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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

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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 www.elsevier.com/locate/humpath 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. 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 601 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). 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. 602 H. Mesa et al. 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
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