Largely unchanged for decades, treatment methods for this potentially disabling condition are finally evolving

Introduction

Our ability to treat adverse medical conditions has long been an admixture of miraculous accomplishment and frustrating stagnation. For every disease such as osteogenic sarcoma, where recent therapeutic breakthroughs have lifted cure rates from 10-20% to over 90%, there is a condition like pulmonary adenocarcinoma, where cure rates approach their historic controls. The management of dermatologic disorders has not offered any particular exception to this rule. There recently have been profound advances in the medical management of inflammatory conditions of the skin; however, many of those advances are the first since the advent of artificial corticosteroids several decades ago. Moreover, an effective means of medically managing some of the most aggressive neoplastic processes of skin remain elusive. For instance, the prognosis attributed to melanoma and Merkle cell tumor, once they’ve spread beyond their site of origin (stage 3 or 4), remains exceedingly poor.
The management of psoriasis has remained largely unchanged for decades. With a better understanding of the pathogenesis of this potentially disabling condition, investigators have begun targeting the precise factors that lead to its progression. This new focus has shown extraordinary promise for long term medical management, particularly in severe cases. In this report we will discuss the clinical findings, diagnosis, and evolving methods of treatment for psoriasis with special attention given to those cases that present themselves in the lower extremity.

Epidemiology

The overall prevalence of psoriasis approaches 2 percent internationally; however, it has been estimated to be as high as 4.6% in the United States.1 The prevalence in children is significantly lower, calculated at 0.5% in a large series of kids ages 12-16.2 The incidence in persons of color is lower than that in Caucasians, affecting roughly 0.7 percent of Africans and Asians.1 There is no particular gender predilection.1 The epidemiological features of palmoplantar (acral) psoriasis has remained somewhat obscure; however, in our experience, neither palmoplantar pustulosis nor the chronic plaque form of acral psoriasis can be considered rare in a podiatric practice, and both are certainly under-diagnosed. There is a much rarer form of pustular psoriasis which is limited to the digits and/or nails. This rare variant is designated “acrodermatitis continua”.

There are two peak ages for the development of psoriasis, the first being during early adulthood and the second being during the sixth decade of life.3 Cases manifesting in childhood are far more likely to be associated with HLA-Cw6 and a positive family history.4,5 Such patients also trend toward more severe forms of psoriasis with associated arthritis and nail involvement.6

Pathogenesis

The pathogenesis of psoriasis has been much studied and fairly well characterized; however, the precise inciting event in its initial development remains unclear.7 Psoriasis is fundamentally an autoimmune disease whereby CD4-positive, and to a lesser extent CD8-positive, T lymphocytes are stimulated to evoke a type-1 inflammatory response. Central to this response is the liberation of pro-inflammatory cytokines such as TNF-alpha which have the net result of producing local tissue damage and increasing the turnover rate of the epidermis. Where normal squamous epithelial cells require roughly 15 days to traverse from the basal layer to the stratum corneum, in affected epithelium, this rate is reduced to 1-3 days.8 This rapid turnover of affected squamous epithelium results in the silver scale that is characteristic of psoriasis. Due to the rapidity with which affected epidermis turns over, there is insufficient time for the formation of a normal stratum corneum. In some instances there may be a genetic component to the development of psoriasis. Its inheritance is in the form of a polygenic trait, resulting in the development of psoriasis in 14% of offspring when 1 parent has the disease, and 41% of offspring if both parents are affected.9 There are associations with various major histocompatibility complex phenotypes, particularly HLA B13, HLA B17, and HLA Cw6. The presence of HLA Cw6 antigens confers a relative risk of 13 for the development of psoriasis in the Caucasian population.10 A major gene involved in the development of psoriasis has recently been mapped to chromosome 6p21.3 and has been designated as PSORS1.11 Psoriasis may come and go without apparent reason; however, there are also numerous potential triggers. In many instances, psoriasis is distributed upon sites that are most affected by friction or pressure. This is the result of Koebner phenomenon, a term used to denote conditions that may be catalyzed by external trauma. Other triggers include alcohol ingestion, HIV infection, streptococcal pharyngitis (guttate variant of psoriasis), certain drugs (lithium, glucocorticoids, and beta-blockers), and emotional stress.12,13 Emotional stress plays a particularly important role as a trigger in children.12

Clinical Findings

In the most common form of psoriasis, well-delineated geographic plaques form upon sites that are predisposed to external trauma. This common form of psoriasis has been aptly designated as the chronic plaque form of psoriasis vulgaris. When following its classic pattern of distribution, the knees, elbows, sacral region, and scalp are affected. Focal involvement of the skin is not uncommon even in the chronic plaque form of psoriasis, where approximately 2/3’s of patients develop a limited and relatively mild expression of the disease.14

Chronic plaque form psoriasis is characteristically covered by “silver” scales which when removed produced small areas of pin-point bleeding (Auspitz sign). Such silver scales are less prominent on the palms and soles. When the volar surfaces of the hands and/or feet are involved, the condition is referred to as acral psoriasis. This form of psoriasis breaks with the pattern of distribution exhibited by routine psoriasis vulgaris. Acral psoriasis is unique in that it is limited to the volar surfaces of the hands and/or feet in 81% of cases.9 It is patients with this form of psoriasis that most commonly present themselves to podiatric clinicians. There are many potential nail manifestations in psoriasis. Such pathologic changes vary from the classically described pitting and “oil spots” to onycholysis and frank keratinizing nail dystrophy. Though one set of investigators estimated the frequency of nail involvement to be roughly 80%, the true rate of involvement is probably slightly lower.15 Such overestimation is probable because bona fide keratinizing psoriatic nail unit dystrophy may be clinically indistinguishable from onychomycosis, idiopathic onycholysis, and traumatically induced dystrophy. In the lower extremity, keratinizing nail unit dystrophy (resembling onychomycosis) (Figure 1) is much more likely to be identified than is pitting of the nail plate or “oil spots” (Figure 2).16

In addition to the chronic plaque-forming type of psoriasis, there are more eruptive or acute forms of psoriasis, descriptively designated as pustular and guttate (drop-like) psoriasis. Although these forms of psoriasis may precede or co-exist with chronic plaque psoriasis, due to their unique clinical presentations, they are by convention considered separately. The topic of acral psoriasis warrants separate discussion because of its tendency toward unusual clinical presentations, its recalcitrance to first line therapeutic modalities, and its common role as a diagnostic pitfall. Acral psoriasis includes those cases of psoriasis that predominantly involve the volar (non-hair-bearing) surfaces of the hands and/or feet. As aforementioned, in the overwhelming majority of cases, persons with this variant of psoriasis lack skin involvement beyond the acral surfaces. While chronic plaque form psoriasis may affect the feet in its classic form (Figure 3a and 3b), acral plaques are often less likely to demonstrate the characteristic silver scales that are typical of chronic plaque psoriasis when it arises in non-acral sites (Figure 4a and 4b).

In some cases, acral psoriasis presents as a diffuse keratoderma with thick gray keratotic plaques (Figure 5). Alternatively, acral plaques may be poorly demarcated with erythematous fine scale (Figure 6), or might present as round coinshaped lesions that are reminiscent of nummular dermatitis (Figure 7). Regarding distribution, some cases may not only involve, but are also limited to, the forefeet (Figure 8).

In many instances, psoriasis will consist of only one or a few discrete plaques, while in others it involves the entire plantar surface. There may, or may not, be associated nail involvement and such nail involvement may be seen in isolation (Figure 9).

The differential diagnosis is limited when chronic plaque psoriasis vulgaris presents in its classic distribution; however, because the acral variant may lack many of the distinguishing features for psoriasis (characteristic distribution, silver scale, well-demarcated plaques, Auspitz sign), it may be confused with a wide variety of dermatological conditions. Focal presentation of the chronic plaque form of psoriasis may be confused with conditions as disparate as eczematous (nummular) dermatitis, chronic allergic contact dermatitis, lichen simplex chronicus, or squamous cell carcinoma in-situ (Bowen’s disease). Acral psoriasis may also be mistaken for chronic T. rubrum dermatophytosis, acral lichen planus, pityriasis rubra pilaris, mycosis fungoides, or chronic dyshidrotic dermatitis. Finally, the pustular form of acral pustular psoriasis is commonly misdiagnosed as tinea pedis, resulting in the institution of errant anti-fungal treatment regimens for extended periods of time.

Diagnosis

There is no diagnostic dilemma when psoriasis presents with all its classic clinical features. Well demarcated plaques with silver scale over extensor surfaces may be considered psoriasis until proven otherwise.

Other helpful clinical clues to the diagnosis of psoriasis are Auspitz sign (pinpoint bleeds upon the removal of silver scales) and Koebner phenomenon (psoriatic lesions following the path of external trauma). Unfortunately, acral psoriasis usually lacks the classic topographic distribution that is so often attributed to chronic plaque psoriasis. In addition, acral plaques often fail to exhibit the clinical features that allow them to be easily classified as psoriatic in nature. A potentially helpful sign, particularly when dealing with acral lesions, is the presence of individual lesions in various stages of development, e.g., the presence of acute lesions (pustules), subacute lesions (resolving pustules), and chronic plaques in the same individual.

When the diagnosis of psoriasis is in doubt, biopsies may be indicated. The technique of choice for the diagnosis of psoriasis and virtually all other inflammatory conditions of skin is punch biopsy. On the shoe-covered skin of the foot, many clinicians prefer to perform two 2mm punches biopsies rather than 1 large punch. This technique
allows for more rapid wound healing, better sampling, and reduced risk of infection. From a histopathologic perspective, performing two small punches allows for better sampling of the lesion in question. Sampling surface keratin alone will allow clinicians to rule out dermatophytosis, and may provide histopathologic features that are sugges tive of psoriasis; however, it rarely provides for a definitive diagnosis.

Treatment

Since the dawn of modern medicine, breakthroughs in the medical management of psoriasis have been few and far between. Advancements were delayed for centuries because of widespread confusion between psoriasis vulgaris and leprosy. Psoriatic patients were often expelled from society along with persons harboring Mycobacteria leprae infections in an errant attempt to ward off disease transmission. It wasn’t until the nineteenth century that these conditions were distinguished from each other, thereby allowing the development of effective therapeutic modalities for psoriasis. The oldest legitimate treatment for psoriasis was sunlight (ultra violet light). Many subsequent attempts at therapy were developed and subsequently abandoned during the 19th, 20th and early 21st centuries. As late as 1925 many purely nonsensical treatments were advocated for the treatment of psoriasis; amongst them: intramuscular mercury or bacterial antigen injections, non-protein diets, tonsil removal, and tooth extractions! 17 Despite the frightening nature of some of these early therapeutic approaches, others such as salicylic acid, coal tar, and ultra violet light (UVB) remain in use today.

When designing a treatment regimen for an affected patient, several factors must be taken into account. The clinical extent of involvement, the patient’s perception of that involvement, the subtype of psoriasis, the impact that some therapeutic modalities will have on lifestyle, and the potential side effects that medication might have.

The lower extremities may be affected by either non-acral chronic plaque form psoriasis, or acral (palmoplantar)
psoriasis. For the clinician, the significance of subtype relates to the potential for anatomic progression, the degree to which the condition will be debilitating, and its tendency toward recalcitrance. Although acral psoriasis
(cases limited to the non-hair-bearing surfaces of the sole and digits) may remain limited with regard to its anatomic scope, this variant may be extremely disabling and recalcitrant to traditional first line therapeutic
approaches. Chronic plaque form psoriasis is often less disabling, though more widespread, and tends to be more responsive to first line therapy.

Topical Corticosteroids

As a general rule, when less than 5% of the body surface area is affected by newly diagnosed psoriasis, the first line therapy should be limited to topical modalities. The possible exception would be for cases where the condition has already proven debilitating. For decades, the first line treatment of choice for limited psoriasis has been topical corticosteroids.18

Steroids of medium to suprahighpotency (Stoughten-Cornell classification 4, 5, 6, 7) are most commonly used for limited lower extremity psoriasis, either alone, or in combination with additional topical agents. Although there is technically a potential for iatrogenic Cushings syndrome after extended courses, the expression of such is rare and typically minimal. The same is true for the development of hypothalamic-pituitaryadrenal axis suppression and significant cases are usually limited to small children due to their low relative body surface area.19 Side effects such as skin atrophy and fragility are much more common complications of topical steroid therapy. Similar to other potential complications, clinically significant cases usually require long term use; in addition, those effects are most prominent on the steroid-sensitive skin of the face and intertriginous areas.18 Newer products such as fluticasone propionate have demonstrated great promise in further reducing the risk of adverse side effects. 20 Tachyphylaxis (decreased effectiveness with continued use) is a common problem with topical high potency corticosteroids; however, its presentation may be delayed or diminished by defaulting to a pulse-dosing regimen after an initial 2-3 course of b.i.d. applications. Such pulse-dosing may be accomplished by limiting applications to 3 application times over a 24 hour period once weekly.21 An alternate method of pulse dosing involves twice daily application for two consecutive days followed by 5 days off. In addition to the emergence of improved corticosteroid formulations, new vehicles such as gels and foams are gaining broad acceptance.

Vitamin D3 Analogues

Vitamin D3 analogues were introduced into the market for the treatment of psoriasis relatively recently, but have quickly joined corticosteroids as a first line therapy for the treatment of limited psoriasis. Within affected skin, vitamin D3 analogues inhibit epidermal proliferation, promote normal stratum corneum development, and inhibit neutrophil and monocyte/ histiocyte activity.22 Possibly the best known and most studied vitamin D3 analogue is calcipotriol  (Dovonex); however, other examples include calcitriol (Silkis) and tacalcitol (Curatoderem). Topical calcipotriol has been shown to be effective for the treatment of limited chronic plaque form psoriasis, including the palmoplantar variant. The efficacy of the vitamin D3 analogues is roughly comparable to that of a class 2 corticosteroid such as betamethasone dipropionate.23
Dosing for calcipotriol (50ug/g) is twice daily; however, night-time application twice weekly under occlusion has been shown to be equally effective in the treatment of palmoplantar psoriasis.24 The most significant clinical improvement is usually seen in the initial 6-8 weeks after initiating therapy.

Corticosteroid—Vitamin D Analogue Combinations

Recently, formulations combining high potency corticosteroids and vitamin D analogues have been shown to be of clinical benefit. One such formulation combines betamethasone dipropionate and calcipotriol (Dovobet®). Such products are typically applied topically once daily and their long-term use has been shown to have a high safety
profile (see below).

Topical Retinoids

Tazarotene is a topical retinoid that was recently approved for the treatment of psoriasis. This product is available in 0.05% and 0.1% concentrations with a gel or cream base. Similar to vitamin D analogues, tazarotene lacks the side effects that are associated with long term corticosteroid therapy; however, this product has its own local side effects, in particular, irritation at the treatment site. This irritation may be managed by using combination therapy with a topical corticosteroid (see below). Due to the combination of its efficacy and its local side effects, tazarotene is usually considered a form of second line therapy in the management of limited psoriasis.

Roughly half the patients using the 0.05% or 0.1% gel describe a 50% improvement in their symptoms after 6 weeks.25 Tazarotene gel has proven more effective than fluocinonide 0.05% cream.26

Combination Regimens Using Topical Corticosteroids

As previously alluded to, corticosteroids are not simply effective as a form of monotherapy; they are widely used in combination with, or as an adjunct to, other topical medications. When used in combination with a keratolytic agent such as 5-10% salicylic acid or 40-50% urea, the therapeutic effects of the steroid are potentiated. In addition, corticosteroids decrease irritation that may be caused by salicylic acid. Corticosteroids also calm the local irritation that might be seen in association with retinoids and vitamin D3 analogues. In one study, improved efficacy and decreased irritation was noted when combining either tazarotene with a class IV (mometasone furoate 0.1% cream) or class II (fluocinonide 0.05% cream).27 The application of tazarotene 0.1% gel on Mondays, Wednesdays, and Fridays in alternation with clobetasol propionate ointment (class I) on Tuesdays and Thursdays, has led to the remission of psoriasis in 73% of patients with psoriasis.28 The combination of calcipotriol (vitamin D3 analogue) and betamethasone dipropionate (class I corticosteroid) has also been shown to be safe and effective.29

Ultra Violet Light and PUVA

Ultra violet light may be used in the treatment of psoriasis as either ultraviolet B phototherapy or photochemotherapy using UVA in combination with systemic or topical psoralen. Soaking affected feet in baths containing 8-methoxypsoralen followed by immediate UVA irradiation has shown to be particularly beneficial for the treatment of palmoplantar psoriasis while limiting side effects.30 The use of a topical cream containing 8- methoxypsoralen followed by UVA irradiation has proven equally effective in at least one small series.31 A recent publication noted the effectiveness of light emitted from a xenon-chloride excimer laser (308 nm) in the management of palmoplantar psoriasis.32

Systemic Therapeutic Modalities

Many of the traditional forms of systemic therapy for severe psoriasis have remained unchanged—namely, Methotrexate and Cyclosporine remain staples in the management of patients with extensive disease. One new class of drugs that has shown notable promise for the management of psoriasis has been designated as the “biologics”. The biologic agents act at the cellular level to target specific processes that are involved in the development of a particular disease. Because their mechanisms of action are specific they tend not to have the widespread side effects that are associated with more globally acting agents such as corticosteroids.

The three principle forms of biologics are monoclonal antibodies, fusion proteins, and recombinant cytokines or growth factors.33
The generic names of the monoclonal antibodies and fusion proteins have been standardized to reflect their mechanism of action. For instance, the generic names of monoclonal antibodies have the suffix -mab, and receptor–antibody fusion proteins have the suffixcept.34 Biologic agents have one or more of 4 principle mechanisms of action. They 1) reduce the number of pathogenic T-cells (alefacept), 2) inhibit T-cell activation or migration (efalizumab), 3) modulate the immune system (Ilodecakin), or 4) block the activity of pro-inflammatory cytokines such as tumor necrosis factor (etanercept).34

The prototypical T-cell reducing agent is alefacept, which, as its name implies, is a protein fusion product combining a T-cell binding site (Lymphocyte Function Antigen- 3) and sequences from the constant region of IgG’s heavy chain. This agent functions by binding with CD2, which is an antigen that is found on the surface of T-cell lymphocytes, particularly memory effector T-cells.35 After binding, this agent induces cell death through apoptosis.35 Current dosing regimens call for consecutive weekly intramuscular injections of 15mg alefacept for 12 weeks followed by 12 weeks of rest. At the conclusion of the rest period, a second course may be considered. A baseline CD4+ lymphocyte count should be performed prior to the initiation of therapy and then biweekly thereafter. In one study, 57% of patients experienced at least a 50% reduction in the Psoriasis Area Severity Index (PASI). This agent has also shown to provide a durable remission. Seventy four percent of those patients that experience >50% reduction in PASI, maintain that such for the 12 week rest period.35

The second strategy for biologic agents used in the treatment of psoriasis involves the inhibition of Tcell activation and migration. Efalizumab is an antibody that is directed against T-cell surface antigen CD11a (a subunit of Lymphocyte Function Antigen-1). Lymphocyte function antigen-1 (LFA-1) functions in T-cell activation and T-cell adhesion to keratinocytes and vascular endothelium.36 By interfering with the function of LFA-1, efalizumab inhibits both T-cell activation and the migration of T-cells into affected skin. The treatment regimen for efalizumab consists of the subcutaneous injection of 0.7mg/kg for the initial dose followed by 1mg/kg per week thereafter.

Higher dosage regimens and extended treatment periods are currently under  investigation. Initial results suggest added benefit with successive treatment cycles and higher dosages of 2mg/kg.37 The effectiveness of efalizumab appears to be slightly shy of that achieved by alefacept with a similar tendency toward durable remissions. The third mechanism by which biologic agents may influence the development and/or progression of psoriasis is through immune system modulation. Immune system modulation may be achieved by guiding the differentiation of T-cells toward forms that play a less pathogenic role in the development of psoriasis. In general, lymphocytes may follow one of two paths of differentiation. Lymphocytes that are CD4+ may differentiate into either Th1 or Th2 phenotype and CD8+ lymphocytes may differentiate into either Tc1 or Tc2 phenotype. Lymphocytes may be pushed toward the Th1 and Tc1 phenotypes through the influence of cytokines Il-12 and INF-gamma or toward the Th2 and Tc2 phenotypes through the modulatory effects of IL-4, IL-6, and IL-10. Psoriasis has been shown to be driven by the cytokines that are liberated by Th1 and Tc1 phenotypes.38 Chief amongst those instigating cytokines are IFN-gamma, IL- 2, and TNF-alpha.38

Interleukin-10 is a type 2 cytokine which is under-expressed in psoriatic patients. This substance actively
opposes the type 1 cytokine response; therefore it could conceivably curtail the progression of psoriasis by reducing the liberation of cytokines IFN-gamma, IL-2, and TNFalpha. Subcutaneous injections of recombinant human IL-10 (Ilodecakin) have been shown to prolong remissions when administered in combination with other products39;however, the effectiveness of this cytokine as a stand-alone therapeutic agent in the treatment of psoriasis has thus far been disappointing. 40 Oprelvekin is a human recombinant IL-11 that has shown initial promise when administered daily in subcutaneous injections.41 This cytokine also diminishes the type 1 cytokine response; however, it further acts by decreasing both intraepidermal T-cells and keratinocyte adhesion molecules that facilitate the action of T-cells on affected epidermis. 41 Studies are currently ongoing to further assess the usefulness of these agents.The final, and possibly most promising, class of biologics for the treatment of psoriasis includes those that directly or indirectly block the activity of type-1 inflammatory cytokines. A chief target of this class of biologics is tumor necrosis factor-alpha (TNF-alpha). Tumor necrosis factor-alpha is a type-1 cytokine that functions as a potent mediator of inflammation and keratinocyte hyperproliferation. This factor plays a pivotal role in the development and escalation of psoriasis due to its excess production and liberation by T-cell lymphocytes, keratinocytes, and mast cells.42 In theory, reducing the activity of this cytokine should impede the pathogenesis of psoriasis. Etanercept (Enbrel) is a recombinant TNF-alpha receptor which is fused with the fc portion of IgG1. This product binds with TNF-alpha on cell membranes, thereby neutralizing it. This drug is administered weekly by subcutaneous injection in 25mg or 50mg doses. Investigators have shown that higher dosages and longer therapeutic regimens provide further benefit.43 In one study nearly 50% of all patients experienced either “complete clearing” or “almost complete clearing” of their lesions after 12 weeks of biweekly 50mg injections compared to 5% in the placebo group.44 An added benefit of etanercept therapy is the durability of disease remissions in those that respond favorably. The median time until relapse (<50% PASI) was 85 days with 25% of patients not experiencing relapse for 141 days.
Infliximab (Remicade) is an additional agent that inhibits the function of TNF-alpha; however, unlike etenercept, infliximab is a monoclonal antibody. Infliximab is a composite human-mouse antibody that targets TNF-alpha directly by binding it on the surface of cells. This agent is administered intravenously at 2-4 week intervals in a concentration of 5mg/kg. As many as 80% of patients with moderate to severe psoriasis can expect to have a 75% reduction in PASI and most will experience such benefits within 4 weeks.45
As a group, the biologics are extremely new agents and thus their safety profiles remain in flux. Injection site complications are the most common adverse effects associated with etanercept and tend to diminish with ongoing therapy.46 Another potential complication is an increased risk of infection, particularly upper respiratory infections. Chronic fatigue, liver toxicity, leukocytopenia and lymphopenia are rarely reported complications.46

Summary

Psoriasis is a chronic autoimmune condition of skin which may have unconventional features when arising on the acral surfaces of the hands and/or feet. Due to its less specific appearance when arising on the skin of the feet, misdiagnoses are common, potentially resulting in inappropriate treatment. First line therapy in the treatment of limited psoriasis remains topical corticosteroids; however, there is increasing use of Vitamin D3 analogues in this context. Combination therapy using both corticosteroids and Vitamin D3 analogues or corticosteroids and retinoids have been proven affective as a second line therapy. Advances in our understanding of the athogenesis of psoriasis has allowed for the introduction of a new class of effective therapeutic agents designated as the “biologics”. These medications exert their effects by directly inhibiting the function or production of the cytokines that are responsible for the development and propagation of psoriasis. Further long-term studies are needed; however, in initial research the biologics have demonstrated tremendous promise for the treatment of moderate to severe psoriasis, including debilitating variants such as that which affects the acral surfaces.

Largely unchanged for decades, treatment methods for this potentially disabling condition are finally evolving.

This is a technique for the evaluation of small fiber peripheral neuropathy

Introduction

Epidermal nerve fiber density (ENFD) testing is not a new technique; however, it is new to the diagnostic armamentarium of many in the podiatric community. This analysis is quickly becoming recognized as the gold standard among clinicians when assessing for the presence, and the degree, of small fiber peripheral neuropathy.

Though podiatric clinicians have only recently discovered this test, it has been an important diagnostic procedure for neurologists for many years. In fact, ENFD analysis has been widely used by neurologists in the United States and in Europe, to qualify and quantify small fiber peripheral neuropathy since the 1990’s.

In the following article we will discuss the meaning of “epidermal nerves” and small fiberperipheral neuropathy. We will further elaborate upon the definition of ENFD analysis, its evolution as a testing modality, its advantages over other tests, and technical aspects regarding the technique itself. Emphasis will be placed upon the feafeatures that make this test unique, and its potential uses in podiatric medicine.

What are “Epidermal Nerve Fibers”?

Peripheral nerves may be classified according to their function,  their size, or their conduction velocity. In most instances, nerve size is directly related to conduction velocity. Nerves that have been classified based on their diameters and conduction velocities have been given specific designations. The largest myelinated peripheral nerves have been designated as A-alpha and A-beta nerves, while mediumsized myelinated nerves are, by convention, labeled A-gamma nerves. The most terminal end branches of sensory nerves represent the smallest peripheral nerves. These nerves are exceedingly  small, being composed of only a few axons bundled together. By convention, these “small fibers”  have been designated “A-delta” and “C” fibers, and when they terminate in the epidermis, they are called epidermal nerves (Table 1).

The majority of epidermal nerves are unmyelinated C-fibers. There is a much smaller subset of epidermal nerves, which possess a myelin sheath. This minor population of epidermal nerves is referred to as the “A-delta fibers”. As nerves course more superficially from the reticular dermis to the papillary dermis, and then on to the epidermis, they become progressively smaller (Figure 1).

The C and A-delta fibers branch off of small nerve fascicles that course within the papillary dermis. After emerging from larger branches within the papillary dermis, these small nerve fibers run toward the skin surface, eventually entering into the viable epidermis (Figure 2).

As aforementioned, once small fibers enter into the surface epithelium, they are designated as “epidermal nerve fibers”, or more appropriately , “intra-epidermal nerve fibers.”

Paul Langerhans postulated the presence of intra-epidermal nerves a century ago; however, their presence could not be substantiated until the advent of electron microscopy in the early 1970’s. Though electron microscopy showed that intra-epidermal nerves genuinely existed, because ultra-structural examination is most effective when analyzing tissue fragments measuring only 1mm in diameter and only a few microns in thickness, this science is of limited value when trying to calculate an average density across a relatively large area of skin. The calculation of intra-epidermal nerve fiber density over a large area became possible 20 years later with the emergence of immune-histochemistry. Investigators at Johns Hopkins Medical Center performed much of the research that led to the perfection of this technique. As alluded to above, intra-epidermal nerve fibers cannot be readily visualized in routine stainedsections; rather, due to their extremely small size, they require special staining techniques. A single Epidermal nerve fiber consists of a mere 1-3 axons that are bound together with scant myelin. They have been shown to play a role in nociception (pain), temperature perception, and autonomic regulation. As they course through the epidermis, intra-epidermal fibers are thought to interact with poorly understood intra-epidermal neuro-endocrine cells known as “Merkel cells”. Roughly 90% of all intra-epidermal nerve fibers are unmyelinated Cfibers, and the remaining 10% are myelinated A-delta fibers.

In normal subjects, intra-epidermal nerves course between the squamous epithelial cells of the epidermis toward the skin surface. This intercellular path gives them a less than linear  ppearance as they pass superficially (Figure 3).

The number of intra-epidermal nerves per a unit area of skin is termed the “epidermal nerve density.” This density is relatively constant throughout life, and between genders; however, it varies widely depending on the anatomic site being tested. In general, the skin’s average fiber density decreases as one moves further from the dorsal root ganglia. In other words, the average intra-epidermal nerve fiber density is higher at the trunk than it is at the thigh, and the density in the skin of the thigh will be higher than in the skin of the distal leg.

Because the normative range of epidermal fiber density varies depending on the anatomic location, to accurately assess the meaning of the epidermal nerve fiber density at any particular site, there must be an established/published normative value for that distinct site. This is one reason that the distal leg (10cm proximal to the lateral malleolus) and proximal thigh (10cm distal to the greater trochanter of the femur) are the most commonly sampled sites. There is a massive amount of data defining the normative ranges in these locations.

What Is Small Fiber Peripheral Neuropathy?

For research purposes, intra-epidermal nerves may be locally eliminated, or “knocked out”, using exposure to low-energy shock waves or topical capsaicin. A more widespread loss of intra-epidermal nerves, leading to bonafide small fiber neuropathy, may be iatrogenic-induced through treatment with some chemotherapeutic agents. Small fiber neuropathy may arise as an occupational hazard secondary to solvent exposure or persistent vibratory forces. The latter of these commonly affects the hands of jackhammer operators and the feet of those who spend extended periods of time operating heavy equipment. Chronic alcohol abuse may precipitate small fiber peripheral neuropathy, as might systemic amyloidosis and some forms of vasculitis. Finally, patients infected with HIV commonly develop small fiber peripheral neuropathy, sometimes quite early in the onset of AIDS (Table 2).

The term “peripheral neuropathy” often has been long used as a waste-basket diagnosis into which all forms of symptomatic peripheral neuropathy have been thrown. Peripheral neuropathy is actually not a single condition, but rather, it is a descriptive term connoting any disease state, which at least in part. compromises the function of the peripheral nervous system. Peripheral neuropathy may manifest in various patterns, and may arise as the result of a wide spectrum of predisposing conditions. Small fiber peripheral neuropathy represents a large and distinct subset of the cases of peripheral neuropathy. Patients with pure small fiber peripheral neuropathy exhibit demonstrable pathology that is limited to the “small fibers” (A-delta and C fibers). This form of neuropathy may be focal, but more often involves peripheral nerves in a length-dependent pattern, meaning that the earliest and most severely affected nerves are those that are furthest away from the dorsal root ganglia. This length-dependent pattern of nerve fiber involvement gives rise to its characteristic stocking and/or glove-like distribution.

Most cases of peripheral neuropathy that are seen in a podiatric practice are of the small fiber variety; however, in some instances, large fibers may be foremost affected. It is the large fibers that are affected in patients with compression neuropathy and in demyelinating neuropathies. Even some forms of diabetic neuropathy may foremost involve large nerve fibers, as in large fiber mononeuropathy or poly-neuropathy. These large fiber neuropathies may not initially exhibit an associated small fiber component. It is the function of large fibers, not small fibers, that is tested by nerve conduction studies. This explains why some patients with frank clinical evidence of peripheral neuropathy may display normal conduction studies. Such patients suffer from pure small fiber peripheral neuropathy.

Small fiber peripheral neuropathy may be caused by a wide array of medical conditions or exposures. It has been long known that the most common causes are diabetes mellitus (types I and II) and idiopathic; however, the significance of each has been somewhat in flux in recent years. Investigators have recently revealed that many patients who were formerly thought to have idiopathic small fiber neuropathy may actually suffer from metabolic syndrome, e.g. “pre-diabetic” persons with an altered glucose metabolism.

Epidermal Nerve Fiber Density Analysis

How are epidermal nerve fibers visualized?

As aforementioned, intra-epidermal nerve fiber density analysis usually uses the science of immunehistochemistry. Immuno-histochemical studies take advantage of the fact that the cells which make up the human body have on their surfaces antigens (usually proteins) that are relatively specific for that particular cell type. To wit, endothelial cells express an antigen designated as CD31, which is somewhat specific for the cells that line vessels, and melanocytes express a relatively specific antigen called S100 protein. It just so happens that nerves possess a particular antigen on them that has been designated as PGP 9.5. This antigen provides a specific target on intraepidermal nerves that can be used for the purpose of identification and subsequent analysis. As their name implies, immune-histochemical studies take advantage of the specific binding of antibodies (“immuno”) to identify a particular antigenic target. For intra-epidermal nerve density analysis, a small amount of PGP 9.5 is introduced into the blood of a rabbit. As a physiologic response to the presence of the foreign antigen, the rabbit will form antibodies that are highly specific for PGP 9.5. These new antibodies are then retrieved from the rabbit serum for laboratory use.

For intra-epidermal nerve fiber density analysis, clinicians most commonly perform one or two 3mm punch biopsies of skin. Once in the lab, the punch biopsy is sliced into 50μm thick sections, and each is placed in its own well within a testing tray. The rabbit-derived antibodies are then applied to each of the tissue sections. The PGP 9.5-specific rabbit antibodies will identify nerve fibers that are present within the tissue sections. The final step consists of the application of goat-derived anti-rabbit antibodies and pigment. Many goatderived anti-rabbit antibodies will bind to each of the previously applied PGP 9.5-specific rabbit antibodies. As the antibodies continue to bind, they will form a dense coat of antibody and pigment around each intra-epidermal nerve fiber. The net result is that even the tiniest nerve fibers will become visible using light microscopy.

How are epidermal nerve fiber morphology and density assessed?

Once the fibers can be visualized, the next steps are to discern the health of the fibers by reviewing their structure and integrity, and to calculate their “density” within the epidermis. To assess nerve morphology (structure), several parameters are reviewed. Signs of degeneration include features as disparate as excessive branching, nerve thinness, nerve segmentation, poor staining, fiber varicosities, and axonal swellings (Figure 4).

When quantifying the intra-epidermal nerve fibers, first all the fibers are counted across five randomly selected 50 micron-thick tissue sections using 400X magnification. The number of intra-epidermal nerves from each representativesection is summed up. To obtain nerve density, the breadth of the epidermal surface of each crosssection is measured using image analysis software. By dividing the number of nerves by the length of the epidermal surface (in millimeters), an average density is established. The density is then expressed as the number of nerve fibers per millimeter (fibers/mm). Once the density falls below a “normal” threshold, the patient is said to have small fiber neuropathy, which may be quantified depending on the severity (Figure 5).

It is important to note that there are several different methods of counting intra-epidermal nerves. Some labs count only those nerves that are seen crossing the epidermal basement membrane. Some investigators have found that a better counting method involves adding both those nerves that are seen crossing the basement membrane and those that are noted higher within the epidermis. This is the method used at institutions such as Johns Hopkins Medical Center and Bako Pathology Services.

Much less commonly-used techniques employ software in an attempt to create three-dimensional reproductions of the tissue to be tested. The particular manner of counting employed by a lab is of great importance because the disparate methods are not interchangeable. Each technique will elicit its own normative curve in any anatomic location. In other words, a normal count using one method might be >4 fibers/mm, but for an alternate lab (using a different counting method), the normal threshold might be >7 fibers/mm. For this reason, some consistency is warranted when monitoring a particular patient, particularly when looking for small changes secondary to oral therapy.

How does epidermal nerve fiber density analysis differ from routine pathology?

In addition to the method of staining that is used (immune-histochemistry versus routine stains), ENFD differs from routine anatomic pathology in many other ways. Whereas routine biopsies are fixed in formalin, tissue taken for ENFD analysis is not. In fact, formalin inhibits the binding of antibodies to PGP 9.5, thereby rendering the tissue useless. Biopsies taken for ENFD analysis are fixed in Zamboni’s fixative or PLP (the pros and cons of these fixatives will be discussed later). An additional difference between tissue prepared for ENFD testing, and that destined for routine pathology, is that punch biopsies being prepared for ENFD analysis are frozen and then cut into 50 micron-thick sections; in contrast, neuropatissues taken for routine pathology are dehydrated and then infused/embedded in paraffin wax so that they may be cut into 3-5 micronthick slices. Due to the greater complexity of the technique itself, from the time of receipt until the completion of slide preparation, ENFD analysis will take at least three days, whereas routine histopathology slides may be prepared in several hours.

What are the ideal biopsy sites and why?

For a particular epidermal nerve fiber density quantification to have clinical significance, there must be an established normative range at the location where that sample was taken. Thankfully, potential variables such as gender and age have little influence on fiber density between individuals. The most significant single variable among normal subjects is anatomic location. This means that for a biopsy from any particular anatomic location to be useful, there must be an established normative range from that site. By far, the most studied single anatomic location on the human body is the calf at 10cm proximal to the lateral malleolus (Figure 6), followed by the proximal thigh at 10 cm distal to the greater trochanter of the femur. Normative values have been established at these locations using a variety of analytical methods.

To ensure that punches are not damaged when being picked up, only atraumatic forceps should be used.

Small fiber peripheral neuropatissues thy is a length-dependent process; meaning distal anatomic sites are affected more severely than more proximal locations. For this reason, there is an advantage to taking biopsies from both the proximal thigh and distal leg, when possible. The distal biopsy is most important, offering clinicians an excellent reflection of the extent of the disease process at any point in time. It is there that the fiber density has optimal significance, and where the test is most sensitive.

In contrast, because small fiber neuropathy is length-dependent, biopsies obtained from more proximal sites may be expected to be less severely affected than distal samples in bona-fide small fiber neuropathy. In this vein, an abnormal distal biopsy and a normal proximal biopsy is evidence of length dependence, and therefore further militates in favor of small fiber neuropathy.

There is no published evidence that biopsies taken from cutaneous sites which are near to, or adjacent to, each other (such as the lower calf and dorsal foot) establish length dependence. In fact, in our experience, the second punch rarely offers enough additional information to justify the expense. Arguably, more information might be derived from performing bilateral punches. We have found that small fiber neuropathy is not always a symmetrical process, as evidenced by density values that are often not identical.

Additional sites where normal values are either being studied or pending publication are the skin posterior to the fibula, at the level of the talar dome, and over the dorsum of the foot at the level of the fourth metatarsal-cuboid articulation.

How are punch biopsies for the purpose of epidermal nerve fiber density testing obtained?

Punch biopsies taken for epidermal nerve fiber density testing are taken in a manner similar to those obtained to assess other conditions of skin with four important differences:

1) biopsies must be performed at one of a few specific anatomic locations,

2) as a standard, 3mm punch biopsies are used,

3) special care must be taken not to crush or distort the surface epithelium,

4) the biopsy is NOT fixed in formalin.

With regard to punch size, the overwhelming majority of the research on the subject has used 3mm punches. For the purposes of the test, larger punches also may be used; however, because such biopsies may require sutures and carry with them a higher complication rate, they are difficult to justify. It is primarily with differences 3 & 4 that, in our experience, clinicians may run into trouble, because in each case, if mismanaged, the very integrity of the analysis may be compromised.

When performing punch biopsies for the purpose of epidermal nerve fiber density analysis, it is important to avoid introducing artifact into the specimen, which might artificially alter the nerve fiber density. To avoid such artifacts, the punch blade should cut through the skin by rotating the punch back and forth between the thumb and index finger. The punch should never be forced through the skin exclusively using vertical pressure; rather, only slight downward pressure should be applied. Forcing the punch down through the skin will create a mushroom-shaped biopsy and may result in crush artifact along the biopsy’s peripheral edges. Such an artifact will likely decrease the resultant nerve fiber density.

An additional source of crush artifact is secondary to rough handling when samples are removed from the biopsy site. To ensure that punches are not damaged when being picked up, only atraumatic forceps should be used. In most instances, such forceps are included within the biopsy transport kit. The surface epidermis should never be cross-clamped when handling the biopsy. To avoid handling the surface epithelium, the sample should be gripped by the dermal soft tissue, rather than the surface epithelium. To help accomplish this, after the skin has been punched and the punch blade has been removed, the opposing limbs of the forceps may be pressed down on the skin on either side of the biopsy site. Pushing down on the surrounding skin in this manner will cause the punch itself to rise out relative to the adjacent skin. When the sample rises above the surrounding skin, the underlying dermis is exposed. It is this deeper “beefy” tissue which should be grasped with the forceps. Once the deep tissue has been grasped, the sample may be lifted out and the connective tissue base may be cut using curved scissors. An on-line demonstration of this biopsy technique may be viewed at www.bakopathology.com.

How are biopsy specimens fixated for epidermal nerve fiber density testing?

Almost all tissue that is obtained for pathologic analysis is placed in formalin; however, there are three notable exceptions, namely: tissue taken for microbiologic culture (kept fresh), tissue taken for crystal analysis due to suspected gout (placed in dehydrated alcohol), and punches taken for epidermal nerve fiber density analysis (placed in Zamboni’s fixative or PLP). Although formalin is an excellent fixative for routine anatomic pathology specimens, in tissue that is destined for epidermal nerve fiber density testing, it binds with the PGP 9.5 antigen on the surface of neurons and prevents the attachment of the anti-PGP 9.5 antibody. This lack of antibody-antigen binding will result in an erroneously diminished number of epidermal nerve fibers, even in normal subjects, and possibly lead to a falsepositive diagnosis of small fiber neuropathy.

There are two common fixatives that may be used to prepare tissue for epidermal nerve fiber analysis: PLP and Zamboni’s fixative. Each method of fixation has its advantages and disadvantages. The first fixative to be used for epidermal nerve fiber density analysis was PLP. This is an excellent fixative, but has its disadvantages. The principle disadvantages of PLP are that it has limited shelf life and must be kept refrigerated. Over the course of a few weeks following its preparation, the constituents of PLP will precipitate out, leaving the formulation ineffective for tissue fixation. This means that the kit cannot be ordered until a patient is actually scheduled for a procedure. If for any reason there is a significant delay before performing the biopsy, following the receipt of the biopsy kit, new PLP must be requested. In contrast to PLP, Zamboni’s fixative is stable at room temperature and has a much longer shelf life. When using PLP, medical practices may require a dedicated refrigerator to house their biopsy kits. This is virtually never a requirement when using Zamboni’s fixative. In addition, delays prior to performing the procedure are rarely problematic because of its long shelf life. A potential drawback to the use of Zamboni’s fixative is that it is a mild acid. Because of its corrosive properties, the biopsy specimen can only be exposed to Zamboni’s for a limited amount of time. For sufficient fixation, the specimen should be submerged in Zamboni’s fixative for at least six to eight hours; however, the analysis is best performed when the exposure time is less than 24 hours. Ideally, the biopsy should be placed in Zamboni’s fixative overnight, and then a quick rinsing step is performed early the following morning. This rinsing step removes and neutralizes the Zamboni’s fixative; it is simple to do, and takes a staff member no more than two minutes.

To perform the rinsing step, the vial containing the biopsy and Zamboni’s fixative is opened at the beginning of the day following the biopsy. The yellow Zamboni’s fixative is then poured off (leaving the skin sample in the vial). The initial vial, which should now contain only the biopsy, is then filled with phosphate buffer. The phosphate buffer is then poured off, again leaving the skin sample in its original vial. To be sure to remove or neutralize all Zamboni’s fixative, the tissue is again rinsed with phosphate buffer. Finally the vial containing the rinsed skin sample is filled with a cryoprotectant to protect the specimen during transportation. This rinsing step will ensure that the specimen is safe, even if there are short delays during shipping. Again, this biopsy rinse and transfer technique is easy to do and takes only a moment. A demonstration may be viewed online at www.bakopathology.com.

How is the biopsy for epidermal nerve fiber density handled/shipped?

Most labs that perform epidermal nerve fiber density analysis supply the materials that are essential for obtaining and shipping the sample. A basic transport kit will contain a sterile barrier, sterile punch, curved scissor, alcohol and Betadine wipes, a transport cooler and cool-pack, and prepaid overnight shipping labels. Prior to performing the biopsy procedure, the cool pack should be placed in a freezer for use during return shipping (particularly important during Jasummer months). When Zamboni’s fixative is used, the sample is fixed overnight in that fixative, then rinsed as describe above. The biopsy is then packed for shipping.

When packing the specimen, place the cool-pack into the cooler first, cover with the Styrofoam divider, and then insert the vial containing the biopsy. If Zamboni’s fixative is used, and the rinse and transfer step is deferred, clinicians must ensure that the specimen is received at the lab with 24 hours. Moreover, the sample must be received during that lab’s hours of operation so that the rinse and transfer step can be performed by laboratory personnel within the 24- hour exposure period. To minimize the chance of over-exposure, when biopsies are to be shipped in Zamboni’s fixative, they should be obtained during the afternoon hours, and then shipped to the lab on the same day for delivery the following morning.

What are the indications for epidermal nerve fiber density?

The full utility of epidermal nerve fiber density analysis in the management of our diabetic patient populations is only now becoming appreciated. Though for much of the last two decades this test has been used mostly in the context of research, it has now become a highly specific, and sensitive, method to qualify and quantify small fiber peripheral neuropathy. In fact, this test itself has played a crucial role in the research that has allowed this pattern of peripheral neuropathy to be characterized.

There are multiple angles from which epidermal nerve fiber density (ENFD) analysis will allow us to approach the management of our diabetic patients: namely, as a confirmatory diagnostic tool, as a prospective or predictive tool, and as a tool to gauge the effectiveness of medical management. The most obvious use for this examination is to definitively diagnose suspected small fiber peripheral neuropathy, particularly when a predisposing condition is not apparent.

For years, many of us in the podiatric profession have lumped all patients with burning, tingling, or numbness into a single wastebasket diagnosis, e.g., “peripheral neuropathy.” By not defining the precise pattern of neuropathy, we made it impossible to assess the effectiveness of emerging therapies on specific patient populations. This can be likened to treating every bacterial infection with penicillin and then believing the antibiotic is useless because, in some instances, it doesn’t work. By precisely characterizing the form of neuropathy affecting our patients, we can look at and judge specific therapeutic modalities in their appropriate light.

Perhaps the most interesting use of this test, and the most intriguing facet of its potential future use, involves its role as a predictive tool in patients who are at risk, or who are exceedingly early in the development of small fiber peripheral neuropathy. Because ENFD analysis may reveal degenerative changes that precede an actual drop of nerve fiber density, and in some cases a decrease in epidermal nerve fiber density may precede the symptoms of neuropathy, this test may in time become a standard means of determining which patients should be placed on preventive medication prior to their development of overt symptomatology. This approach has the potential to curb the number of patients who eventually become neuropathic.

Like most testing methods, the specificity of ENFD analysis increases when the results are nearer to the extremes of the bell curve.

Finally, ENFD analysis is widely used to monitor the effectiveness of various therapeutic modalities in diabetic (and non-diabetic) patients. In recent years, we have seen several prolific public speakers in podiatry, such as Allen Jacobs, DPM, Mackie Walker, DPM,

and Lawrence Didominico, DPM, lecture on this topic. Epidermal nerve fiber density analysis allows us to document an objective baseline prior to the initiation of therapy. We can then repeat the exam after a specific interval (6-12 months) to assess disease progression, or alternatively, disease regression. It is this manner of use that is allowing for the development and refinement of specific therapeutic options for small fiber peripheral neuropathy. It is also here that the podiatry profession can play a major, if not pivotal, role in ongoing clinical and pharmaceutical research.

What is the specificity and sensitivity of epidermal nerve fiber density analysis?

Like most testing methods, the specificity of ENFD analysis increases when the results are nearer to the extremes of the bell curve. For instance, the threshold for what should be considered a “low” ENFD, using the Bako method of counting, is 7.1 epidermal nerves fibers per millimeter. At this threshold (the 10th percentile), the specificity of ENFD is about 90%; however, at 3.8 fibers per millimeter (the 5th percentile), the specificity rises to 97%. The sensitivity of ENFD analysis is roughly 70% if no effort is made to screen out cases where there is large fiber involvement; however, if a simple tuning fork is used to assess vibratory sensation (diminished sensation is indicative of large fiber involvement), the sensitivity can be increased to roughly 90%.

What are the advantages of epidermal nerve fiber density analysis over other testing methods?

The principle advantages of ENFD over other testing methods are three-fold. Foremost, ENFD is an objective analysis, meaning it is not affected by the inherent flaws that plague subjective tests such as

Semmes-Weinstein monofilaments.  Secondly, ENFD analyses exhibit high sensitivity and specificity when specifically assessing for the presence of small fiber peripheral suspectneuropathy. Many common testing methodologies, such as nerve conduction studies, measure predominantly large fiber abnormalities, and have little relation to small fiber disease. The same may also be said for sural nerve biopsy. Finally, many of the available testing methods are not readily available in an office setting; rather, they mandate that the patient report to a major academic center for testing. The biopsy used for epidermal nerve fiber density analysis may be obtained in a few minutes in an office setting.

Conclusion

In summary, epidermal nerve fiber density is a test which allows for the diagnosis of small fiber peripheral neuropathy in an objective manner, based on the analysis of a common punch of skin. The biopsy technique has subtle differences from a standard punch biopsy, some of which are crucial to the integrity of the test, namely, the biopsy size, site, handling, and fixation. This test has proven to be highly specific and sufficiently sensitive in the diagnosis of small fiber neuropathy, and an ideal method for monitoring the disease process in patients who are undergoing medical management. An additional use as a predictive modality shows great potential and will certainly be a subject of future research.