AMACR/34βE12/p63鸡尾酒双染对诊断前列腺癌及癌前病变的价值

目的探讨AMACR／34βE12／p63鸡尾酒双染对诊断小灶性前列腺癌及癌前病变的价值。方法从2005年6月起对3个月内临床连续送检的105例前列腺穿刺活检标本,6例前列腺癌根治标本和19例经尿道和耻骨上摘除的良性前列腺增生标本总计130个病例,1030个组织块中需要用免疫组织化学辅助诊断的262个组织块分别作AMACR／34βE12／p63鸡尾酒双染,同时作这3个抗体的单项染色,并结合HE切片和临床资料观察结果作出诊断。结果鸡尾酒双染切片中前列腺癌和高级别上皮内瘤变（HGPIN）上皮细胞呈蓝黑色,良性腺体的基底细胞呈红色。癌的蓝黑色腺上皮周围无红色基底细胞围绕,HGPIN的蓝黑色腺上皮周围有间断或连续的红色基底细胞。共在214个（82％）组织块中发现前列腺癌,包括31处小灶性癌。64个（24％）组织块中发现HGPIN,包括局灶性HGPIN和小腺泡性HGPIN。1个组织块有不典型性腺瘤样增生。未发现上皮细胞AMACR阳性同时基底细胞34βE12和p63阴性的良性腺体。结论鸡尾酒双染有助于提高小灶性前列腺癌和HGPIN检出率。     

Using an AMACR (P504S)/34betaE12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.     

Alpha-methylacyl-CoA racemase (P504S)/34betaE12/p63 triple cocktail stain in prostatic adenocarcinoma after hormonal therapy

Alpha-methylacyl-CoA racemase (AMACR) has recently been shown to be a highly sensitive marker for the diagnosis of prostate cancer. However, there is limited information concerning its utility as a marker for prostate carcinoma after hormonal therapy. Our current investigation was conducted to evaluate the expression of AMACR in patients with prostate carcinoma after hormonal therapy and assess its diagnostic utility in combination with p63 and high molecular weight cytokeratin (34betaE12) staining. Prostate tissues from 49 patients who had been treated with hormonal therapy were immunohistochemically analyzed for AMACR, 34betaE12, and p63 expression by a triple antibody cocktail stain. The staining intensities and the percentages of positively staining tumor cells were recorded. The correlations between AMACR expression and metastatic status, associated hormonal therapy regimens, and the extent of hormone therapy effect were analyzed. All malignant acini were completely negative for both basal cell markers (34betaE12 and p63). Tumor cells failed to demonstrate expression of AMACR in 14 (29%) of 49 cases. In the remaining 35 cases (71%), positive immunostaining for AMACR was noted, but with variable intensities and percentages of cells stained. Positive staining for AMACR in benign glands was not seen in any case. In all cases, basal cells were strongly stained by p63 in benign acini with a mean positive percentage of 96%. Similarly, basal cells in benign acini displayed moderate staining intensities for 34betaE12 in 3 (7%) of 41 cases and strong immunostaining for this marker in the remaining 38 cases (93%); the mean percentage of positive cells was 92%. alpha-methylacyl-CoA racemase expression may be substantially diminished or entirely lost in prostate carcinoma after hormonal therapy. This variation in AMACR expression does not correlate with the metastatic status, the modality of hormonal therapy, or the extent of therapy-related effect. It is important that pathologists be aware that some hormonally treated prostate carcinomas do not express AMACR, and that immunostaining in such cases must be interpreted with caution. A triple cocktail stain using AMACR, 34betaE12, and p63 can be helpful in evaluating prostate specimens for the presence of residual or recurrent carcinoma after hormonal therapy for cancer.     

Differential diagnosis of glandular proliferations in the prostate

 A variety of small acinar lesions of the prostate can mimic prostate cancer in punch biopsies and in transurethral resection material. The first part of this review deals with differential diagnostic problems of the central and transition zone, including atypical adenomatous hyperplasia of the prostate, atrophic processes, sclerosing adenosis, basal cell hyperplasia, and low-grade adenocarcinoma. The second part deals with differential diagnostic problems in the peripheral zone: prostatic intraepithelial neoplasia, postatrophic hyperplasia, Cowper’s glands, seminal vesicles, and ductal and intraductal carcinoma. Finally, atypical and small acinar proliferations are described. Diagnostic perspectives are discussed. Received: 23 June 1998 / Accepted: 13 August 1998     

An update on atypical large glandular proliferations of the prostate

The differential diagnosis of prostatic atypical large gland proliferations includes several benign and malignant entities. This review focusses on issues relevant to the practising pathologist, particularly around areas of controversy such as high-grade prostatic intraepithelial neoplasia (HGPIN) and intraductal carcinoma of the prostate (IDCP). HGPIN is a putative precursor of prostate cancer, but its clinical relevance is as a surrogate marker of unsampled prostate cancer, thereby identifying patients who would benefit from a prompt repeat biopsy. The incidence of missed prostate cancer is much lower in contemporary practice due to pre-biopsy MRI and extended sampling protocols so HGPIN is currently less important. It is however important to distinguish HGPIN from PIN-like carcinoma and IDCP. PIN-like carcinoma is considered a histological subtype/variant of acinar prostate carcinoma and should be graded as Gleason pattern 3. A diagnosis of cribriform HGPIN should not be made in needle biopsies as such a proliferation may represent IDCP. This review discusses controversies related to the diagnosis, reporting and management of IDCP. A personalized approach to management of patients with isolated IDCP in needle biopsies that is based on the histological and radiological features of an individual case is outlined.     

Diagnostic utility of a p63/alpha-methyl-CoA-racemase (p504s) cocktail in atypical foci in the prostate.

Prostatic needle biopsy is the preferred method for diagnosing early prostate cancer, providing specific information. In cases of histological cancer mimics, a diagnosis of atypical small acinar proliferation suspected of but not diagnosed as malignancy can be made. In such cases, and in small focus carcinomas, pathologists use 34betaE12, cytokeratin (CK) 5/6 or p63 immunostaining to label basal cells, and alpha-methylacyl-CoA racemase (AMACR/p504s) immunostaining as a positive prostate cancer marker on two distinct slides. However, in cases of small foci, ambiguous lesions might disappear. The purpose of our study was to improve the sensitivity of a cocktail of two antibodies (p63/p504s) with a sample incubation on 260 prostatic specimens, in order to help make a decision in conjunction with standard histology and CK 5/6 immunostaining. We tested 101 small focus prostatic cancers, 104 atypical small acinar proliferation, 19 high-grade prostatic intraepithelial neoplasia, two atypical adenomatous hyperplasia and 34 benign mimics of cancer. After p63/p504s immunostaining, the final diagnoses retained were as follows: 154 prostatic cancers, 14 atypical small acinar proliferation, 30 high-grade prostatic intraepithelial neoplasia, three atypical adenomatous hyperplasia and 62 benign mimics of cancer. To differentiate malignant from benign lesions, we used the criteria of greater sensitivity to p504s/p63 (95%) than to CK 5/6 (57%) or p63 (86%), and higher specificity for p504s/p63 (95%) than for CK 5/6 (88%) or p63 (81%). With the p504s/p63 cocktail, 89% of the ambiguous lesions were classified vs 53% for CK 5/6. Combined use of the two antibodies, one (p504s) as a positive marker and the other (p63) as a negative marker, with a simple immunostaining procedure, may improve diagnostic performance, sensitivity and specificity, leading to a reduction in the risk of false negatives; this technique in cases of atypical small acinar proliferation should reduce the percentage of residual ambiguous lesions and the need for additional biopsies.     

P504S、34βE12双重免疫组织化学染色在前列腺癌诊断中的应用

前列腺癌是老年男性常见的恶性肿瘤之一。近年来发病率逐渐上升。随着微创技术的发展,穿刺组织中微小癌的诊断越来越多,美国近期发现的前列腺癌的平均直径在1cm左右。前列腺癌的诊断主要依赖于光镜病理检查,但高分化前列腺癌和前列腺上皮内肿瘤（PIN）的差别非常小,有时单凭HE染色难以鉴别,     

Limiting the diagnosis of atypical small glandular proliferations in needle biopsies of the prostate by the use of immunohistochemistry

Prostatic biopsies containing small glandular formations suspicious of, but not diagnostic for, carcinoma represent a diagnostic dilemma, as they cannot be definitely identified as either benign or malignant. The term 'atypical small acinar proliferation' (ASAP) in the differential diagnosis of carcinoma has recently evoked considerable discussion. This study has tried to assess the biological potential of ASAP by further immunohistochemical (IHC) analysis. Biopsy-proven cases of ASAP (n=114) were analysed, in which consecutive sections still contained the suspicious lesion. IHC studies were undertaken with anti-cytokeratin 34betaE12 and the proliferation marker MIB-1. Staining with 34betaE12 revealed a complete basal cell layer in 25 cases (21.9%), a fragmented layer in 58 cases (50.9%), and absence of basal cells in 31 cases (27.2%). MIB-1 labelling indices (LIs) in these three groups were significantly higher than in benign prostatic tissue (p<0.001) and reached the level of low-grade prostatic carcinoma (p>0.05). The suspicious morphology of ASAP on haematoxylin and eosin-stained slides was supported by the finding of elevated proliferative activity. Subgroups were revealed by immunohistochemical assessment of basal cell status and cases without basal cells were diagnosed as carcinoma. Nevertheless, rebiopsy is recommended if radical surgery is planned, to exclude insignificant cancer. Cases with a complete or fragmented basal cell layer were regarded as non-malignant. Whether a fragmented basal cell layer reflects a technical artefact or transition to carcinoma is unknown, but the proliferative activity of both lesions was increased and corresponded to that of low-grade prostatic carcinoma. In these cases, therefore, at least clinical follow-up is strongly recommended and repeat biopsies are encouraged.     

Comparison of 34betaE12 and P63 in 100 consecutive prostate carcinoma diagnosed by needle biopsies.

P63, a homologue of p53, was recently identified as a useful basal cell-specific marker. We compared the sensitivity and specificity of p63 with the widely used high-molecular-weight keratin 34betaE12 for the diagnosis of prostate carcinoma in needle biopsies. We selected 100 consecutive prostate carcinoma diagnosed by needle biopsies with an adequate number of cancerous glands on the slide. We chose 1 representative hematoxylin and eosin-stained slide from each case and gave it a Gleason score. The same paraffin block was retrieved for 34betaE12 and p63 stains. We compared staining patterns of 34betaE12 and p63 on both malignant glands and benign glands and recorded basal cell density (percentage of basal cells with positive staining in the benign glands). The cases were divided into 3 groups according to the Gleason score: 5 to 6 (31 cases), 7 (46 cases), and 8 to 10 (23 cases). In 20 cases, focal and patchy staining in a basal cell distribution in malignant glands (range, 1%-20%; mean, 6.6%) was demonstrated (19 by both stains and 1 by 34betaE12 only). In 1 case with a Gleason score of 9, the cancer cells, not the basal cells, were stained focally by p63 but not by 34betaE12. Higher-grade tumors demonstrated higher numbers of malignant glands with basal cell staining (1.65% for Gleason 7, 1.26% for Gleason 8-10, compared with 0.42% for Gleason 5-6). The overall specificity of the absence of basal cell staining in the malignant glands for 34betaE12 and p63 was 98.63% and 98.60%, respectively. In 17 cases, both stains revealed total absence of basal cell staining in some benign glands (range, 1%-10%; mean, 3.5%). The overall sensitivity in identifying basal cells in benign glands was 99.48% and 99.44% for 34beta12 and p63, respectively. Basal cell density was higher for 34betaE12 in comparison with p63 (92% vs. 87%). For diagnosing prostate carcinoma in the needle biopsies, p63 is as specific and sensitive Hospital as 34betaE12 and therefore can be used as a complementary basal cell-specific stain for 34betaE12 in difficult cases.     

Negative 34betaE12 staining in a small focus of atypical glands on prostate needle biopsy: a follow-up study of 332 cases

Atypical glands on prostate needle biopsy with a negative 34betaE12 (cytokeratin 903; CK903) immunostain, indicating a lack of a basal cell layer, are typically diagnostic of prostate cancer. However, in certain cases a negative 34betaE12 immunostain in a small focus of atypical glands is still not convincing enough to make the diagnosis of cancer. This study is the first report to evaluate the incidence of prostate cancer on follow-up biopsy in individuals with this diagnosis. A total of 543 men who had prostate core biopsy specimens diagnosed as a small focus of atypical-appearing glands with a negative 34betaE12 immunostain between January 1, 1997 and December 31, 2000 were selected for study. Some 61% of these 543 individuals (n = 332) had undergone at least one follow-up biopsy procedure. Of these, 43% of repeat biopsy cases (n = 142) were diagnostic of prostate cancer. A total of 46 individuals had at least 2 follow-up biopsy procedures, with 48% of these (n = 22) being diagnosed as cancer. The Gleason grades of the detected carcinomas were broken down as follows: Gleason grade 3 + 2 = 5, 6%; grade 3 + 3 = 6, 86%; grade 3 + 4 = 7, 1%; grade 4 + 3 = 7, 4%; and grade 4 + 4 = 8, 3%. The median amount of time to the first follow-up biopsy was 79 days, with 52% of follow-up biopsies performed within 90 days. A negative 34betaE12 immunohistochemical stain in a small focus of atypical glands is not associated with an increased prediction of prostate cancer on follow-up biopsy (43%), compared with previously published data for "small focus of atypical glands" alone (approximately 45%). Because 48% of men with an initial negative biopsy and multiple follow-up biopsy procedures were found to have cancer, more than one repeat biopsy session or more extensive sampling on the first repeat biopsy procedure may be necessary to maximize the identification of cancer. This finding is similar to that found in men with atypical diagnoses in general, without a negative 34betaE12 immunohistochemical stain. Only half of all individuals with a diagnosis of 34betaE12-negative focus of atypical glands underwent repeat biopsy within 3 months. Urologists need to be educated as to the significance of an atypical diagnosis and the need for repeat biopsy. In a small focus of atypical glands on prostate biopsy, negative staining for 34betaE12 should not necessarily lead to a definitive malignant diagnosis in all cases, because almost half of these biopsies on follow-up sampling are benign.     

Prostate-specific antigen, high-molecular-weight cytokeratin (clone 34betaE12), and/or p63: an optimal immunohistochemical panel to distinguish poorly differentiated prostate adenocarcinoma from urothelial carcinoma

An optimal immunohistochemical panel to distinguish poorly differentiated prostate (PCa) from urothelial (UCa) carcinoma was selected from a panel consisting of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP), high-molecular-weight cytokeratin (HMWCK), clone 34betaE12, cytokeratin (CK) 7, CK20, p63, and alpha-methylacyl-coenzyme A racemase. The pilot group was composed of poorly differentiated UCa (n = 36) and PCa (n = 42). PSA and PAP stained 95% of PCa vs 0% and 11% of UCa cases, respectively. HMWCK and p63 stained 97% and 92% of UCa vs 2% and 0% of PCa cases respectively. CK7/CK20 coexpression was noted in 50% of UCa cases, whereas 86% of PCa cases were negative with both. A panel of PSA, HMWCK, and p63 was optimal for separating 95% PCa (PSA+/HMWCK and/or p63-) vs 97% UCa (PSA-/HMWCK and/or p63+). This panel was used on 26 diagnostically challenging cases and resolved 81% of cases as UCa vs PCa. The majority of PCa cases retain PSA. Negative PSA with positive HMWCK and/or p63 establishes a diagnosis of UCa.     

细胞角蛋白34βE12在鉴别乳腺良、恶性病变中的意义

目的 探讨高分子量细胞角蛋白34βE12作为良性病变的标记物对鉴别乳腺病变的意义。方法收集90例有随访活检和组织病理学诊断对照的乳腺细针吸取细胞学(FNAC)资料：良性病变50例,包括非增生性病变30例和增生性病变20例、导管内癌10例和浸润癌30例,对其FNAC涂片和相应的石蜡切片作34βE12的抗生物素蛋白-生物素-过氧化酶复合(ABC)法免疫组织化学分析。利用SPSS10．0软件进行统计学分析。结果 (1)34βE12在良性非增生和增生性病变组中的表达差异无显著性。(2)34βE12在良性病变和癌组中的表达差异具显著性,34βE12在癌组中,FNAC涂片和相应的石蜡切片分别为66．7％和63．3％的病例表现为完全阴性或散在1 的肿瘤细胞胞质阳性;在良性病变组中,FNAC涂片和相应的石蜡切片分别为100％和78％的病例表现为2 至3 的细胞阳性,且在石蜡切片中34βE12表现为完整的细胞膜和细胞质的强阳性,与癌中阳性标本之细胞质颗粒状阳性为主的表达特点不同。(3)34βE12在细胞分化较好的筛孔型、乳头型和实性型导管内癌中为完全阴性和散在细胞胞质阳性,而在细胞分化较差的粉刺型导管内癌中为阴性至3 的细胞阳性。结论 34βE12可作为乳腺病变鉴别诊断中良性病变的标记物,上皮细胞出现34βE12表达缺失时高度提示为癌;大量上皮细胞表达34βE12,且为细胞膜强阳性时,则应多考虑为良性病变。     

p63 expression in assessment of bronchioloalveolar proliferations of the lung.

Discrimination of well-differentiated pulmonary adenocarcinoma from reactive bronchioloalveolar epithelium can be difficult on routine histology, especially with small biopsies. Ancillary studies to help in this distinction are desirable. p63, a p53-homologous nuclear protein, is a marker of reserve cells of the bronchus and terminal lobular unit. In this study, 33 cases of adenocarcinoma (20 open lung and 13 transbronchial/percutaneous biopsies) and 43 cases of benign lungs with fibrosis and metaplasia (22 open lung and 21 transbronchial/percutaneous biopsies) were studied for nuclear p63 expression by immunohistochemistry (Dako, Carpinteria, CA, USA). Five additional cases each of atypical adenomatous hyperplasia and adenosquamous carcinoma and three cases of squamous carcinoma (all open lung biopsies) were also stained. The diagnostic categories of benign lung conditions were usual interstitial pneumonia, parenchymal scar, cryptogenic organizing pneumonia and diffuse alveolar damage. In neoplastic cases, p63 positivity was calculated as percentage of all tumor cells examined. In areas of normal lung, p63 decorated the reserve cells of large and small airways and occasional cells of the distal lobular unit. In fibrotic reactive processes, an interrupted but distinct pattern of nuclear staining was present in all cases, with staining of basal cells of the airways as well as bronchiolar- and squamous-metaplastic epithelium (43/43, 100%). p63 immunoreactivity was less uniform in areas of acute lung injury within these cases. One adenocarcinoma and two cases of atypical adenomatous hyperplasia showed strong immunoreactivity (>80%), while three adenocarcinomas highlighted only rare tumor nuclei (<5% of tumor cells). Morphologic areas where p63 immunostaining was not helpful included the junction of normal lung and lepidic growth of adenocarcinoma, and retrograde spread of adenocarcinoma into small airways. Our results highlight the differential expression of p63 across various bronchioloalveolar lesions. Moreover, p63 may be helpful in distinguishing reactive from neoplastic glandular proliferations in the lung.     

Diagnostic usefulness of monoclonal antibody P504S in the workup of atypical prostatic glandular proliferations

We stained 37 prostate needle biopsies and 3 transurethral resections (TURP) containing atypical foci and 20 morphologically unequivocal prostate cancer biopsies, including 4 with foamy features, with P504S. Of 20 biopsies with unequivocal cancer, 18 showed variable P504S staining (sensitivity, 90%); 1 minute cancer and 1 foamy cancer lacked P504S staining. Of 40 cases with atypical foci (biopsies, 37; TURP, 3), 9 were diagnosed as high-grade prostatic intraepithelial neoplasia (HGPIN), 2 were excluded, and 29 had foci of atypical small glandular proliferation. Of these 29 cases, 7 were highly suggestive of cancer, 2 of which lacked P504S staining. In 22 cases with benign atypical foci, 11 were diagnosed as postatrophic hyperplasia (none expressed P504S) and 7 as atypical adenomatous hyperplasia (AAH; 1 showed focal weak P504S staining). Of 9 HGPIN specimens, 8 showed predominantly diffuse, moderate P504S staining. P504S has slightly lower sensitivity for detection of prostate cancer than found previously. Heterogeneous expression patterns may explain negativity in some biopsy specimens with minute cancer. In atypical small glandular proliferations, diffuse positive P504S staining in atypical glands strongly supports a cancer diagnosis, but negative staining does not exclude it. P504S seems to have low sensitivity for detecting foamy prostate cancer. Most HGPINs show diffuse moderate P504S staining. AAH may show focal P504S staining. We recommend using P504S along with morphologic examination and conventional basal cell markers.     

High molecular weight cytokeratin antibody (clone 34betaE12): a sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer

AIMS: There is no well-established positive immunomarker for urothelial carcinoma. We evaluated the diagnostic utility of high molecular weight cytokeratin (HMWCK) antibody clone 34betaE12 in differentiating high-grade invasive urothelial carcinoma from prostate cancer. METHODS AND RESULTS: Formalin-fixed paraffin-embedded sections from 28 cases of high-grade invasive urothelial carcinoma (20 not otherwise specified (UC-NOS), eight with glandular differentiation) and 20 cases of poorly differentiated prostate carcinoma were immunostained with a monoclonal antibody to carcinoembryonic antigen (CEA), clone 85A12 and with HMWCK antibody clone 34betaE12 after microwave pretreatment or protease 24 predigestion. All cases of UC-NOS expressed HMWCK on 34betaE12 immunostaining after microwaving or enzyme predigestion. Immunoreactivity was intense and diffuse in all the cases after microwave pretreatment, whilst with enzyme predigestion immunoreactivity was sometimes patchy with <50% tumour cells positive in 20% of cases. In comparison with 34betaE12, 85A12 was insensitive with 15% of UC-NOS cases totally CEA-negative and <50% tumour cell immunoreactivity in 60% of cases. Rare positive cells were present in two (10%) cases of prostate cancer with monoclonal anti-CEA and 34betaE12 on microwaved sections, but all the cases were HMWCK-negative using 34betaE12 on sections pretreated by enzyme digestion. CONCLUSIONS: HMWCK antibody clone 34betaE12, particularly when used with microwave heat retrieval, is a very sensitive positive marker for high-grade invasive urothelial carcinoma.     

p63/AMACR antibody cocktail restaining of prostate needle biopsy tissues after transfer to charged slides: a viable approach in the diagnosis of small atypical foci that are lost on block sectioning

We assessed the utility of using a p63/a-methylacyl-coenzyme-A racemase (AMACR) antibody cocktail on destained H&E-stained sections. We transferred 61 stored (7-11 months old) and 10 recent (<1 month old) H&E-stained sections of prostate needle biopsy tissues to charged slides and subsequently stained them with a p63/AMACR immunohistochemical antibody cocktail. The AMACR and p63 staining intensities were compared with those obtained with the same antibody cocktail performed on sections recut directly from the paraffin block. Transfer of sections and subsequent immunohistochemical staining was successful in 69 (97%) of 71 cases. For stored cases, there were similar AMACR and p63 staining intensities in destained and recut sections in 55 (90%) and 11 (18%) of 61 cases, respectively. In recent sections, AMACR and p63 staining intensities were almost identical by both methods. We conclude that p63/AMACR cocktail immunostaining of destained H&E-stained sections is a viable approach in the workup of small "suspicious" foci in recently sectioned prostate needle biopsy tissues. This approach is best used when 2 or more H&E-stained sections harbor the suspicious focus, as we always recommended preservation of at least 1 H&E-stained section.