Özgün Araştırma/Original Article doi: 10.5146/tjpath.2012.01126 Digitally Reinforced Polarization of Hematoxylin-Eosin in the Diagnosis of Renal Amyloidosis Renal Amiloidoz Tanısında Dijital Güçlendirilmiş Hematoksilen Eozin Polarizasyonu Sait ŞEN, Banu SARSIK KumbaraCI Department of Medical Pathology, Ege University, Faculty of Medicine, İZMİR, TURKEY The summary of this study was presented at 24th Congress of Pathology held in Prague on 8-12 September 2012 ABSTRACT ÖZ Objective: Systemic amyloidosis is a rare disorder, characterized by Amaç: Sistemik amiloidozlar, hematoksilen-eozin boyamada amorf extracellular accumulation of Congo red positive fibrillar amyloid eozinofilik görülen, Kongo kırmızısı ile boyanan fibriller amiloid protein deposits that have an amorphous, eosinophilic appearance proteinlerin ekstrasellüler birikimiyle karakterize nadir hastalıklardır. on hematoxylin-eosin stained preparations. The kidney is the Böbrekler sistemik amiloidozlardan en sık etkilenen organdır. Kongo most commonly affected organ by systemic amyloidosis. Congo kırmızısı, zayıf birefrenjant boyanmamış amiloidin birefranjansını red staining increases the positive birefringence of the weakly artırır. Bu çalışmada, böbrek biopsilerinin rutin hematoksilen eozin birefringent unstained amyloid. In this study, we investigated the kesitlerinde dijital güçlendirilmiş birefrenjansın potansiyel tanısal potential diagnostic power of digitally reinforced birefringence of gücünü araştırdık. routine hematoxylin-eosin stained slides from renal biopsies. Gereç ve Yöntem: Hematoksilen-eozin boyalı 130 preparat Material and Method: We reviewed 130 hematoxylin-eosin stained polarizasyon için değerlendirildi. Altmış beş yeni amiloidoz olgusuna slides for polarization. Sixty-five new amyloidosis cases were böbrek biyopsisi ile tanı konuldu. Tüm böbrek biopsileri ışık ve diagnosed by renal biopsy. All renal biopsies were evaluated by light immünflöresan mikroskop ile değerlendirildi. Preparatlar kör olarak, microscopy and immunofluorescence. Slides were reevaluated blindly polarizasyon filtreleri eklenmiş ve digital kamera (Olympus DP21, using a microscope (Olympus BX51) that was attached polarization SAL) bağlanmış mikroskopla (Olympus BX51) değerlendirildi. filters and connected to a digital camera (Olympus DP21, SAL). Dijitalize mikroskopi ile yeşil birefrenjans gösteren birikimler pozitif Deposits that showed green birefringence on hematoxylin-eosin olarak değerlendirildi ve Kongo kırmızısı ile birikimler doğrulandı. preparations with digitalized microscopy were considered positive Bulgular: Kongo kırmızısı ile doğrulanmış 65 amiloid pozitif and the results were confirmed using Congo red. biopsinin 61’i hematoksilen-eozin ile yeşil birefrenjans gösterdi. Results: Of the 65 Congo red confirmed amyloid positive biopsies, Kongo kırmızısı ile doğrulanmış, 65 amiloid birikimi olmayan 61 showed green birefringence with hematoxylin-eosin. Of the olgunun 2’si yalancı pozitif olarak değerlendirildi. Duyarlılık, 65 Congo-red confirmed amyloid negative biopsies, two were özgüllük, pozitif ve negatif prediktif değerler sırasıyla %94, %97, %97 considered to be false positive. The sensitivity, specificity, and positive ve %94 olarak hesaplandı. and negative predictive values were estimated as 94%, 97%, 97% and Sonuç: Renal amiloidoz için polarize edilmiş hematoksilen eozin 94% respectively. kesitler dijital olarak hızlı ve ilk basamak tanı metodu olarak Conclusion: We concluded that polarized hematoxylin-eosin sections kullanılabilir. can be used digitally as a fast and first step diagnostic method for Anahtar Sözcükler: Amiloid, Amiloidozis, Kongo kırmızısı, renal amyloidosis Hematoksilen Key Words: Amyloid, Amyloidosis, Congo Red, Hematoxylin (Turk Patoloji Derg 2012, 28:204-212) Correspondence: Sait Şen Department of Medical Pathology, Ege University, Faculty of Medicine, Received : 27.04.2012 Accepted : 14.06.2012 İZMİR, TURKEY E-mail: [email protected] Phone: +90 533 424 16 69 204 ŞEN S and SARSIK KUMBARACI B: Polarization of Hematoxylin-Eosin Türk Patoloji Dergisi/Turkish Journal of Pathology INTRODUCTION including diabetic glomerulosclerosis (25 cases), segmental sclerosis and/or glomerulonephritis associated with Amyloidosis is caused by the systemic or localized fibrinoid necrosis and ANCA (20 cases), and myeloma accumulation of at least 27 different proteins (1). Systemic cast nephropathy (15 cases) in the study. In addition, 15 forms involve the internal organs at different rates, and the cases, including previously published ones, diagnosed with kidneys are important within these affected organs in terms amyloidosis localized to the seminal vesicle determined in of morbidity and mortality. Incidence in kidney biopsies in the adult population is under 2% in American and European cases evaluated for prostate adenocarcinoma were included publications, but reaches 10% in our country (2-7). as non-renal positive control cases (12). Amyloid deposits appear pink under light microscopic The clinical and laboratory data of the cases with a investigation of routine hematoxylin-eosin (H&E) sections diagnosis of amyloidosis that were included in the study in kidney biopsies and can be confused with findings were investigated from the hospital archive records. such as hyalinosis and sclerosis that can cause a similar Light microscopic examination: Renal needle biopsies appearance (8). Staining of the eosinophilic deposits with were evaluated with three 3-4 micron thick H&E stained Congo red (CR) and making the diagnosis with apple green slides of different levels as well as JMS, MT, PAS, and polarization is accepted as the gold standard for amyloidosis Putchtler’s modified CR method during the histochemical diagnosis (1, 8-11). Unstained amyloid deposits show examination (11). Antibodies marked with fluorescent weak birefringence and the CR staining increases this isothiocyanate were used for IgG, IgM, IgA, kappa, lambda, birefringence (9). Histochemical staining methods such C1q, C3c, and fibrinogen for the immunofluorescence as Schiff’s periodic acid (PAS), Masson’s trichrome (MT) microscopic investigation. Amyloidosis was diagnosed by and Jones’s methenamine silver (JMS) used during routine the deposits staining CR positive with the Putchler staining evaluation of kidney biopsies are helpful in the differential method showing apple-green polarization on the polarizing diagnosis (8). However, amorphous eosinophilic deposits microscope and these cases were evaluated as systemic in biopsies of cases where CR is not used routinely and do amyloidosis. In cases with amyloidosis, scoring and not have a clinical suspicion of amyloidosis may not receive staging were performed according to the renal amyloidosis an amyloidosis diagnosis. histological classification (6). Kidney needle biopsies and needle or endoscopic biopsies Typing of amyloid deposits: Immunohistochemical (IHC) of other organs are small in size, and the optimal evaluation investigation with antibodies against kappa, lambda, pre- and preservation of tissue are therefore important. albumin (TTR), fibrinogen, lysozyme and AA was used Preparing new sections for methods requiring a thick to type the amyloid preliminary proteins of the cases section such as CR staining can also deplete the tissue with amyloidosis. IHC staining was performed with a in paraffin blocks in small biopsies and there may not be fully automated device (Benchmark XT, Ventana Medical enough tissue for further investigations. Therefore, the Systems, Tucson, USA) (Table I). IHC staining in amyloid optimum evaluation of small biopsies that create differential deposit areas was scored as negative (0) or positive (in 4 diagnosis difficulties and require advanced investigation is values as +, ++, +++, ++++ according to the severity extremely important. During the evaluation of cases with of staining). In non-AA amyloidosis cases, concurrent amyloidosis, we observed that the amyloid deposits show minimal positivity with anti-AA antibody was not taken very weak polarization under strong light in routine H&E into account for typing. Typing was performed together stained preparations. We therefore planned this study to with clinical and laboratory findings in cases where we were determine whether H&E polarization can be used as an not able to perform reliable typing with multiple positive additional method in kidney needle biopsies for amyloidosis IHC results. diagnosis and whether its enhancement by digitalization will contribute to the diagnosis of amyloidosis. Digital imaging method: The main feature of this study is digital imaging and photography of H&E polarization. MATERIAL and METHODS The light intensity of the microscope and the setting of We included 65 cases diagnosed with amyloidosis at Ege the camera were optimized as they are important for University Medical Faculty Pathology Department between the imaging method. The polarization of the amyloid 2008 and 2012 and a total of 65 cases with amorphous deposits is difficult to recognize under microscope ocular eosinophilic deposits suspicious in terms of amyloid in in H&E stained sections during normal light microscopic the differential diagnosis following renal needle biopsy, examination. The amyloid deposit polarization is best Cilt/Vol. 28, No. 3, 2012; Sayfa/Page 204-212 205 Türk Patoloji Dergisi/Turkish Journal of Pathology ŞEN S and SARSIK KUMBARACI B: Polarization of Hematoxylin-Eosin Table I: Antibodies and dilutions used for the immunohistochemical typing of amyloid proteins