The Mystery of the Vanishing Reinke Crystals☆,☆☆ Hector Mesa MD A,⁎, Scott Gilles MD B, Sophia Smith MD B, Susan Dachel HT A, Wendy Larson HT A, J

Human Pathology (2015) 46, 600–606

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Original contribution The mystery of the vanishing Reinke crystals☆,☆☆ Hector Mesa MD a,⁎, Scott Gilles MD b, Sophia Smith MD b, Susan Dachel HT a, Wendy Larson HT a, J. Carlos Manivel MD a,b aDepartment of Pathology and Laboratory Medicine, Veterans Administration Health Care Service, Minneapolis, MN 55417 bDepartment of Pathology and Laboratory Medicine, University of Minnesota Medical School, Minneapolis, MN 55455

Received 18 November 2014; revised 19 December 2014; accepted 2 January 2015

Keywords: Summary Reinke crystals (RC) are pathognomonic of Leydig cells (LCs); they are thought to be rare in Human; normal testes and to occur only in approximately one third of LC tumors. We noticed that crystals present in Reinke crystal; touch imprint and frozen sections of an LC tumor disappeared after tissue fixation. This phenomenon led us Leydig cell; to hypothesize that their reported low frequency in normal and neoplastic LCs may be secondary to Histology; degradation/dissolution of the crystals after formalin fixation. Our review of the literature also led us to Special stain; hypothesize that RC are better preserved after air-drying and alcohol fixation. We collected testicular Immunohistochemistry samples from 21 autopsies including air-dried cytologic preparations and tissue samples that were fixed in alcohol or formalin. We found that RC are common in normal LC but dissolve rapidly in formalin and slowly and only partially in alcohol. The composition of RC is unknown; however, they have been reported to stain specifically for nestin, an intermediate filament expressed mainly in neural and muscle tissue. Because the crystals have only been described in androgen-producing cells, we hypothesized that the crystals may represent a crystallized form of androgenic hormones, hormone complexes, or enzymes involved in their synthesis. We performed immunostains for androgens and enzymes involved in androgenesis. We also performed nestin immunostain to confirm the previous study. The crystals stain specifically with antibodies anti–3β-hydroxysteroid dehydrogenase and are negative for the remaining androgenic enzymes, androgenic hormones, and nestin. Published by Elsevier Inc.

1. Introduction 65-year-old man during workup for a recurrent retroperitoneal liposarcoma. A frozen section and touch imprints of the mass were performed showing bland oncocytoid cells with numerous A frozen section and touch imprints were performed on intracellular and extracellular rectangular and rhomboid crystals an incidental 1.2-cm hypoechogenic testicular mass found in a and lipofuscin-like pigment consistent with Leydig cells (LCs) containing Reinke crystals (RC) (Fig. 1A-C). After fixation, the crystals were no longer apparent (Fig. 1D). The “mystery of ☆ Competing interests: The authors do not have conflicts of interest the vanishing RC” motivated us to do some research about to declare. this phenomenon. Reinke crystals are considered to be rare in ☆☆ Funding/Support: The authors have no financial disclosures. normal testes and to occur only in approximately one third of ⁎ Corresponding author. Minneapolis VA-Health Care Service, Office LC tumors [1,2]. However, the original description by Reinke BB-104, 1 Veterans Drive, Minneapolis, MN 55417. E-mail addresses: [email protected] (H. Mesa), [email protected] in 1896 described crystals in 9 of 11 autopsies; he used absolute (S. Gilles), [email protected] (S. Smith), [email protected] (S. Dachel), alcohol as a fixative and Weigert fibrin stain [3]. Previous [email protected] (W. Larson), [email protected] (J. C. Manivel). publications have described that crystals are numerous in air- http://dx.doi.org/10.1016/j.humpath.2015.01.004 0046-8177/Published by Elsevier Inc.

Fig. 1 Testicular LC tumor. A, Touch imprint (Diff-Quick stain, original magnification ×100). Oncocytoid cells with granular intracytoplasmic pigment and numerous rectangular and club-shaped RC. B and C, Frozen section, hematoxylin and eosin stain (H&E), ×40 and ×100, respectively. Numerous RC are easily identifiable in the cytoplasm of LC. D, Permanent sections (H&E, ×40). The crystals are no longer apparent after formalin fixation. dried and alcohol-fixed needle aspirates from LC tumors [4-6]. blocks were stained with hematoxylin and eosin for micro- These findings led us to hypothesize that RC are common and scopic semiquantitation of RC. Special stains included that their reported low frequency in normal and neoplastic LCs periodic acid–Schiff, periodic acid–Schiff–diastase, tri- is because of their degradation/dissolution after formalin chrome, Giemsa, toluidine blue, Gram, Fite, iron, reticulin fixation. The biochemical nature of the crystals remains a and elastic fibers. Immunohistochemical stains included mystery. Because the crystals have only been described in antibodies for the androgens testosterone (GeneTex, Irvine, androgen-producing cells, we also hypothesized that the CA; product no. GTX72779; dilution, 1:125), androstenedione crystals may represent a crystallized form of androgenic (US Biological, Salem, MA; product no. 151581; dilution, hormones, hormone-complexes, or enzymes involved in their 1:100), and dehydroepiandrosterone (US Biological; product synthesis. We performed comprehensive immunohistochemi- no. 140041; dilution, 1:50) and the androgen-specific cal studies to test this hypothesis. steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD; Santa Cruz Biotechnology, Dallas, TX; product no. sc-100466; dilution, 1:250), 17α-hydroxylase/17,20-lyase (CYP17A1; Santa Cruz Biotechnology; product no. sc-46084; 2. Materials and methods dilution, 1:110), 17β-hydroxysteroid dehydrogenase (Abcam Inc, Boston, MA; [EP1682Y] ab51045; dilution, 1:50), and Testicular samples from 21 autopsy cases were collected 5α-reductase (Santa Cruz Biotechnology; product no. sc- prospectively. None of the specimens showed tumors on 20400; dilution, 1:50) and nestin (Thermo Fisher Scientific, gross examination. From each case, 5 air-dried touch imprints Vernon Hills, IL; catalog no. MA1-110; dilution, 1:300). from the serially sectioned testes were prepared and stained Cytochemical stains and immunostains were performed in with Diff-Quick (Fisher Diagnostics, Middletown, VA); 5 selected formalin-fixed cases with the highest number of representative sections were fixed in 10% formalin and 5 in crystals. Immunohistochemistry was performed on a Leica absolute ethanol. The formalin-fixed samples underwent BOND-III automated stainer (Leica Biosystems, Melbourne, conventional tissue processing; the alcohol-fixed specimens Australia) using citrate and EDTA buffer antigen retrieval were processed replacing the formalin by anhydrous ethanol in protocols. Optimal dilutions and retrieval methods were the tissue processor. The fixation time was variable and optimized in preliminary studies. All procedures were contingent on avoiding interference with the daily functioning performed in compliance with institutional guidelines of the laboratory. Tissue sections of the paraffin-embedded (Veterans Health Administration handbook 1200.05).

3. Results with short fixation times (≤1 day). There was no correlation between age (range, 61-100 years), time from death to autopsy Crystals were not apparent in the air-dried touch imprints (range, 0-4 days), exposure to corticosteroids, days of alcohol from normal testes. Nineteen (90%) of 21 alcohol-fixed fixation (range, 1-53 days), and number of crystals. The specimens showed crystals (Fig. 2A);7(33%)of21 alcohol-fixed cases with the longest fixation times (N41 days) formalin-fixed specimens showed crystals (Fig. 2B). The 2 showed variable degeneration of the crystals with a subset alcohol-fixed specimens without crystals were globally appearing hollow, fragmented, or faded (Fig. 2D). The crystals atrophic and devoid of LC; these patients had been treated were conspicuous with Giemsa (gray blue), trichrome (red), with the anti-androgens finasteride and enzalutamide (Fig. 2C). and Gram (black) stains. They were visible but not highlighted The number of crystals per slide varied from a few to with the remaining stains (Fig. 3). These results suggest that innumerable and was arbitrarily semiquantified as scant, less RC have a basic/cationic nature and do not contain a significant than 5; moderate, 5 to 20; and abundant, greater than 20. amount of carbohydrates. Special stains were not superior Semiquantitative comparison of alcohol-fixed versus formalin- to hematoxylin and eosin in staining the crystals. Immunohisto- fixed specimens showed scant, 6 of 21 alcohol versus 3 of 21 chemical stains for androgenic hormones stained specifically the formalin; moderate, 6 of 21 alcohol versus 3 of 21 formalin; LC in a granular, cytoplasmic pattern but not the crystals. and abundant 7 of 21 alcohol versus 2 of 21 formalin (Table). Immunohistochemical stains for the androgen-specific The number of crystals per cell varied from single to multiple; steroidogenic enzymes showed variable results: anti- the length and width showed marked variability; most were in CYP17A1 stained specifically all LC but not the crystals, the cytoplasm, a few were intranuclear/juxtanuclear, and very anti–17β-hydroxysteroid dehydrogenase stained specifi- rarely, they appeared to be extracellular. The crystals were cally a small subset (b10%) of LC but not the crystals, better defined and brightly eosinophilic in the alcohol-fixed anti–5α-reductase was negative in all testicular cells and specimens and pink in the formalin-fixed ones. Reinke crystals RC, and anti–3β-HSD stained uniformly the LC and appeared oblong in longitudinal sections and hexagonal to Sertoli cells (SC) and variably the RC. Although SC globular when cut transversely. Frequently, observation of showed a homogeneous staining pattern, LC showed the entire crystals required focusing up and down. In the variable intensity. The crystals stained partially with a formalin-fixed specimens, the crystals were more numerous grumous pattern. Increasing concentrations of the antibody

Fig. 2 Nontumoral testes, effect of fixative, and time of fixation. A and B, One-day fixation; A, absolute ethanol; B, 10% formalin (H&E, ×100). After short fixation time, RC are visible in both alcohol-fixed and formalin-fixed specimens; however, the crystals are better defined and brighter in the alcohol-fixed specimens. C, Testes devoid of RC (H&E, ×20). The only 2 alcohol-fixed cases without RC showed burnout morphology. D, Absolute ethanol, 53-day fixation (H&E, ×100). The RC (arrows) are still recognizable; however, most show a hollow/ ground-glass morphology.

Table Demographic characteristics and variables studied affecting the presence or absence of RC in testicular autopsy specimens N Age Days to autopsy Days of fixation Androgens Steroids Antiandrogens No. of crystals, alcohol No. of crystals, formalin 1 61 1 1 No No No Abundant Abundant 2 100 2 10 No No No Scant Absent 3 74 1 8 No No No Scant Absent 4 70 1 7 No No No Scant Absent 5 86 2 2 No Yes No Abundant Moderate 6 64 2 1 No No No Abundant Abundant 7 85 1 28 No No No Moderate Absent 8 63 1 4 No Yes No Moderate Absent 9 76 0 24 No No No Moderate Absent 10 75 3 18 No No No Abundant Scant 11 65 1 4 No No No Moderate Moderate 12 62 2 4 No Yes No Moderate Absent 13 89 1 15 No No No Abundant Absent 14 78 1 4 Yes Yes Finasteride Absent Absent 15 99 1 4 No No No Moderate Scant 16 64 1 2 No Yes No Abundant Moderate 17 67 2 4 No No No Scant Absent 18 81 2 53 No Yes No Abundant Scant 19 69 3 46 No Yes No Scant Absent 20 66 1 41 No Yes Enzalutamide Absent Absent 21 76 4 4 No No No Scant Absent NOTE. Number of crystals per slide: scant, less than 5; moderate, 5 to 20; abundant, greater than 20. showed more uniform staining not only of the crystals but 4. Discussion also the cytoplasm of LC, making visualization more difficult. Anti–nestin antibodies stained strongly the Friedrich Reinke described in 1896 the presence of endothelial cells, moderately the SC and smooth muscle polygonal crystals within testicular interstitial stromal cells cells, and minimally the LC; RC were negative (Fig. 4). previously described by Franz Leydig in 1850 [3]. Reinke

Fig. 3 Nontumoral testes, special stains (×100). Reinke crystals appear blue-gray with Giemsa (A), bright red with trichrome (B), stain partially black with Gram (C), and are visible but not highlighted by periodic acid–Schiff (D).

Downloaded for Anonymous User (n/a) at VISN 23 - Minneapolis VA Health Care System from ClinicalKey.com by Elsevier on September 18, 2017. For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved. 604 H. Mesa et al. crystals are pathognomonic of LC and presumed to be a Our study confirms the original observations by Reinke protein [7]. Ultrastructurally, they have been shown to consist that the crystals are common, probably ubiquitous in normal of hexagonal prisms of 5-nm-thick filaments in a honeycomb testicles, but the number is highly variable. Reinke crystals pattern and to lie freely in the cytoplasm [7,8];however,their also show marked variation in size; they have been reported function and biochemical composition have remained a to be up to 20 μm [1]; however, we found crystals twice as mystery [1,2].Reinkecrystalshavebeenreportedtobemore long without much effort (Fig. 5A) and up to 16 μm in width. numerous in cryptorchid testes; it has been hypothesized that The crystals show amphiphilic properties: they dissolve the increased intra-abdominal temperature facilitates their rapidly in aqueous solutions (10% formalin) and very slowly formation and that they may represent waste/degenerative and incompletely in alcohol. The effect of time was products. No experimental support has been provided for these serendipitously identified in our study while trying not to hypotheses [7-9]. Reinke crystals have been observed in only a interfere with the functioning of the laboratory. The frozen few animal species; they are thought to be rare in normal section case that prompted this study had been in formalin human testes and to occur only in approximately one third of over the weekend before processing, which explains why the LC tumors [1,2,8]. However, the original publication by crystals had vanished on the permanent sections. These Reinke described the presence of crystals in all autopsy findings also explain the discrepancy between Reinke’s specimens from individuals with normal reproductive function study and current notions about their frequency. These (9/11 cases) [3]. In contrast to current processing methods, he findings very likely apply to LC tumors as well; however, used absolute alcohol as a fixative and Weigert fibrin stain. verification of this would require the fixation of LC tumor

Fig. 4 Nontumoral testes, immunohistochemistry, formalin-fixed specimens. Immunohistochemical stains for testosterone (×40), dehydroepiandrosterone (DHEA; ×40), and androstenedione (ASD; ×40) stain specifically but variably the LC, with a granular pattern. Immunostain for CYP17A1 (×20) stains specifically and homogeneously all LC. Anti–17β-hydroxysteroid dehydrogenase (17β-HSD; ×20) stains specifically a small subset (b10%) of LC. Anti–5α-reductase (5α-R; ×20) was negative in all testicular cells. Anti–3β-HSD (×20) stained specifically LC with marked variation in intensity and SC with similar intensity. Nestin (×20) stained strongly endothelial cells, moderately SC and smooth muscle cells, and weakly LC. Reinke crystals stained specifically but only partially with 3β-HSD (×100) and were negative for the remaining immunostains.

Downloaded for Anonymous User (n/a) at VISN 23 - Minneapolis VA Health Care System from ClinicalKey.com by Elsevier on September 18, 2017. For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved. The mystery of the vanishing Reinke crystals 605 samples in absolute alcohol and replacing the formalin by This finding is similar to what has been found in Charcot- alcohol in the tissue processor; given the relative rarity of this Leyden crystals, which are the result of autocrystallization of tumor, it would be difficult to perform such studies. Reinke an enzyme complex between galectin 10 and eosinophil crystals were not identified in our air-dried cytologic phospholipase [14]. Lobo et al [9] studied the expression of preparations from normal testes, probably because LCs are nestin, an intermediate filament predominantly expressed by sparse and do not shed easily. Their presence in cytologic stem cells of neural and muscle lineages, in normal and specimens of a testicular neoplasm provides compelling abnormal human testes by immunohistochemistry and evidence of an LC tumor. Western blot. In normal testes, nestin was expressed in the There is ample indirect evidence that RC are related to cytoplasm of endothelial cells, a few LC, and a few androgenesis: they appear only after puberty, are present peritubular cells presumed to represent LC precursors. In only in men with active testosterone production, and have LC with RC, the crystals stained strongly, whereas the been reported in androgen-producing tumors of the adrenal cytoplasm appeared negative. The crystals were negative for gland and ovarian hilus [3,8,10-13]. However, the precise other intermediate filaments suggesting that the reaction was nature of this relationship has not been elucidated [1-3,8]. specific. Based on the size of the Western blot bands Our immunohistochemical studies for androgens and obtained in testes with RC, which were smaller than intact enzymes involved in androgenesis addressed this question. nestin, the authors hypothesized that RC may consist of or Immunostains for 3β-HSD stained specifically the crystals, contain degradation products of nestin. We repeated the whereas all other antibodies were negative. The crystals study with antibody against nestin and partially confirmed stained partially with a grumous pattern suggesting that the the results obtained by Lobo et al. In our study, expression of epitopes recognized by the antibodies cannot be consistently nestin was strong in endothelial cells, moderate in smooth reached within the crystals probably because of steric muscle cells and SC, and minimal in LC; however, the hindrance. The cells containing the crystals also showed crystals were negative. Lobo et al used a polyclonal anti= the strongest expression of 3β-HSD suggesting that RC nestin antibody that is no longer commercially available; we represent autocrystallized forms of this enzyme or a used a monoclonal antibody that, although more specific, is compound associated with this enzyme and that the probably less sensitive. Of all stains tested in this study, phenomenon requires high concentrations of the enzyme CYP17A1 was the best for discriminating LC from all other (Fig. 5B-D). This also explains why the crystals are not testicular elements because it was consistently and specifi- observed in SC, which also expressed 3β-HSD but at lower cally expressed in all LC in a cytoplasmic homogeneous and more consistent levels. pattern. The anti-androgen stains also stained specifically

Fig. 5 A, Forty-micrometer long RC (H&E, ×100). B-D, 3β-HSD, ×100. Reinke crystals stain specifically but variably for 3β-HSD suggesting steric hindrance.

LC, but the expression was granular and showed marked [2] Rosai J. Rosai and Ackerman's surgical pathology, 10th ed., vol. 1. variability; anti-testosterone showed the strongest expression. New York: Mosby Elsevier; 2011. p. 1355-7. [3] Christensen KA. A history of Leydig cell research. The Leydig cell in These stains can potentially be used to separate LC tumors health and disease. Totowa, New Jersey: Humana Press; 2007. p. 3-30. from other nonandrogenic mimics such as sex cord stromal cell [4] Gupta SK, Sheikh ZA, al-Rubah NA, Das DK. Intranuclear Reinke's tumors with ambiguous morphology, hyperplastic ectopic crystals in a testicular Leydig cell tumor diagnosed by aspiration cytology. adrenal tissue, and metastatic adrenocortical carcinomas. A case report. Acta Cytol 1994;38:252-6. [5] Jain M, Aiyer HM, Bajaj P, Dhar S. Intracytoplasmic and intranuclear Reinke's crystals in a testicular Leydig-cell tumor diagnosed by fine- needle aspiration cytology: A case report with review of the literature. 5. Conclusions Diagn Cytopathol 2001;25:162-4. [6] Hribar KP, Warner NE, Sherrod AE. Cytologic identification of Reinke crystals are common, probably ubiquitous in Reinke crystalloids in scrapings and imprints of fresh testicular tumors: a simple and rapid technique for intraoperative use. Arch Pathol Lab normal testicles. The crystals show amphiphilic properties: Med 2005;129:e65-6. they dissolve rapidly in aqueous solutions (10% formalin) [7] Nagano T, Ohtsuki I. Reinvestigation of the fine structure of Reinke's andslowlyinalcohol-fixedspecimens.Reinkecrystalsand crystals in the human testicular interstitial cell. J Cell Biol 1971;51: crystals in general are not common in cytologic prepara- 148-61. tions from normal testes; their presence in a testicular [8] Kozina V, Geist D, Kubinova L, et al. Visualization of Reinke's crystals in normal and cryptorchid testis. Histochem Cell Biol 2011;135:215-28. neoplasm should suggest the diagnosis of LC tumor. The [9] Lobo MV, Arenas MI, Alonso FJ, et al. Nestin, a neuroectodermal crystals stain specifically with 3β-HSD suggesting that they stem cell marker molecule, is expressed in Leydig cells of the human represent an autocrystallized form of this enzyme or an testis and in some specific cell types from human testicular tumors. enzyme complex. The previously reported positivity for Cell Tissue Res 2004;316:369-76. nestin in RC could not be confirmed in our study; however, [10] Pollock WJ, McConnell F, Hilton C, Lavine RL. Virilizing Leydig cell adenoma of adrenal gland. Am J Surg Pathol 1986;10:816-22. we used a monoclonal antibody in contrast to the now [11] Ryan JJ, Rezkalla MA, Rizk SN, Peterson KG, Wieve RH. discontinued polyclonal antibody used in the previous Testosterone secreting adrenal adenoma that contained crystalloids study. Immunostains for testosterone, CYP17A1, and of Reinke in an adult female patient. Mayo Clin Proc 1995;70:380-3. androgens are specific for LCs and may be used to separate [12] Paraskevas M, Scully RE. Hilus cell tumor of the ovary. A LC tumors from nonandrogenic mimics. clinicopathological analysis of 12 Reinke crystal-positive and nine crystal-negative cases. Int J Gynecol Pathol 1989;8:299-310. [13] Vasiloff J, Chideckel E, Boyd C, Foshag L. Testosterone-secreting adrenal adenoma containing crystalloids characteristic of Leydig cells. References Am J Med 1985;79:772-6. [14] Ackerman J, Liu L, Kwatia MA, et al. Charcot-Leyden crystal protein [1] Ulbright TM, Young RH. AFIP Atlas of tumour pathology. Tumours (Galectin-10) is not a dual function galectin with lysophospholipase of the testis and adjacent structures. Fourth series fascicle. Silver activity but binds a lysophospholipase inhibitorin a novel structural Spring, MD: ARP Press; 2013. p. 271-86. fashion. J Biol Chem 2002;277:14859-68.