逆转录聚合酶链反应检测石蜡包埋组织中细胞周期蛋白D1 mRNA对套细胞淋巴瘤的诊断价值

目的 探讨用逆转录聚合酶链反应（RT-PCR）法和竞争性RT-PCR法检测套细胞淋巴瘤（MCL）石蜡包埋组织中细胞周期蛋白D1（cyclin D1）蛋白和mRNA在常规病理工作中的可行性及其诊断和鉴别诊断价值。方法 收集淋巴结内MCL38例、对照组包括结内小B细胞淋巴瘤58例（B小淋巴细胞性淋巴瘤14例,淋巴浆细胞性淋巴瘤3例,滤泡性淋巴34例,淋巴结边缘区B细胞淋巴瘤7例）和淋巴结反应性增生病例20例,用免疫组织化学EnVision法和RT-PCR法、竞争性RT-PCR法检测cyclin D1蛋白及其mRNA的表达,以看家基因PGK作为内对照检测RNA。结果 （1）38例结内MCL中,cyclin D1蛋白阳性率为71．1％（27／38）,对照组均为阴性。（2）116例标本中,可检出内对照PGK基因mRNA表达103例（88．8％）。38例MCL中PGK阳性36例（94．7％）。（3）38例结内MCL中,34例可检出cyclin D1 mRNA表达,去除PGK和cyclin D1 mRNA均阴性的2例,MCL中cyclin D1 mRNA表达的阳性率为94．4％（34／36）。对照组中B小淋巴细胞性淋巴瘤1例检出cyclin D1 mRNA表达,其余病例均未检出cyclin D1 mRNA表达。PCR结果全部经测序证实。（4）用竞争性RT-PCR,38例结内MCL中27例可检出cyclin D1 mRNA高表达,去除2例PGK也为阴性的病例,MCL中cyclin D1 mRNA高表达率为75．0％（27／36）。对照组小B细胞恶性淋巴瘤及淋巴结反应性增生无1例有cyclin D1 mRNA高表达。结论 RT-PCR方法和竞争性RT-PCR方法可在石蜡包埋组织中检测cyclin D1 mRNA的表达,均可用于MCL的诊断。     

套细胞淋巴瘤的临床病理特征和细胞周期蛋白D1在诊断中的意义

目的 观察套细胞淋巴瘤的临床病理学特征及细胞周期蛋白D1染色在诊断中的意义。方法 对8例淋巴结套细胞淋巴瘤作临床病理观察及随访,LSAB法做免疫表型分析（CD45RO、CD5、CD20、细胞周期蛋白D1、Ki-67、bcl-2）。结果 患者年龄43-78岁（平均年龄57岁）,男女3：1。组织学特点为：（1）淋巴结结构破坏并被单一的淋巴样细胞所取代,淋巴细胞以套区增生性、结节性、弥漫性三种模式增生。（2）淋巴样细胞核有一定的不规则性,染色质中等致密,核分裂象少见,类似中心细胞。其中有3例转变为高度侵袭性的母细胞样变型。所有的病例都呈cyclinD1与bcl-2阳性、CD20阳性、CD45RO阴性、CD5阳性。结论 套细胞淋巴瘤有其特征的形态改变及免疫表型。根据组织病理学特征及cyclin D1阳性,可与其它类型的小B细胞淋巴瘤相鉴别。套细胞淋巴瘤的母细胞样变型也应当与其它变型区别。     

Antibody selection in immunohistochemical detection of cyclin D1 in mantle cell lymphoma

An assessment in Nordic immunohistochemical Quality Control (NordiQC) revealed that only 23% participant laboratories performed optimal staining for detection of cyclin D1 (CyD1) in mantle cell lymphoma (MCL). False-negative results were secondary to suboptimal protocols. We compared the 5 anti-CyD1 antibodies (monoclonal SP4, P2D11F11, and DCS-6 and polyclonal CP236 and 06-137) used in the Scandinavian laboratories. Evaluated were 31 MCLs, 16 other malignant lymphomas, and 19 samples of normal tissues. Sensitivity was as follows: CP236, 100%; SP4, 95%; P2D11F11, 90%; DCS-6, 84%; and 06-137, 53%. SP4 produced the strongest staining. Correlation of CyD1 with the proliferative index was best with polyclonal antibodies CP236 and 06-137. The use of heat-induced epitope retrieval in an alkaline buffer such as 1/10 mmol/L of Tris-EDTA buffer, pH 9, seemed mandatory. For the optimal detection of CyD1 expression, both SP4 and CP236 antibodies should be available in the laboratory.     

Differentiation of osteoblasts in three-dimensional culture in processed cancellous bone matrix: quantitative analysis of gene expression based on real-time reverse transcription-polymerase chain reaction

Processed bovine cancellous bone (PBCB) is an attractive material for tissue engineering of bone. It is biocompatible, osteoconductive, nonimmunogenic, and porous and its biomechanical properties are close to those of native bone. In this study, differentiation of primary rat osteoblasts (rOBs) incubated on PBCB was investigated in vitro. rOBs were isolated and expanded in two-dimensional culture. Expanded rOBs were seeded into PBCB disks and cultured either in basal medium (BM) or differentiation medium (DM) containing ascorbic acid, beta-glycerol phosphate, and dexamethasone. Alkaline phosphatase (ALP) activity and RNA expression of ALP, bone sialoprotein (BSP), collagen type I (COL1), osteocalcin (OC), and osteopontin (OPN) were assessed by chemiluminescence assay and quantitative real-time RT-PCR over 14 days. Histologic analysis was performed on day 14. ALP increased over the observation period independent of stimulation. OPN and BSP expression was significantly higher in the DM group whereas COL1 and OC expression was significantly higher in the BM group. Matrix calcification was detectable only in the DM group by von Kossa stain. The observed expression patterns suggest a physiological response of rOBs to the differentiation stimulus. PBCB is a suitable matrix for in vitro differentiation of osteoblasts. Cell-seeded PBCB is a potential osteogenic construct for in vivo application.     

Catalyzed signal amplification for cyclin D1 detection in mantle cell lymphoma.

Mantle cell lymphoma is characterized by a t(11;14)(q13;q32) translocation resulting in cyclin D1 protein overexpression. Immunohistochemical detection of the latter, therefore, is a useful marker for the diagnosis of mantle cell lymphoma. Nevertheless, interpretation of results is often hampered by the weak immunoreactivity obtained with routine detection techniques. This problem can be overcome by resorting to highly sensitive catalyzed signal amplification methods based on peroxidase-catalyzed deposition of a biotinylated phenolic compound. The present study compares the results obtained with catalyzed signal amplification, labeled streptavidin biotin, and dextran polymeric conjugate (EnVision+) techniques in cyclin D1 demonstration in mantle cell lymphoma. The study was performed on formalin-fixed, paraffin-embedded archival tissue from 20 mantle cell lymphoma cases. Ten cases of small lymphocytic lymphoma and 10 instances of follicular center cell lymphoma were used as controls. Antigen retrieval was done by autoclaving under controlled pressure (2 bar) and temperature (120 degrees C) conditions. The best results were obtained after 1 minute of exposure with catalyzed signal amplification and after 6 minutes with other detection systems. Regarding cyclin D1 expression in mantle cell lymphoma cases, 17 (85%) were weakly positive and 3 (15%), moderately positive with labeled streptavidin biotin, whereas 15 (75%) were weakly positive and 5 (25%) moderately positive with EnVision+. In contrast, all 20 mantle cell lymphoma cases were strongly cyclin D1 positive with catalyzed signal amplification. No evidence of cyclin D1 immunostaining was obtained in any of the small lymphocytic lymphoma and follicular center cell lymphoma instances with any of the three methods used. In conclusion, catalyzed signal amplification methods provide a very useful tool for cyclin D1 demonstration in cases in which other immunohistochemical techniques yield inconclusive results.     

LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法的建立

目的 人副流感病毒1,2,3型是呼吸道感染的主要病原.本研究建立了特异、快速、灵敏的多重荧光定量RT-PCR方法用于人副流感1,2,3型病毒临床标本检测.方法 针对人副流感1,2,3型病毒设计特异性引物探针,优化荧光RT-PCR反应条件.应用体外转录方法分别制备人副流感1,2,3型病毒的标准品.验证荧光定量RT-PCR方法的特异性,敏感性和稳定性.结果 该方法对人副流感1,2,3型病毒核酸检测有高度特异性,检测的灵敏度HPIV1为10个拷贝,HPIV2为100个拷贝,HPIV3为100个拷贝.可从临床患者鼻咽吸出物标本中直接检出.结论 本研究建立的LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性.适用于临床早期诊断和实验室病原谱筛查.     

SOX11可表达于Cyclin D1阴性母细胞样套细胞淋巴瘤

近期的研究表明SOX11表达有助于套细胞淋巴瘤(MCL)的诊断,包括具有典型形态学改变的cyclin D1阴性MCL。本文作者探讨了SOX11在B细胞非霍奇金淋巴瘤(B-NHL)某些亚型中的表达模式及其特异性,并将其作为免疫表型特征分析cyclin D1阴性的母细胞样MCL(该组病例系首次报道)的识别价值。作者应用免疫组化方法评估了     

Morphological spectrum of cyclin D1-positive mantle cell lymphoma: study of 168 cases

Immunostaining for cyclin D1 is essential for reliable diagnosis of mantle cell lymphoma (MCL). However, a small number of cyclin D1-positive lymphomas other than MCL have been encountered. Our goal was to investigate the morphological spectrum of MCL as a disease entity, based on cyclin D1 overexpression. We reviewed 181 biopsy specimens obtained from 168 cases of cyclin D1-positive MCL. Typical findings were the presence of nodular (53.9% of cases) or diffuse (46.1%) histological patterns, containing mantle zone patterns (16.8%), naked germinal centers (33.5%) and perivascular hyaline deposition (83.2%). Unusual findings of residual germinal centers with a mantle cuff (four cases) and follicular colonization (two cases) were seen. High magnification showed a monotonous proliferation of tumor cells with cytological diversity including small (3.0%), intermediate (43.1%), medium (34.1%), medium-large (13.2%) and large (6.6%) cells. Pleomorphic and blastic/blastoid variants were encountered in 9.6 and 7.2% of cases, respectively. Three cases had foci of cells of considerable size, with a moderately abundant pale cytoplasm resembling marginal zone B cells. Two cases showed an admixture of cells which appeared transformed and mimicked the histology of chronic lymphocytic leukemia/small lymphocytic leukemia. In one, neoplastic mantle zones were surrounded by sheets of mature plasma cells, resembling the plasma cell type of Castleman's disease. An admixture of areas characteristic of MCL and of other larger cells, indicating histological progression or a composite lymphoma, were observed in seven cases. In high-grade lesions of five cases, nuclear staining of cyclin D1 was rarely detected. In our experience, cyclin D1 expression was also found in nine lymphomas other than MCL (five plasma cell myelomas, three Hodgkin's disease and one anaplastic large cell lymphoma). The application of cyclin D1 staining prompted us to recognize the broad morphological spectrum of MCL. MCL can be diagnosed with the application of cyclin D1 immunostaining, if careful attention is given to architectural and cytological features.