J Cutan Pathol 2009: 36: 560–564doi: 10.1111/j.1600-0560.2008.01116.xJohn Wiley & Sons. Printed in Singapore

Copyright # 2009 John Wiley & Sons A/S

Journal of

Cutaneous Pathology

Simple non-staining method todemonstrate urate crystals informalin-fixed, paraffin-embeddedskin biopsies

Background: Gouty tophi classically occur as nodules over joints andthe helix. The ideal fixative for preservation of gout crystals hastraditionally been alcohol because the crystals are formalin and watersoluble. However, most biopsies are submitted in formalin fixative,which results in dissolution of urate crystals leaving behind a non-specificpale amorphous area. Although complex staining methods to showurate crystals in tissue have been described, the present study elucidatesa simple non-staining method utilizing a thick unstained coverslippedmicroscopy slide that allows detection and confirmation of birefringenceof urate crystals in formalin-fixed, paraffin-embedded tissue.Methods: Twenty-nine cases of cutaneous gouty tophi were evaluatedusing a hematoxylin-eosin (H&E)-stained section, a 10-mm unstainedcoverslipped section and a 4-mm unstained coverslipped section. In allcases, the specimen was received and submitted in formalin.Results: Polarizable crystals were not identified in any of the H&Esections. The use of the thicker unstained coverslipped section was moresensitive than the standard 4 mm section by recognizing thecharacteristic urate crystals in 48% and 38% of the cases, respectively.Conclusions: This inexpensive adjunctive tool can be used todocument gout crystals in almost half of skin biopsies.

Weaver J, Somani N, Bauer TW, Piliang M. Simple non-stainingmethod to demonstrate urate crystals in formalin-fixed, paraffin-embedded skin biopsies.J Cutan Pathol 2009; 36: 560–564. # 2009 John Wiley & Sons A/S.

Joshua Weaver1, NajwaSomani1,2, Thomas W. Bauer1

and Melissa Piliang1,2

1Division of Pathology and LaboratoryMedicine, and 2Division of Dermatology,Cleveland Clinic, Cleveland Clinic LernerCollege of Medicine of Case Western ReserveUniversity, Cleveland, OH, USA

Joshua Weaver, MD, Department of AnatomicPathology/L25, Cleveland Clinic, 9500 EuclidAvenue, Cleveland, OH 44195, USATel: (216) 444 2828Fax: (216) 445 3707e-mail: weaverj3@ccf.org

Accepted for publication June 17, 2008

Gout classically manifests in the skin during thechronic tophaceous stages as a nodule(s) on the lowerand upper digits and helical rims of the ears of oldermen (Fig. 1). The nodules are formed by thedeposition of monosodium urate (MSU) crystalsduring bouts of hyperuricemia. They appear grosslyas white chalky material. Histologically, when prop-erly fixed in alcohol, classic gouty tophi appear asdeposits of brown, negatively birefringent, needle-shaped crystals in the dermis or subcutaneous tissues.However, the clinical diagnosis of gout is not always

suspected, and skin biopsies are usually submitted informalin fixative. Urate crystals are thought to beextremely soluble in formalin and therefore dissolveout of small skin biopsies leaving behind onhematoxylin-eosin (H&E)-stained section a dermalor subcutaneous deposition of amorphous materialsurrounded by mononuclear histiocytes, foreign bodygiant cells, fibrosis and chronic inflammation. Diag-nostic difficulties occur when characteristic morpho-logic findings are sparse or lack classical appearance.In some cases, overlying or adjacent reactive changes

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such as epidermal hyperplasia and angioplasia similarto that seen in chondrodermatitis nodularis helices(CNH) may mask subtle deposits. Documentation ofthe diagnostic negatively birefringent urate crystals isrequired in these cases to accurately alert clinicians tothe pathogenesis of the patient’s findings, particularlybecause gouty tophi can be the presenting symptomofsystemic hyperuricemia. Complex-specific and non-specific staining methods to show urate crystals intissue have been described.1,2 A variation of thismethod has previously been described in orthopedicand general pathology textbooks for use in localizinguric acid deposits in bone and peri-articular tissues;however, these sources do not comment on thethickness of the unstained section or the fact thata coverslipmust beused.3,4Thepresent studydescribesa simple non-staining method utilizing a thickunstained and coverslipped microscopy slide thatallows detection and confirmation of birefringence ofurate crystals in formalin-fixed, paraffin-embeddedskin biopsy tissue sections under light microscopy.

Materials and methods

After receiving institutional review board approval,31 cases of formalin-fixed and paraffin-embeddedskin biopsies diagnosed as gout, gouty tophus or goutytophi were identified from the archives of theDepartment of Anatomic Pathology at the ClevelandClinic from 1992 to 2008. Each case received one 10-mm unstained coverslipped section, one 4-mmunstained coverslipped section and one standard4-mm recut H&E-stained section. Each H&E-stainedsection was independently reviewed by two dermato-pathologists (M. P. and N. S.) for the characteristicmorphologic changes consistent with gout includingbrown needle-shaped crystals, deposition of ampho-philic amorphous material in the dermis or

subcutaneous tissue, peripherally located mononu-clear histiocytes, giant cells, fibrosis, chronic inflam-mation and calcification. Two cases were excludedfrom the study because the histologic findings werenot diagnostic for gout on addition H&E recutsbecause the amorphous material was exhausted fromthe blocks (n ¼ 2). Each of the H&E and unstainedslides were polarized for identification of the diag-nostic negatively birefringent needle-shaped uratecrystals. Negative birefringence of MSU crystals isdefined by the generation of a yellow color when thelong axis of the crystal is oriented parallel to thepolarized light. Rotating the crystals 90 degreeschanges the interference color to blue.

Results

Twenty-nine cases were identified (Table 1). Thegouty tophi predominantly occurred in men (male tofemale ratio, 6.8 : 1) with a mean age of 62 years. Thenodules presentedmost often on the digits (45%), helixof the ears (45%) or other locations (10%). The clinicaldiagnosis submittedon the pathology requisition formswas variable. In some cases, the diagnosis consideredwas gout (34%), but the biopsies were still received informalin. Other clinical diagnoses included calcinosiscutis (24%), cyst (10%), CNH (7%), squamous cellcarcinoma (SCC) (3%) and others (17%).

Table 1. Summary of clinical features

Case Age (years) Sex Site Clinical diagnosis

1 69 M Right upper helix Gout2 50 M Right ear CNH3 92 M Left 1st finger Gout4 50 M Left ear None provided5 51 F Left thigh Calcinosis cutis6 67 M Left ear CNH7 26 M Left ear Gout8 69 M Left ear Milia9 74 M Left toe Gout10 85 F Right 3rd toe Cyst11 59 M Right ear Infection12 66 F Right 4th toe Wart13 59 M Right ear Squamous cell

carcinoma14 52 M Left ear Cyst15 66 M Right elbow Gout16 57 M Right ear Cyst17 69 F Right 2nd finger Calcinosis cutis18 70 M Right 1st finger Calcinosis cutis19 42 M Right leg Calcinosis cutis20 54 M Left ear None provided21 70 M Right 2nd finger Gout22 74 M Right 2nd finger Gout23 85 M Right 3rd finger Gout24 54 M Right ear Gout25 56 M Left 1st finger Lobular capillary

hemangioma26 55 M Right 3rd finger Calcinosis cutis27 55 M Right 3rd finger Calcinosis cutis28 40 M Right ear Gout29 67 M Right 1st finger Calcinosis cutis

CNH, chondrodermatitis nodularis helices; F, female; M, male.

Fig. 1. Chronic tophaceous stage of gout presenting clinically as

nodules on a distal upper extremity digit.

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Microscopically, all the skin biopsies displayeddeposits of amorphous material in the dermis orsubcutaneous tissue with a variable degree ofsurrounding granulomatous inflammation (Figs. 2and 3); however, some of the deposits were focal andsubtle. Seven (24%) cases lacked the characteristicforeign body giant cells, and many cases hadsecondary reactive changes such as fibrosis, angio-plasia and pseudoepitheliomatous hyperplasia mim-icking pathologic findings of CNH.

All H&E-stained sections lacked brown needle-shaped crystals, and none of the specimens revealednegatively birefringent needle-shaped crystals underpolarized light. Eleven cases (38%) on 4-mmunstainedcoverslipped sections displayed the diagnostic brownnegatively birefringent needle-shaped MSU crystalsarranged in short intersecting fascicles, while 14 cases(48%) exhibited the diagnostic crystals while evaluat-ing the thick 10-mm unstained coverslipped sections(Figs. 4–7).

Discussion

Gout manifests cutaneously during the chronictophaceous stage where MSU crystals deposit in thedermis and subcutaneous tissue of the digits and helix

of the ear creating nodules. Dermatopathology andgeneral pathology textbooks recommend that biopsiesfor gout be submitted in alcohol fixative so that theMSU crystals do not dissolve away.5 However, thediagnosis is often not expected because the tophus ortophi may be the initial findings of hyperuricemia orbecause they are clinically confused with clinicalsimulators, such as CNH, SCC or calcinosis cutis,and therefore placed in formalin. Thirty-four percentof our cases were thought to be gout clinically and allwere still submitted in formalin fixative, suggesting thatalcohol fixative is not easily available tomost clinicians.Alcohol fixative is thought to preserve crystals

because it lacks water, long thought to be the mediumthat dissolved MSU crystals.6 Simkin et al. showed ina simple experiment that not only water but alsoformalinwas responsible for dissolving urate crystals.7

Using a gradient of different amounts of urate crystal,

Fig. 2. Low magnification hematoxylin-eosin photomicrograph of

an acral skin biopsy displaying deposits of amorphous material in

the dermis with a surrounding granulomatous inflammation.

Fig. 3. Higher magnification hematoxylin-eosin photomicrograph

of the dermal deposits of amorphous material surrounded by

mononuclear histiocytes, giant cells and fibrosis.

Fig. 4. Low magnification view of a 10-mm unstained coverslipped

section displaying the diagnostic brown monosodium urate crystals

deposited in the dermis.

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Simkin et al. qualitatively assessed the presence of theurate crystals after 72 h in 10 ml of three differentsolvents – formalin (37% formaldehyde in water),water and ethanol. Formalin dissolved all the uratecrystals up to a mass of 200 mg. Urate crystals did notbecome visibly detectable until after adding 500 mgof urate crystals in 10 ml of formalin. By contrast,water could only dissolve up to 2 mg urate crystals,and only 5 mg of urate crystals in 10 ml of water wassufficient to detect residual crystals after 72 h. Visibleurate crystals were always observed in the ethanolsolvent even at a minimum urate crystal mass of0.5 mg. These findings suggest that MSU crystals are10 and 1000 times more soluble in water andformalin, respectively, compared with alcohol fixa-tive. Although this study further supports water asa solvent for urate crystals, it shows the greater

importance of formaldehyde in dissolving the uratecrystals.

After initial formalin fixation, the skin biopsy is notexposed to formalin but rather to water during thestaining stage of preparation of an H&E slide.Shidham et al. developed a non-aqueous stainingmethod to show the urate crystals that may havesurvived the formalin fixation.1 The non-aqueousstaining prevents the dissolution of the remainingurate crystals from the thin tissue sections. Shidhamet al. divided their known gout biopsies into fourgroups based on size of tissue core (smaller or largerthan 2 mm) and time fixed in formalin (less than ormore than 12 h). They discovered that their non-aqueous staining method detected urate crystals bylight microscopy or polarization most frequently intissue specimens larger than 2 mm and/or withformalin fixation times less than 12 h. For specimenslarger than 2 mm, urate crystals were detectable in100% of cases fixed for less than 12 h vs. 33% of casesfixed for greater than 12 h. For tissue specimens lessthan 2 mm in size, urate crystals were detectable in20% of cases fixed for less than 12 h, but whenformalin fixationwas less than 6 h, urate crystals weredetected in 100%. In contrast to the non-aqueousstaining method, none of the standard H&E-stainedsections in any of the four groups displayed lightmicroscopic or polarizable evidence of urate crystals,including those cases fixed in alcohol fixative. In thelatter instance, despite lack of exposure to formalinfixative, routine aqueous H&E staining is sufficient todissolve the minute quantities of urate crystals foundin thin tissue sections.8 The conclusion drawn fromthe study of Shidham et al. emphasizes that exposureto aqueous solutions during either fixation or stainingcontributes to crystal dissolution. Uric acid crystals

Fig. 7. Diagnostic negatively birefringent monosodium urate crys-

tals arranged in short intersecting fascicles generating a blue color

when their long axis is oriented perpendicular to the polarized light

(arrow depicts direction of polarized light).

Fig. 6. Diagnostic negatively birefringent monosodium urate crys-

tals arranged in short intersecting fascicles generating a yellow color

when their long axis is oriented parallel to the polarized light (arrow

depicts direction of polarized light).

Fig. 5. Higher magnification view of the diagnostic refractile lightly

brown monosodium urate crystals arranged in short intersecting

fascicles under normal light microscopy.

Urate crystals in skin biopsies

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are more likely to dissolve from small samples andfrom those that remain in formalin for long periods oftime when compared with larger samples and thosewith short fixation intervals.

The prior two paragraphs have described studiesshowing (i) that water and formalin are responsible forthe dissolution of urate crystals in tissue specimens and(ii) that thewater present in routine aqueousH&E staintechniques is also sufficient to dissolve all the uratecrystals from tissue even if they are submitted in alcoholfixative.Our study shows that a simple and inexpensiveunstained 10-mm thick coverslipped tissue sectionbypasses exposure of the tissue to the aqueous H&Estep, thereby preserving the urate crystals, whichappear as short intersecting fascicles of brown needle-shaped structures under light microscopy and nega-tively birefringent under polarized light.

The enhanced crystal preservation obtained fromalcohol fixed over formalin-fixed specimens is welldocumented in both the dermatopathology and theorthopedic pathology textbooks.3,5 However, to ourknowledge, the use of unstained sections to avoidaqueous dyes has been described only in orthopedicpathology and not in dermatopathology textbooks.Nonetheless, the orthopedic pathology textbooks failto mention that the unstained tissue section must becoverslipped and that thick sections may producea higher yield.

Our study shows that more difficult and costly non-aqueous staining methods are unnecessary to detecturate crystals in skin biopsies. Rather the simple andthe inexpensive use of a 10-mmunstained coverslippedtissue section shows negatively birefringent brownneedle-shaped pathognomonic urate crystals innearly 50% of cases. The sensitivity of this simpleadjunctive tool is lower than what might be expectedbecause skin biopsies taken from the helix of the earand the digits are often extremely small (, 2 mm). Itis likely that all the urate crystals are dissolved with

longer durations of exposure to 10% bufferedformalin such that no crystals are detectable even onthicker unstained coverslipped sections. It must beremembered that a more time-consuming and costlynon-aqueous staining method would also fail topreserve the urate crystals under conditions of longerformalin fixative exposure times.In summary, a 10-mmcoverslipped unstained tissue

slide is a useful adjunctive tool to document andpreserve residual urate crystals from skin biopsiesfixed in 10% buffered formalin or alcohol in cases ofsuspected cutaneous gouty tophi. The detection ratefor crystals may be enhanced by shorter durations offormalin fixation, and clinicians should take this intoaccount when submitting their biopsy specimens.

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