Eniko Bagdi

Follicular dendritic cells in reactive and neoplastic lymphoid tissues: A reevaluation of staining patterns of CD21, CD23, and CD35 antibodies in paraffin sections after wet heat-induced epitope retrieval

Structural alterations in the meshwork of follicular dendritic cells (FDCs) are frequently found in malignant lymphomas. Formaldehyde fixation and paraffin embedding, however, have long prevented consistent detection of FDCs. Wet heat-induced epitope retrieval in Dako Target Retrieval Solution (TRS) (pH 6.0) enabled the reliable detection of FDCs through CD21, CD23, and CD35 antigens in routinely processed tissues from 11 reactive and 69 neoplastic lymphoproliferations, thus allowing the distribution of the FDCs to be reevaluated. Germinal center FDCs in lymphoid hyperplasias and expanded FDC meshworks in the 8 mantle cell lymphomas, 7 low-grade MALT lymphomas, and 6 low-grade follicular lymphomas were intensely stained with all these markers. In 6 cases of B cell chronic lymphocytic leukemia, tumor cells were CD23+. In four cases of nodular lymphocyte predominance Hodgkins disease (HD), expanded FDC meshworks sharply delineating negative tumor cells and their rosetting T cell, were revealed mainly with the CD21 and CD35 antibodies. Follicular dendritic cells were also demonstrated in 11 cases of grade I nodular sclerosing HD, including follicular HD. Striking dendritic cell clusters were revealed with all 3 antibodies in 9 angioimmunoblastic T cell lymphomas. Sparse or no FDC meshworks were detected in the 4 cases of grade II nodular sclerosing HD, 5 follicular lymphomas with high-grade transformation, and 5 T cell–rich B cell lymphomas. CD35 immunostaining showed the most consistent labeling in the four FDC sarcomas studied in the current article. Reproducible demonstration of FDCs in routinely processed paraffin sections with CD21, CD23, and CD35 antibodies, as presented here, provides invaluable pieces of information in the diagnosis of lymphoproliferative disorders.

Transient upregulation of connexin43 gap junctions and synchronized cell cycle control precede myoblast fusion in regenerating skeletal muscle in vivo

The spatio-temporal expression of gap junction connexins (Cx) was investigated and correlated with the progression of cell cycle control in regenerating soleus muscle of Wistar rats. Notexin caused a selective myonecrosis followed by the complete recapitulation of muscle differentiation in vivo, including the activation, commitment, proliferation, differentiation and fusion of myogenic cells. In regenerating skeletal muscle, only Cx43 protein, out of Cx-s 26, −32, −37, −40, −43 and −45, was detected in desmin positive cells. Early expression of Cx43 in the proliferating single myogenic progenitors was followed by a progressive upregulation in interacting myoblasts until syncytial fusion, and then by a rapid decline in multinucleate myotubes. The significant upregulation of Cx43 gap junctions in aligned myoblasts preceding fusion was accompanied by the widespread nuclear expression of cyclin-dependent kinase inhibitors p21waf1/Cip1 and p27kip1 and the complete loss of Ki67 protein. The synchronized exit of myoblasts from the cell cycle following extensive gap junction formation suggests a role for Cx43 channels in the regulation of cell cycle control. The potential of Cx43 channels to stimulate p21waf1/Cip1 and p27kip1 is known. In the muscle, proving the involvement of Cx43 in either a direct or a bystander cell cycle regulation requires functional investigations.

Optimization of PCR Amplification for B- and T-cell Clonality Analysis on Formalin-fixed and Paraffin-embedded Samples

In many cases, particularly in retrospective studies, only formalin-fixed and paraffin-embedded (FFPE) tissue samples are available for molecular studies. DNA recovered from FFPE tissues generally consists of fragmented small target sequences with chemical alterations. Clonality analysis is not easy on FFPE samples, in fact, it requires even more experience than that of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes. In our study, we have performed a multi-parameter PCR evaluation investigating immunoglobulin heavy chain gene (IgH) and T-cell receptor gamma gene (TCRy) rearrangements on non-purified crude lysates of FFPE samples, in order to establish the significance of different variables on performance of PCR amplification. The results showed that a slight decrease in the concentration of primers in combination with a slight increase in MgCl2 andTaq polymerase concentrations, as well as the use diluted crude template and a standard amount of dNTPs can be the modifications of choice while adjusting IgH and TCRy clonality tests on poor quality DNA FFPE samples. Using our improved protocol, 74% (17/23) of the tested B-cell lymphomas and 68% (31/46) of the tested T-cell lymphomas demonstrated monoclonal PCR product, proving the applicability of our optimized method. Our experience may be of help during the optimization process in technically difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive results.

How we process trephine biopsy specimens: Epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only ∼30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5′→3′ DNA polymerase and 3′→5′ exonuclease activity

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (rTth DNA Polymerase, XL) providing both 5′→3′ polymerase and 3′→5′ exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The rTth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the rTth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

Nagy, granulált citoplazmájú lymphocytás leukaemia. Ritka betegség, személyre szabott kezelési lehetoségekkel

Bevezetes: A nagy, granulalt lymphocytas leukaemia ritka, legtobbszor kronikus lefolyasu betegseg. Vezető tunete a neutropenia, de okozhat mas immun medialta cytopeniat is. A kezeles megkezdeset a tunetek sulyossaga indokolja. Nincs egyseges kezelesi ajanlas, a valasztasanal a klinikai tuneteket kell figyelembe venni. Celkitűzes: A szerzők sajat tapasztalataik alapjan osszefoglaljak a diagnozis fontos lepeseit es a kezeles lehetőseget, illetve szuksegesseget. Modszer: Osszesen 17 nagy, granulalt lymphocytas leukaemias beteg adatait dolgoztak fel retrospektiv modon. Eredmenyek: A 17 beteg kozul 7 esetben volt szukseg kezelesre, kozuluk 4 esetben indokolta ezt a transzfuziot igenylő anaemia, a tobbi betegnel neutropenia miatt indult kezeles. Negy esetben kortikoszteroid adasat kezdtek, egy esetben ciklosporinnal kiegeszitve. Anti-CD52-, kis dozisu metotrexat- es kombinalt kemoterapia egy-egy betegnel tortent. A kezelesek hatasara 5 betegnel sikerult reszleges valaszt elerni, mig ket beteget szepszis kovetkezteben elveszitettek. Kovetkeztetesek: A diagnozis felallitasat kovetően a betegek kisebb hanyada igenyel kezelest. Az immunszuppressziv kezeles eredmenyes lehet, de a hatas gyakran atmeneti. A legsulyosabb szovődmeny a szepszis, amely neutropenias esetekben tovabbra is jelentős mortalitasi kockazattal jar. Orv. Hetil., 2014, 155(11), 414–419. | Introduction: Large granular lymphocyte leukemia is rare, mainly chronic disease. The most common complication is neutropenia, but other immune-mediated cytopenia may also occur. There are no unified treatment recommendations and initiation of treatment mainly depends on the severity of the symptoms. Aim: The aim of the authors was to analyze the main steps of the diagnosis and the necessity and outcome of treatment in their patients diagnosed with large granular lymphocyte leukaemia. Method: The authors retrospectively analyzed the data of 17 large granular lymphocyte leukemia patients. Results: Of the 17 patients, 7 patients required treatment because of transfusion dependent anemia (4 patients) or neutropenia (3 patients). In 4 patients corticosteroid was given (supplemented with cyclosporine in one patients), while the other patients received anti-CD52 (one patient), low dose methotrexate (one patient) and combined chemotherapy (one patient). Five patients achieved partial response, and two patients died in sepsis. Conclusions: In this cohort only a smaller proportion of patients required therapy. Immunosuppression can be successful, but the effect in most cases was temporary. The most serious complication was sepsis, which is associated with a significant risk of mortality in cases with neutropenia. Orv. Hetil., 2014, 155(11), 414–419.INTRODUCTION Large granular lymphocyte leukemia is rare, mainly chronic disease. The most common complication is neutropenia, but other immune-mediated cytopenia may also occur. There are no unified treatment recommendations and initiation of treatment mainly depends on the severity of the symptoms. AIM The aim of the authors was to analyze the main steps of the diagnosis and the necessity and outcome of treatment in their patients diagnosed with large granular lymphocyte leukaemia. METHOD The authors retrospectively analyzed the data of 17 large granular lymphocyte leukemia patients. RESULTS Of the 17 patients, 7 patients required treatment because of transfusion dependent anemia (4 patients) or neutropenia (3 patients). In 4 patients corticosteroid was given (supplemented with cyclosporine in one patients), while the other patients received anti-CD52 (one patient), low dose methotrexate (one patient) and combined chemotherapy (one patient). Five patients achieved partial response, and two patients died in sepsis. CONCLUSIONS In this cohort only a smaller proportion of patients required therapy. Immunosuppression can be successful, but the effect in most cases was temporary. The most serious complication was sepsis, which is associated with a significant risk of mortality in cases with neutropenia.

Nazális NK/T-sejtes lymphoma hosszú túlélése

The nasal NK/T cell lymphoma is a rare, extranodal non-Hodgkin lymphoma in western civilizations, which has poor prognosis. The Epstein-Barr virus can be detected in tumor cells in nearly all cases. There are no definite treatment guidelines in our days. There is no significant difference in survival between radiotherapy and chemotherapy according to Asian studies. In this case study we show our diagnostic procedures, our treatment options and we present the summary of this illness based on the data found in the literature.

Refrakter sprue mint az enteropathia típusú T-sejtes lymphoma prekurzor laesiója - Klinikopatológiai esetismertetés

INTRODUCTION Refractory sprue is characterised by distinctive morphologic alterations and the emergence of clonal intraepithelial lymphocytes. AIM In this case report the authors emphasize the importance of histopathology in the diagnosis of refractory sprue. METHODS The sequential biopsies from this patient have been investigated with routine histology, immunohistochemistry and molecular genetics for T-cell clonality analysis. RESULTS The severely cachectic patient presenting with malabsorption syndrome has been diagnosed with celiac disease through a duodenal biopsy, and the CD8 negativity of the intraepithelial lymphocytes suggested the possible diagnosis of refractory sprue. Azathioprine and glucocorticoid therapy was administered due to the failed jejunal feeding and gluten-free diet, resulting in clinically complete, morphologically partial remission. Intestinal T-cell lymphoma developed in the ileocecal region within two years after the first clinical presentation. DISCUSSION Refractory sprue and the enteropathy-type T-cell lymphoma constitute a disease spectrum. The reported case shows how a simple method can provide crucial information in the diagnosis of refractory sprue.