epidermalnervefiber densitytesting: anoverview · august 2010 • podiatry management 143 not...

IntroductionEpidermal nerve fiber density

(ENFD) testing is not a new tech-nique; however, it is new to the di-agnostic armamentarium of manyin the podiatric community. Thisanalysis is quickly becoming recog-nized as the gold standard amongclinicians when assessing for thepresence, and thedegree, of smallfiber peripheraln e u r o p a t h y .Though podiatricclinicians haveonly recently dis-covered this test,it has been an im-portant diagnos-tic procedure forneurologists formany years. Infact, ENFD analy-sis has been wide-ly used by neurol-ogists in the Unit-ed States and inEurope, to qualifyand quantify small fiber peripheralneuropathy since the 1990’s.

In the following article we willdiscuss the meaning of “epidermalnerves” and small fiber peripheralneuropathy. We will further elabo-rate upon the definition of ENFDanalysis, its evolution as a testingmodality, its advantages overother tests, and technical aspectsregarding the technique itself. Em-phasis will be placed upon the fea-

tures that make this test unique,and its potential uses in podiatricmedicine.

What are “Epidermal NerveFibers”?

Peripheral nerves may be classi-fied according to their function,their size, or their conduction veloc-ity. In most instances, nerve size isdirectly related to conduction veloc-

ity. Nerves thathave been classi-fied based on theirdiameters andconduction veloc-ities have beengiven specific des-ignations. Thelargest myelinatedperipheral nerveshave been desig-nated as A-alphaand A-beta nerves,while medium-sized myelinatednerves are, byconvention, la-beled A-gammanerves. The most

terminal end branches of sensorynerves represent the smallest periph-eral nerves. These nerves are exceed-ingly small, being composed of onlya few axons bundled together. Byconvention, these “small fibers”have been designated “A-delta” and“C” fibers, and when they terminatein the epidermis, they are called epi-dermal nerves (Table 1).

The majority of epidermalnerves are unmyelinated C-fibers.

There is a much smaller subset ofepidermal nerves, which possess amyelin sheath. This minor popula-tion of epidermal nerves is referredto as the “A-delta fibers”. As nervescourse more superficially from the

By Bradley W. Bakotic, DPM, DO

Continued on page 142

Epidermal Nerve FiberDensity Testing:An OverviewHere’s a technique for the evaluation of small fiber peripheral neuropathy.

AUGUST 2010 • PODIATRY MANAGEMENTwww.podiatrym.com 141

Figure 1: A photomicrograph of normalskin, demonstrating its constituent lay-ers. The reticular dermis has beenmarked with the letters RD, the papil-lary dermis has been annotated withPD, and the epidermis has been anno-tated with E. (50X)

Figure 2: A photomicrograph demon-strating a normal epidermal nerve fiberdensity test. A-delta and C-fibers areseen coursing from larger branches ofthe papillary dermis, into the overlyingepidermis. (400X)

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.

tissue fragments measuring only1mm in diameter and only a fewmicrons in thickness, this science isof limited value when trying to cal-culate an average density across arelatively large area of skin. The cal-culation of intra-epidermal nervefiber density over a large area be-came possible 20 years later withthe emergence of immune-histo-chemistry. Investigators at JohnsHopkins Medical Center performedmuch of the research that led to

the perfection ofthis technique.

As alluded toabove, intra-epi-dermal nervefibers cannot bereadily visualizedin routine stainedsections; rather,due to their ex-tremely smallsize, they requirespecial stainingtechniques. A sin-gle epidermal

nerve fiber consists of a mere 1-3axons that are bound together withscant myelin. They have beenshown to play a role in nociception(pain), temperature perception, andautonomic regulation. As theycourse through the epidermis,intra-epidermal fibers are thoughtto interact with poorly understoodintra-epidermal neuro-endocrine

reticular dermis to the papillarydermis, and then on to the epider-mis, they become progressivelysmaller (Figure 1).

The C and A-delta fibers branchoff of small nerve fascicles thatcourse within the papillary dermis.After emerging from larger branch-es within the papillary dermis,these small nerve fibers run towardthe skin surface, eventually enter-ing into the vi-able epidermis(Figure 2). Asaforementioned,once small fibersenter into thesurface epitheli-um, they are des-ignated as “epi-dermal nervefibers”, or moreapp rop r i a t e l y ,“intra-epidermalnerve fibers.”

Paul Langer-hans postulated the presence ofintra-epidermal nerves a centuryago; however, their presence couldnot be substantiated until the ad-vent of electron microscopy in theearly 1970’s. Though electron mi-croscopy showed that intra-epider-mal nerves genuinely existed, be-cause ultra-structural examinationis most effective when analyzing

cells known as “Merkel cells”.Roughly 90% of all intra-epidermalnerve fibers are unmyelinated C-fibers, and the remaining 10% aremyelinated A-delta fibers.

In normal subjects, intra-epider-mal nerves course between thesquamous epithelial cells of the epi-dermis toward the skin surface.This intercellular path gives them aless than linear appearance as theypass superficially (Figure 3). Thenumber of intra-epidermal nervesper a unit area of skin is termed the“epidermal nerve density.” Thisdensity is relatively constantthroughout life, and between gen-ders; however, it varies widely de-pending on the anatomic site beingtested. In general, the skin’s averagefiber density decreases as onemoves further from the dorsal rootganglia. In other words, the average

intra-epidermal nerve fiberdensity is higher at thetrunk than it is at the thigh,and the density in the skinof the thigh will be higherthan in the skin of the distalleg.

Because the normativerange of epidermal fiberdensity varies depending onthe anatomic location, toaccurately assess the mean-ing of the epidermal nervefiber density at any particu-lar site, there must be an es-tablished/published norma-tive value for that distinctsite. This is one reason thatthe distal leg (10cm proxi-mal to the lateral malleolus)and proximal thigh (10cmdistal to the greatertrochanter of the femur) arethe most commonly sam-

Epidermal...


142 www.podiatrym.comPODIATRY MANAGEMENT • AUGUST 2010

The number

of intra-epidermal

nerves per a unit area

of skin is termed the

“epidermal nerve

density”.

TABLE 1

Nerves Classified bySize/Conduction Velocity

NERVE FIBERS DESIGNATION FUNCTION

Large myelinated nerves A-alpha Proprioception,A-beta vibratory sensation,

motor

Medium myelinated A-gamma Motornerves

Small Myelinated A-delta Pain, temperature,fibers autonomics

Small unmyelinated C-fibers Pain, temperature,fibers autonomics

Figure 3: A photomicrograph demon-strating the course of an epidermalnerve coursing between epidermal squa-mous cells. (1000X)


not initially exhibit an as-sociated small fiber com-ponent. It is the functionof large fibers, not smallfibers, that is tested bynerve conduction studies.This explains why somepatients with frank clini-cal evidence of peripheralneuropathy may displaynormal conduction stud-ies. Such patients sufferfrom pure small fiber pe-ripheral neuropathy.

Small fiber peripheralneuropathy may becaused by a wide array ofmedical conditions or ex-posures. It has been longknown that the mostcommon causes are dia-betes mellitus (types I andII) and idiopathic; howev-er, the significance ofeach has been somewhatin flux in recent years. Investigatorshave recently revealed that manypatients who were formerlythought to have idiopathic smallfiber neuropathy may actually suf-fer from metabolic syndrome, e.g.“pre-diabetic” persons with an al-tered glucose metabolism.

For research purposes, intra-epi-dermal nerves may be locally elimi-

nated, or “knockedout”, using expo-sure to low-energyshock waves ortopical capsaicin.A more widespreadloss of intra-epi-dermal nerves,leading to bona-fide small fiberneuropathy, maybe iatrogenic-in-duced throughtreatment withsome chemothera-peutic agents.

Small fiber neuropathy may arise asan occupational hazard secondary tosolvent exposure or persistent vibra-tory forces. The latter of these com-monly affects the hands of jackham-mer operators and the feet of thosewho spend extended periods of timeoperating heavy equipment. Chronicalcohol abuse may precipitate smallfiber peripheral neuropathy, as mightsystemic amyloidosis and some formsof vasculitis. Finally, patients infected

pled sites. There is a massiveamount of data defining the nor-mative ranges in these locations.

What Is Small FiberPeripheral Neuropathy?

The term “peripheral neuropa-thy” often has been long used as awaste-basket diagnosis into whichall forms of symptomatic peripheralneuropathy have been thrown. Pe-ripheral neuropathy is actually nota single condition, but rather, it is adescriptive term connoting any dis-ease state, which at least in part.compromises the function of theperipheral nervous system. Periph-eral neuropathy may manifest invarious patterns, and may arise asthe result of a wide spectrum ofpredisposing conditions. Small fiberperipheral neuropathy represents alarge and distinct subset of thecases of peripheral neuropathy. Pa-tients with pure small fiber periph-eral neuropathy exhibit demonstra-ble pathology that is limited to the“small fibers” (A-delta and Cfibers). This form of neuropathymay be focal, but more often in-volves peripheral nerves in alength-dependent pattern, meaningthat the earliest and most severelyaffected nervesare those that arefurthest awayfrom the dorsalroot ganglia. Thislength-dependentpattern of nervefiber involvementgives rise to itsc h a r a c t e r i s t i cstocking and/orglove-like distri-bution.

Most cases ofperipheral neu-ropathy that areseen in a podiatric practice are ofthe small fiber variety; however, insome instances, large fibers may beforemost affected. It is the largefibers that are affected in patientswith compression neuropathy andin demyelinating neuropathies.Even some forms of diabetic neu-ropathy may foremost involve largenerve fibers, as in large fiber mono-neuropathy or poly-neuropathy.These large fiber neuropathies may

with HIV commonly develop smallfiber peripheral neuropathy, some-times quite early in the onset of AIDS(Table 2).

Epidermal Nerve Fiber DensityAnalysis

How are epidermal nerve fibersvisualized?

As aforementioned, intra-epi-dermal nerve fiber density analysisusually uses the science of immune-histochemistry. Immuno-histo-chemical studies take advantage ofthe fact that the cells which makeup the human body have on theirsurfaces antigens (usually proteins)that are relatively specific for thatparticular cell type. To wit, en-dothelial cells express an antigendesignated as CD31, which is some-what specific for the cells that linevessels, and melanocytes express arelatively specific antigen calledS100 protein. It just so happensthat nerves possess a particularantigen on them that has been des-ignated as PGP 9.5. This antigenprovides a specific target on intra-epidermal nerves that can be usedfor the purpose of identificationand subsequent analysis. As theirname implies, immune-histochemi-cal studies take advantage of thespecific binding of antibodies (“im-muno”) to identify a particular

Epidermal...


For intra-epidermal

nerve fiber density

analysis, clinicians

most commonly

perform one

or two 3mm punch

biopsies of skin.

TABLE 2

Some of the MostCommon Causes of

Small Fiber PeripheralNeuropathy

Diabetes Mellitus, Types I and IIMetabolic syndromeIdiopathic/familialAlcohol abuseHuman Immunodeficiency virusAmyloidosisPharmacologic toxins (metronidazole)Solvent exposureChemotherapy-inducedLeprosyVasculitisAutoimmune (various disorders)

bind to each of the previously ap-plied PGP 9.5-specific rabbit anti-bodies. As the antibodies continueto bind, they will form a dense coatof antibody and pigment aroundeach intra-epidermal nerve fiber.The net result is that even the tini-est nerve fiberswill become visi-ble using lightmicroscopy.

How are epider-mal nerve fiberm o r p h o l o g yand density as-sessed?

Once thefibers can be visu-alized, the nextsteps are to dis-cern the health ofthe fibers by re-viewing their structure and integri-ty, and to calculate their “density”within the epidermis. To assessnerve morphology (structure), sev-eral parameters are reviewed. Signsof degeneration include features asdisparate as excessive branching,nerve thinness, nerve segmenta-tion, poor staining, fiber varicosi-ties, and axonal swellings (Figure4). When quantifying the intra-epi-dermal nerve fibers, first all thefibers are counted across five ran-domly selected 50 micron-thick tis-sue sections using 400X magnifica-tion. The number of intra-epider-mal nerves from each representa-tive section is summed up. To ob-tain nerve density, the breadth ofthe epidermal surface of each cross-section is measured using imageanalysis software. By dividing thenumber of nerves by the length ofthe epidermal surface (in millime-ters), an average density is estab-lished. The density is then ex-pressed as the number of nervefibers per millimeter (fibers/mm).Once the density falls below a “nor-mal” threshold, the patient is saidto have small fiber neuropathy,which may be quantified depend-ing on the severity (Figure 5).

It is important to note thatthere are several different methodsof counting intra-epidermal nerves.Some labs count only those nervesthat are seen crossing the epidermalbasement membrane. Some investi-gators have found that a better

antigenic target. For intra-epider-mal nerve density analysis, a smallamount of PGP 9.5 is introducedinto the blood of a rabbit. As aphysiologic response to the pres-ence of the foreign antigen, therabbit will form antibodies that arehighly specific for PGP 9.5. Thesenew antibodies are then retrievedfrom the rabbit serum for laborato-ry use.

For intra-epidermal nerve fiberdensity analysis, clinicians mostcommonly perform one or two3mm punch biopsies of skin. Oncein the lab, the punch biopsy issliced into 50µm thick sections,and each is placed in its own wellwithin a testing tray. The rabbit-de-rived antibodies are then applied toeach of the tissue sections. The PGP9.5-specific rabbit antibodies willidentify nerve fibers that are pre-sent within the tissue sections. Thefinal step consists of the applica-tion of goat-derived anti-rabbit an-tibodies and pigment. Many goat-derived anti-rabbit antibodies will

counting method involves addingboth those nerves that are seencrossing the basement membraneand those that are noted higherwithin the epidermis. This is themethod used at institutions such asJohns Hopkins Medical Center and

Bako PathologyServices.

Much lesscommonly-usedtechniques em-ploy software inan attempt to cre-ate three-dimen-sional reproduc-tions of the tissueto be tested. Theparticular mannerof counting em-ployed by a lab isof great impor-tance because the

disparate methods are not inter-changeable. Each technique willelicit its own normative curve inany anatomic location. In otherwords, a normal count using onemethod might be >4 fibers/mm,but for an alternate lab (using a dif-ferent counting method), the nor-mal threshold might be >7fibers/mm. For this reason, someconsistency is warranted whenmonitoring a particular patient,particularly when looking for smallchanges secondary to oral therapy.

How does epidermal nerve fiberdensity analysis differ fromroutine pathology?

In addition to the method ofstaining that is used (immune-his-tochemistry versus routine stains),ENFD differs from routine anatom-ic pathology in many other ways.Whereas routine biopsies are fixedin formalin, tissue taken for ENFDanalysis is not. In fact, formalin in-hibits the binding of antibodies toPGP 9.5, thereby rendering the tis-sue useless. Biopsies taken for ENFDanalysis are fixed in Zamboni’s fixa-tive or PLP (the pros and cons ofthese fixatives will be discussedlater). An additional difference be-tween tissue prepared for ENFDtesting, and that destined for rou-tine pathology, is that punch biop-sies being prepared for ENFD analy-sis are frozen and then cut into 50micron-thick sections; in contrast,

Epidermal...



By far, the most

studied single

anatomic location

on the human body

is the calf at 10cm

proximal to

the lateral malleolus.

Figure 4: High power magnification,demonstrating large dilatations of epi-dermal nerves. These axonal swellingsare part of the degeneration processwithin diseased epidermal nerves.

Figure 5: High power magnificationshowing several attenuated and seg-mented epidermal nerve fibers. Thetotal count was within the moderatelyneuropathic range.

particular anatomic location to beuseful, there must be an establishednormative range from that site. Byfar, the most studied singleanatomic location on the humanbody is the calf at 10cm proximalto the lateral malleolus (Figure 6),

followed by the proximal thigh at10 cm distal to the greatertrochanter of the femur. Normativevalues have been established atthese locations using a variety ofanalytical methods.

Small fiber peripheral neuropa-

tissues taken for routine pathologyare dehydrated and then in-fused/embedded in paraffin wax sothat they may be cut into 3-5 mi-cron-thick slices. Due to the greatercomplexity of the technique itself,from the time of receipt until thecompletion of slide preparation,ENFD analysis will take at leastthree days, whereas routine histo-pathology slides may be preparedin several hours.

What are the ideal biopsy sitesand why?

For a particular epidermal nervefiber density quantification to haveclinical significance, there must bean established normative range atthe location where that sample wastaken. Thankfully, potential vari-ables such as gender and age havelittle influence on fiber density be-tween individuals. The most signifi-cant single variable among normalsubjects is anatomic location. Thismeans that for a biopsy from any

thy is a length-dependent process;meaning distal anatomic sites areaffected more severely than moreproximal locations. For this reason,there is an advantage to takingbiopsies from both the proximalthigh and distal leg, when possible.The distal biopsy is most impor-tant, offering clinicians an excel-lent reflection of the extent of thedisease process at any point intime. It is there that the fiber densi-ty has optimal significance, andwhere the test is most sensitive.

In contrast, because small fiberneuropathy is length-dependent,biopsies obtained from more proxi-mal sites may be expected to be lessseverely affected than distal sam-ples in bona-fide small fiber neu-ropathy. In this vein, an abnormaldistal biopsy and a normal proxi-mal biopsy is evidence of length de-pendence, and therefore furthermilitates in favor of small fiber neu-ropathy.

There is no published evidencethat biopsies taken from cutaneous

Epidermal...



To ensure that

punches are not

damaged when being

picked up, only

atraumatic forceps

should be used.


sites which are near to, or adjacentto, each other (such as the lowercalf and dorsal foot) establishlength dependence. In fact, in ourexperience, the second punchrarely offers enough additional in-formation to justify the expense.Arguably, more information mightbe derived from performing bilater-al punches. We have found thatsmall fiber neuropathy is not al-

ways a symmetrical process, as evi-denced by density values that areoften not identical.

Additional sites where normalvalues are either being studied orpending publication are the skinposterior to the fibula, at the level ofthe talar dome, and over the dorsumof the foot at the level of the fourthmetatarsal-cuboid articulation.

How are punch biopsies for thepurpose of epidermal nervefiber density testing obtained?

Punch biopsies taken for epider-mal nerve fiber density testing aretaken in a manner similar to thoseobtained to assess other conditionsof skin with four important differ-ences: 1) biopsies must be per-formed at one of a few specificanatomic locations, 2) as a stan-dard, 3mm punch biopsies areused, 3) special care must be takennot to crush or distort the surfaceepithelium, 4) the biopsy is NOTfixed in formalin.

With regard to punch size, theoverwhelming majority of the re-search on the subject has used3mm punches. For the purposes ofthe test, larger punches also may beused; however, because such biop-

Epidermal... sies may require suturesand carry with them ahigher complicationrate, they are difficultto justify. It is primarilywith differences 3 & 4that, in our experience,clinicians may run intotrouble, because ineach case, if misman-aged, the very integrityof the analysis may becompromised.

W h e nperformingp u n c hbiopsies forthe purposeof epidermal nerve fiberdensity analysis, it isimportant to avoid in-troducing artifact intothe specimen, whichmight artificially alterthe nerve fiber density.To avoid such artifacts,the punch blade shouldcut through the skin byrotating the punch back

and forth between the thumb andindex finger. The punch shouldnever be forced through the skinexclusively using vertical pressure;rather, only slight downward pres-sure should be applied. Forcing thepunch down through the skin willcreate a mushroom-shaped biopsyand may result in crush artifactalong the biopsy’s peripheral edges.

Continued on page 148Figure 6: The ideal anatomic location for epidermal nervefiber density testing, 10cm above the lateral malleolus.

Figure 7: A normative value will be published soon for asite within the scope of practice of all podiatric clinicians(at the level of the talar dome, posterior to the fibula).

This lack of antibody-antigen bind-ing will result in an erroneously di-minished number of epidermalnerve fibers, even in normal sub-jects, and possibly lead to a false-positive diagnosis of small fiberneuropathy.

There are two common fixativesthat may be used to prepare tissuefor epidermal nerve fiber analysis:PLP and Zamboni’s fixative. Eachmethod of fixation has its advan-tages and disadvantages. The firstfixative to be used for epidermalnerve fiber density analysis wasPLP. This is an excellent fixative,but has its disadvantages. The prin-ciple disadvantages of PLP are thatit has limited shelf life and must bekept refrigerated. Over the course ofa few weeks following its prepara-

tion, the constituents of PLP willprecipitate out, leaving the formu-lation ineffective for tissue fixation.This means that the kit cannot beordered until a patient is actuallyscheduled for a procedure. If forany reason there is a significantdelay before performing the biopsy,following the receipt of the biopsykit, new PLP must be requested. Incontrast to PLP, Zamboni’s fixativeis stable at room temperature andhas a much longer shelf life. Whenusing PLP, medical practices mayrequire a dedicated refrigerator tohouse their biopsy kits. This is vir-tually never a requirement whenusing Zamboni’s fixative. In addi-tion, delays prior to performing theprocedure are rarely problematicbecause of its long shelf life. A po-tential drawback to the use of Zam-boni’s fixative is that it is a mildacid. Because of its corrosive prop-

Such an artifact will likely decreasethe resultant nerve fiber density.

An additional source of crushartifact is secondary to rough han-dling when samples are removedfrom the biopsy site. To ensure thatpunches are not damaged whenbeing picked up, only atraumaticforceps should be used. In most in-stances, such forceps are includedwithin the biopsy transport kit. Thesurface epidermis should never becross-clamped when handling thebiopsy. To avoid handling the sur-face epithelium, the sample shouldbe gripped by the dermal soft tis-sue, rather than the surface epithe-lium. To help accomplish this, afterthe skin has been punched and thepunch blade has been removed, theopposing limbs of the forceps maybe pressed down on the skin on ei-ther side of the biopsy site. Pushingdown on the surrounding skin inthis manner will cause the punchitself to rise out relative to the adja-cent skin. When the sample risesabove the surrounding skin, theunderlying dermis is exposed. It isthis deeper “beefy” tissue whichshould be grasped with the forceps.Once the deep tissue has beengrasped, the sample may be liftedout and the connective tissue basemay be cut using curved scissors.An on-line demonstration of thisbiopsy technique may be viewed atwww.bakopathology.com.

How are biopsy specimensfixated for epidermal nervefiber density testing?

Almost all tissue that is ob-tained for pathologic analysis isplaced in formalin; however, thereare three notable exceptions, name-ly: tissue taken for microbiologicculture (kept fresh), tissue taken forcrystal analysis due to suspectedgout (placed in dehydrated alco-hol), and punches taken for epider-mal nerve fiber density analysis(placed in Zamboni’s fixative orPLP). Although formalin is an ex-cellent fixative for routine anatom-ic pathology specimens, in tissuethat is destined for epidermal nervefiber density testing, it binds withthe PGP 9.5 antigen on the surfaceof neurons and prevents the attach-ment of the anti-PGP 9.5 antibody.

erties, the biopsy specimen canonly be exposed to Zamboni’s for alimited amount of time. For suffi-cient fixation, the specimen shouldbe submerged in Zamboni’s fixativefor at least six to eight hours; how-ever, the analysis is best performedwhen the exposure time is less than24 hours. Ideally, the biopsy shouldbe placed in Zamboni’s fixativeovernight, and then a quick rinsingstep is performed early the follow-ing morning. This rinsing step re-moves and neutralizes the Zam-boni’s fixative; it is simple to do,and takes a staff member no morethan two minutes.

To perform the rinsing step, thevial containing the biopsy andZamboni’s fixative is opened at thebeginning of the day following thebiopsy. The yellow Zamboni’s fixa-tive is then poured off (leaving theskin sample in the vial). The initialvial, which should now containonly the biopsy, is then filled withphosphate buffer. The phosphatebuffer is then poured off, againleaving the skin sample in its origi-nal vial. To be sure to remove orneutralize all Zamboni’s fixative,the tissue is again rinsed with phos-phate buffer. Finally the vial con-taining the rinsed skin sample isfilled with a cryoprotectant to pro-tect the specimen during trans-portation. This rinsing step will en-sure that the specimen is safe, evenif there are short delays duringshipping. Again, this biopsy rinseand transfer technique is easy to doand takes only a moment. Ademonstration may be viewed on-line at www.bakopathology.com.

How is the biopsy for epidermalnerve fiber density handled/shipped?

Most labs that perform epider-mal nerve fiber density analysissupply the materials that are essen-tial for obtaining and shipping thesample. A basic transport kit willcontain a sterile barrier, sterilepunch, curved scissor, alcohol andBetadine wipes, a transport coolerand cool-pack, and prepaidovernight shipping labels. Prior toperforming the biopsy procedure,the cool pack should be placed in afreezer for use during return ship-ping (particularly important during

Epidermal...



The full utility of

epidermal nerve fiber

density analysis in the

management of our

diabetic patient

populations is

only now becoming

appreciated.

patients with burning, tingling, ornumbness into a single wastebasketdiagnosis, e.g., “peripheral neu-ropathy.” By not defining the pre-cise pattern of neuropathy, wemade it impossible to assess the ef-fectiveness of emerging therapieson specific patient populations.This can be likened to treatingevery bacterial infection with peni-cillin and then believing the antibi-otic is useless because, in some in-stances, it doesn’t work. By precise-ly characterizing the form of neu-ropathy affecting our patients, wecan look at and judge specific ther-apeutic modalities in their appro-priate light.

Perhaps the most interestinguse of this test, and the most in-triguing facet of its potential futureuse, involves its role as a predictivetool in patients who are at risk, or

who are exceedingly early in thedevelopment of small fiber periph-eral neuropathy. Because ENFDanalysis may reveal degenerativechanges that precede an actualdrop of nerve fiber density, and insome cases a decrease in epidermalnerve fiber density may precede thesymptoms of neuropathy, this testmay in time become a standardmeans of determining which pa-tients should be placed on preven-tive medication prior to their devel-opment of overt symptomatology.This approach has the potential tocurb the number of patients whoeventually become neuropathic.

Finally, ENFD analysis is widelyused to monitor the effectivenessof various therapeutic modalitiesin diabetic (and non-diabetic) pa-tients. In recent years, we haveseen several prolific public speak-ers in podiatry, such as Allen Ja-

summer months). When Zamboni’sfixative is used, the sample is fixedovernight in that fixative, thenrinsed as describe above. The biop-sy is then packed for shipping.

When packing the specimen,place the cool-pack into the coolerfirst, cover with the Styrofoam di-vider, and then insert the vial con-taining the biopsy. If Zamboni’sfixative is used, and the rinse andtransfer step is deferred, cliniciansmust ensure that the specimen isreceived at the lab with 24 hours.Moreover, the sample must be re-ceived during that lab’s hours ofoperation so that the rinse andtransfer step can be performed bylaboratory personnel within the 24-hour exposure period. To minimizethe chance of over-exposure, whenbiopsies are to be shipped in Zam-boni’s fixative, they should be ob-tained during the afternoon hours,and then shipped to the lab on thesame day for delivery the followingmorning.

What are the indications forepidermal nerve fiber density?

The full utility of epidermalnerve fiber density analysis in themanagement of our diabetic patientpopulations is only now becomingappreciated. Though for much ofthe last two decades this test hasbeen used mostly in the context ofresearch, it has now become a high-ly specific, and sensitive, method toqualify and quantify small fiber pe-ripheral neuropathy. In fact, thistest itself has played a crucial role inthe research that has allowed thispattern of peripheral neuropathy tobe characterized.

There are multiple angles fromwhich epidermal nerve fiber densi-ty (ENFD) analysis will allow us toapproach the management of ourdiabetic patients: namely, as a con-firmatory diagnostic tool, as aprospective or predictive tool, andas a tool to gauge the effectivenessof medical management. The mostobvious use for this examination isto definitively diagnose suspectedsmall fiber peripheral neuropathy,particularly when a predisposingcondition is not apparent.

For years, many of us in the po-diatric profession have lumped all

cobs, DPM, Mackie Walker, DPM,and Lawrence Didominico, DPM,lecture on this topic. Epidermalnerve fiber density analysis allowsus to document an objective base-line prior to the initiation of thera-py. We can then repeat the examafter a specific interval (6-12months) to assess disease progres-sion, or alternatively, disease re-gression. It is this manner of usethat is allowing for the develop-ment and refinement of specifictherapeutic options for small fiberperipheral neuropathy. It is alsohere that the podiatry professioncan play a major, if not pivotal,role in ongoing clinical and phar-maceutical research.

What is the specificity and sen-sitivity of epidermal nerve fiberdensity analysis?

Like most testing methods, thespecificity of ENFD analysis increas-es when the results are nearer tothe extremes of the bell curve. Forinstance, the threshold for whatshould be considered a “low”ENFD, using the Bako method ofcounting, is 7.1 epidermal nervesfibers per millimeter. At this thresh-old (the 10th percentile), the speci-ficity of ENFD is about 90%; how-ever, at 3.8 fibers per millimeter(the 5th percentile), the specificityrises to 97%. The sensitivity ofENFD analysis is roughly 70% if noeffort is made to screen out caseswhere there is large fiber involve-ment; however, if a simple tuningfork is used to assess vibratory sen-sation (diminished sensation is in-dicative of large fiber involvement),the sensitivity can be increased toroughly 90%.

What are the advantages ofepidermal nerve fiber densityanalysis over other testingmethods?

The principle advantages ofENFD over other testing methodsare three-fold. Foremost, ENFD isan objective analysis, meaning it isnot affected by the inherent flawsthat plague subjective tests such asSemmes-Weinstein monofilaments.Secondly, ENFD analyses exhibithigh sensitivity and specificitywhen specifically assessing for thepresence of small fiber peripheral

Epidermal...



Like most testing

methods, the

specificity of ENFD

analysis increases

when the results are

nearer to the extremes

of the bell curve.

tegrity of the test, namely, the biop-sy size, site, handling, and fixation.This test has proven to be highlyspecific and sufficiently sensitive inthe diagnosis of small fiber neu-ropathy, and an ideal method formonitoring the disease process inpatients who are undergoing medi-cal management. An additional useas a predictive modality shows greatpotential and will certainly be asubject of future research. �

Bibliography1) Ebenezer GJ, Hauer P, Gibbons C,

McArthur JC, Polydefkis M. Assessmentof epidermal nerve fibers: A new Diag-nostic and predictive tool for peripheralneuropathies. J Neuropathol Exp Neurol2007. 66(12):1059-1073.

2) Lauria G, Devigili G. Skin biopsy asa diagnostic tool in peripheral neuropa-thy. Neurology 2007. 3(10):546-557.

3) Sommer C, Lauria G. Skin biopsyin the management of peripheral neu-ropathy. Lancet Neurol 2007. 6:632-642.

4) Gibbons CH, Griffin JW, Polyde-fkis M, et al. The utility of skin biopsyfor prediction of progression in suspect-

neuropathy. Many common testingmethodologies, such as nerve con-duction studies, measure predomi-nantly large fiber abnormalities,and have little relation to smallfiber disease. The same may also besaid for sural nerve biopsy. Finally,many of the available testing meth-ods are not readily available in anoffice setting; rather, they mandatethat the patient report to a majoracademic center for testing. Thebiopsy used for epidermal nervefiber density analysis may be ob-tained in a few minutes in an officesetting.

ConclusionIn summary, epidermal nerve

fiber density is a test which allowsfor the diagnosis of small fiber pe-ripheral neuropathy in an objectivemanner, based on the analysis of acommon punch of skin. The biopsytechnique has subtle differencesfrom a standard punch biopsy,some of which are crucial to the in-

ed small fiber peripheral neuropathy.Neurology 2006. 66:256-258.

5) McArthur JC, Stocks A, Hauer P,Cornblath DR, Griffin JW. Epidermalnerve fiber density. Normal referencerange and diagnostic efficiency. 1998.Arch Neurol 55:1513-1520.

6) Polydefkin M, Hauer P, GriffinJW, McArthur JC. Skin biopsy as a toolto assess distal small fiber innervationin diabetic neuropathy. Diabetes Tech-nol Ther. 2001 3(1):23-28.

Epidermal...


Dr. Bakotic is aFellow of theAmerican Boardof Dermatopathol-ogy, the AmericanBoard of Anatom-ic Pathology, theAmerican Boardof Clinical Pathol-ogy, the College ofAmerican Pathol-ogists, the American Academy of PodiatricPractice Management, and the AmericanProfessional Wound Care Association. Heworks as Chief Executive Officer and Di-rector of Education and Research for BakoPathology Services. Dr. Bakotic can bereached at [email protected].

epidermalnervefiber densitytesting: anoverview · august 2010 • podiatry management 143 not...

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