the bone. Studies with scanning electron microscopy revealed special vascular patterns of nail microcirculation (129). In the eponychium and perionychium, the vascular villi followed the direction of nail growth. In the face of the eponychium in contact with the nail, a wide-mesh

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The venous drainage is achieved by two veins, one on each side of the nail plate, in the proximal nail folds (127). The capillary network is easily seen in the proximal nail fold with a magnifying lens and is seen in more detail with an ophthalmoscopic or capillary microscope. lt is essentially the same as the network of the skin, but the capillary loops are more horizontal and visible throughout their length. Certain diseases (e.g., connective tissue disorders, macroglobulinemia, cryoglobulinemia, psoriasis, antiphospolipid syndrome) can modify its normal structure and a simple clinical examination or use of widefield nailfold microscopy can be very useful as an aid in diagnosis (130,131,132,133). A special vascular formation is present in the distal zone of the nail bed: the glomus bodies. This vascular structure has the mission of regulating the peripheral temperature by means of arteriovenous shunts (134).

Nail Growth

The rate of growth of the nail plate has been studied extensively. Normal nail growth varies between 0.1 and 1.12 mm per day, or 1.9 to 4.4 mm per month (135,136). This growth, however, is not the same in all fingers or toes. For example, fingernails grow faster than toenails. Whereas a normal fingernail grows out completely in approximately six months, a normal toenail takes 12 to 18 months to do the same (83). Nails grow faster when regenerating after avulsion (39).

Several physiological circumstances can cause variations in the nail growth (Table 2.6). Nail growth is quicker in males (39), during the day, during pregnancy (137), in persons who bite their nails (43), and in summer or warm climates (138). Conversely, nails grow more slowly in females, during the night, in toes, in winter, after age 20 (139), and during lactation (137).

Nail growth is also altered in several diseases (Table 2.6) (140,141,142,143,144,145). Nails grow quicker with psoriasis (140), pityriasis rubra pilaris (94), etretinate treatment (141), and hyperthyroidism (39); nails grow slower in cases of immobilization or paralysis (142), local ischemic conditions (135), cytostatic therapy (39,83), denutrition (143), hypothyroidism (139), and yellow nail syndrome (144). In the case of a sudden decrease in nail growth, for example, in acute infections (136), a transverse band will appear afterward, depressed in the proximal line called Beau's line. However, patients with unilateral toenail onychomycosis did not support the hypothesis that slow nail growth rate is a predisposing factor for this onychomycosis (145). The absence of K1 and K10 from healthy nail bed may also be related to the adherence of the overlying nail. In simple terms, the nail plate might be interpreted as the suprabasal layer of the nail bed, containing keratins not produced by the nail bed but affording barrier function or some other properties associated with K1, K10, and Ha1. The presence of these overlying keratins might be responsible for the lack of a granular layer and associated absence of K1 and K10. In diseases such as onychomycosis and psoriasis, where nail plate adherence is lost, a granular layer forms alongside expression of K1 and K10. (99,100,101,145)

The rate of growth of the nail plate is determined by the turnover rate of the matrix cells. Shortly after death, matrix cells do not incorporate tritiated thymidine in their nuclei; the cells appear to be incapable of DNA synthesis and cell division, and, therefore, the nail does not grow (88). Previous reports of nail growth after death are, in fact, erroneous. Apparent growth, caused by severe postmortem drying and shrinking of the soft tissues around the nail plate, is what was observed (146).

The question of where the nail plate is formed is still controversial (147). The first theories at the beginning of the century pointed toward a complete formation by the matrix (148). Years later, Lewis (8), however, concluded that the nail plate was the product of three different matrices on the basis of staining of the nail plate with a silver-protein stain and the morphology of keratinizing cells. Lewis's hypothesis was supported by differential staining of the nail plate (8), by differential interference contrast microscopy (69,70,71,72), and by ultrastructural observation of keratohyalin granules in embryonic nail (69). Lewis's hypothesis, however, has been extensively reviewed. Zaias and Alvarez (88) used radioautography to show that the nail plate was formed exclusively by the matrix in normal conditions; Samman (149) and Norton (147) confirmed it by following the incorporation of H-labeled glycine and thymidine in

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Some recent authors believe that the nail bed epithelium produces a significant 20% portion of the nail plate and thus include the nail bed in the generative portion of the nail (151). Interestingly, indirect immunofluorescence studies in the fetal nail by means of pan-anti-type I and II hair keratin antibodies revealed a positive staining in a broad band of suprabasal cells of the nail bed epithelium. However, exclusion of hair keratins hHa1, hHb5, hHb1, hHb6 and hHa4 from the adult nail bed epithelium clearly identifies the matrix as the sole origin of the nail plate (152). It has previously been speculated that a few so-called horn cells may be added by the nail bed epithelium to the underside of the nail plate. Although our study confirmed the occurrence of clearly visible K5/17-positive nail bed cells in the lower nail plate, we believe that these cells represent sectioning artefacts. Our transverse nail sections, stained for either K5/17 or hHb5, show that the boundary between the nail bed epithelium and the nail plate is extremely undulated and that the resulting narrow-spaced folds and ridges exhibit a distinctly varying height. Therefore, it is evident that already slight deviations from a vertical angle of section through the nail bed region may reveal K5-positive cells in the nail plate, which in reality stem from the tip of an adjacent epithelial fold of the bed epithelium. Collectively these data emphasize that the nail bed epithelium does not actively contribute to the formation of the nail plate. (Figure 2.38).

An important controversy is why nails grow out instead of up. Kligman (153,154) postulated that the cul-de-sac of the proximal nail groove forced the cells of the matrix to grow out. To confirm his theory he transplanted nail matrix to the forearm, producing a vertical cylinder of hard keratin that had histologic characteristies of the nail. Hashimoto et al. (69) stated later that the long axis of matrix cells in embryonic nail was directed upward and distally.

Another important question is why the nail bed accompanies the nail plate in its growth. A well-known fact is that a hemorrhage that occurs between the plate and bed will grow forward with the plate. If the plate merely moved over the bed, the blood would not move; therefore, the upper part of the bed must move out with the plate. Some authors, such as Krantz (155), Kligman (153,154), and Zaias (84), tried to study this phenomenon in an experimental way. Of all theories, the one by Zaias is most acceptable at present: he believes that the proximal nail bed moves out, either by pressure by advancing plate or because of trauma, but that the distal nail bed and hyponychium do not move. Similarly

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Handling and Processing of the Nail

The major problems of processing the nail unit are the difficulty of tissue selection and the need for proper orientation of the specimen. These problems are the reason for the small number of histologic studies.

The first important point is how to take a biopsy of the nail unit (153,154,155,156,157,158,159,160,161,162,163,164,165). The best way of studying a biopsy of the nail is to ascertain that it includes the complete thickness of the nail unit (which means nail plate, bed, and subjacent dermis); these can be sectioned transversely. If a punch biopsy is taken, it should be done by boring firmly through the nail plate and into the underlying tissue to obtain a specimen with the plate attached. If one wishes to eliminate the nail plate before taking the biopsy, special care has to be taken about nail avulsion because if it has been avulsed without care, the epithelium of the bed or matrix may become separated and the undersurface may remain attached to the plate and distort the true histopathologic picture. The ideal biopsy technique for the nail is a longitudinal biopsy (159), which includes the hyponychium, the nail bed, matrix with overlying plate, and the proximal nail fold and cuticle. The specimen may be taken from the center of the nail apparatus or from the lateral edge (161,162), or it may be modified to provide elliptical excision of a tumor of the nail bed (163,164).

The second point is the orientation of the specimen for cutting. In all cases, the surgeon should alert the pathologist on the submission form as to the way the specimen was obtained, whether a particular orientation is needed, and whether a piece of the nail plate is included.

The third point is how to treat the specimen in the laboratory. If the nail plate is present in the specimen, it will be too hard for ready cutting with a microtome unless some method of softening is used. A special fixative of 5% trichloroacetic acid and 10% formalin will leave the plate softer (161). Alternatively, the specimen can be placed in distilled water for a few hours before placing in formalin (162). Another helpful technique for softening the hard keratin of the nail plate was proposed by Luna (165):

• Fix specimen in 10% formalin for 24 hours.

• Place specimens in the following solution until completely dekeratinized. Change solution every 2 days for best results.

  Mercuric chloride	4.0 g
  Chromic acid	0.5 g
  Nitric acid, concentrated	10.0 mL
  Ethyl alcohol, 95%	50.0 mL
  Distilled water	200.0 mL

• Wash in running water for three hours.

• Dehydrate, clear, and impregnate with paraffin, or process as desired.

With this technique, the preservation of the cytologic characteristics of the epithelial cells of the nail matrix and nail bed is not as good as with the previous one, but it is very helpful for softening the hardest nail specimens.

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