774 Arch Pathol Lab Med—Vol 124, May 2000 Urate Crystal Staining Method—Shidham & Shidham State of the Art in Clinical and Anatomic Pathology Staining Method to Demonstrate Urate Crystals in Formalin-Fixed, Parafﬁn-Embedded Tissue Sections Vinod Shidham, MD, MIAC, MRCPath; Ganesh Shidham, MD T he histologic appearance of tophi is usually diagnostic. The tophi typically have a characteristic feathery ap- pearance and foreign body giant cells around aggregates of needle-shaped empty spaces in a basophilic matrix formed by washed urate crystals. 1,2 However, some lesions may have less characteristic histologic features because of minimal crystal deposits, small size of the biopsy speci- men, minimal or absent foreign body giant cell response, abundance of basophilic matrix with scant crystals, or the formation of a prominent palisading granuloma-like pat- tern. In such cases, special techniques are important to demonstrate the presence of urate crystals in tissue. Dif- ferent staining methods are described to demonstrate urate crystals in tissue, including deGalantha stain 3 and Gomori methenamine-silver stain. 4 These stains share the nonspeciﬁcity of routine hematoxylin-eosin (HE) stain without any advantage. Traditionally, alcohol ﬁxation is preferred over formalin ﬁxation. 5 The present study de- scribes a simple method that visualizes urate crystals in formalin-ﬁxed, parafﬁn-embedded tissue sections under a light microscope and allows the conﬁrmation by a polar- izing microscope. MATERIALS AND METHODS We studied 22 blocks of formalin-ﬁxed, parafﬁn-embedded tis- sue from 5 patients with a known diagnosis of gout. The blocks were divided into 4 groups as shown in the Table. Since the size of the specimens was adequate to prepare multiple blocks, all blocks in different groups were from the same 5 patients. Group 1 had 8 blocks of tissue larger than 2 mm core and ﬁxed in formalin for less than 12 hours. Group 2 had 6 blocks of tissue bigger than 2 mm core and ﬁxed in formalin for more than 12 hours. Group 3 had 5 blocks of tissue smaller than 2 mm core and ﬁxed in formalin for less than 12 hours. Group 4 had 3 blocks of tissue smaller than 2 mm core and ﬁxed in alcohol ﬁxative (95% ethyl alcohol) for approximately 6 hours. The larger tissue submitted and left in formalin for less than 1 hour was used to make 2-mm tissue cores for the later group. For groups 1, 2, and 3, the schedule of processing for parafﬁn embedding was 180 minutes in 10% buffered formalin, 90 min- utes in 70% alcohol, 110 minutes in 95% alcohol, 160 minutes in absolute alcohol, 110 minutes in xylene, and ﬁnally 120 minutes in parafﬁn. For group 4, a similar tissue processing schedule without the ﬁrst 2 steps was used. Accepted for publication September 8, 1999. From the Department of Pathology, Medical College of Wisconsin, Milwaukee (Dr V. Shidham), and Department of Medicine, Mount Sinai Services/Queens Hospital Center, Jamaica, NY (Dr G. Shidham). Reprints: Vinod B. Shidham, MD, MIAC, MRCPath, Department of Pathology, Medical College of Wisconsin, 9200 W Wisconsin Ave, Mil- waukee, WI 53226. The sections were stained with routine HE. The respective next level sections from the parafﬁn blocks were stained with our method. The staining method is based on the principle of not passing the tissue section through any aqueous reagent. This pre- vents the dissolution and loss of urate crystals from the thin tis- sue sections. Staining with nonaqueous dye solution (0.5% alco- holic eosin Y [Shandon Inc, Pittsburgh, Pa, catalogue No. 999117] was used in this study) visualizes the tissue architecture, allow- ing the examiner to appreciate the relationship of urate crystals with the surrounding tissue. The glass slides with 5- to 7-m- thick parafﬁn sections were progressively dipped through the various reagents as follows: (1) xylene (6 dips or 20 seconds); (2) 1:1 xylene-absolute alcohol (6 dips or 20 seconds); (3) 0.5% al- coholic eosin Y (Shandon, catalogue No. 999117) (6 dips or 20 seconds); (4) absolute alcohol (6 dips or 20 seconds); (5) absolute alcohol (6 dips or 20 seconds); (6) 1:1 xylene-absolute alcohol (6 dips or 20 seconds); (7) zylene (6 dips or 20 seconds); and (8) coverslip with nonaqueous mounting medium (eg, Permount; Fisher Scientiﬁc, Fair Lawn, NJ). The slides stained with HE and our method were examined with a light microscope for the presence of urate crystals and diagnostic histologic features of gouty tophus. They were also examined with a polarizing microscope for the birefringence of urate crystals. 6–8 RESULTS All the slides in groups 1 and 4 stained with our meth- od demonstrated urate crystals by light microscopy. The crystals were yellow-brown aggregates of needle-shaped structures that simulated haystacks (Figure 1). They dem- onstrated diagnostic negative birefringence under the po- larizing microscope (Figure 2). They could not be detected in any of the sections stained with HE (Table); however, the diagnostic histologic features of gouty tophus were observed in all HE-stained sections. In group 2, only 1 section stained by our method showed urate crystals by light microscopy. In the same group, 2 sections demonstrated urate crystals in the sim- ilarly stained sections by polarizing microscopy. None of the sections from group 3 showed any crystals by light microscopy, but 1 section demonstrated scant urate crys- tals by polarizing microscopy. COMMENT The appearance of tophi in histologic sections is usually diagnostic, and special techniques are not usually needed for a deﬁnitive diagnosis. However, the small amount of tissue present in a biopsy specimen or an unusual histo- logic picture may require ancillary studies. Previously de- scribed techniques 3,4 are not speciﬁc and do not offer ad- ditional information over routine HE sections. The present method can demonstrate urate crystals and differentiate