Lymphoid Tissue Samples Research Articles

The effect of acrylamide on alpha-naphthyl acetate esterase enzyme in blood circulating lymphocytes and gut associated lymphoid tissues in rats

The aim of this study is to determine the functional effects of the acrylamide (AA) administrated by oral gavage on the peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in male Sprague-Dawley rats using alpha-naphthyl acetate esterase (ANAE) demonstration. For this purpose, two separate experiments were performed with Sprague Dawley rats. In Experiment-I rats were gavaged with 0, 30, 45 and 60 mg/kgb.w. AA for five consecutive days and in Experiment-II rats were gavaged with 0, 125, 150, and 175 mg/kg/b.w. AA for single oral dose. Animals were sacrificed 24 h after the last treatments in both experiments by servical dislocations under ether anaesthesia. Blood samples were collected from the heart in heparinized (10 UI heparin/ml(-1) of the blood) tubes before sacrification and lymphoid tissue samples from the ileal Peyer's patches (IPPs) were taken and processed for histochemical demonstration of ANAE following the sacrification. The lymphoid follicles of the IPPs of animals given 125, 150 and 175 mg/kgb.w. AA were markedly reduced in size. Germinal centres (GCs) markedly regressed in AA-treated animals compared with those of controls. ANAE-positive lymphocyte depletion of IPPs was very prominent in the high doses AA-treated animals. In the animals treated with 30, 45, and 60 mg/kg b.w. AA, the IPPs had similar histology to those of the controls. ANAE-positive peripheral blood lymphocyte levels significantly decreased in AA exposed groups in a dose dependent manner (p<0.05). In conclusion, AA has detrimental effects on peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in rats.

Epizootic of dolphin morbillivirus on the Catalonian Mediterranean coast in 2007

BETWEEN 1990 and 1992, thousands of striped dolphins ( Stenella coeruleoalba ) stranded along the Mediterranean coast due to a newly described virus, the dolphin morbillivirus (DMV) (Domingo and others...

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

To establish the utility of flow cytometry (FCM) for screening and diagnosis of B cell non-Hodgkin lymphoma (B-NHL) from lymphoid tissue samples obtained by fine-needle aspiration (FNA). We compared prospectively FCM versus cytology/histology analysis of FNA samples for the diagnostic screening and further World Health Organization (WHO) subclassification of B-NHL. FCM and cytology showed a high degree of agreement (93%); however, diagnosis of reactive processes (RP), B-NHL and T-NHL by FCM showed higher sensitivity than cytology (92-100% versus 64-94%, respectively), without false positive NHL cases. The antibody combination used did not allow a positive diagnosis of Hodgkin lymphoma as distinct from a RP. A high concordance rate was found between FCM and histopathology (74%) in subtyping B-NHL. In this regard, mantle-cell lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma showed the highest degree of agreement (100% concordant rates). In turn, FCM showed higher sensitivity/specificity in classifying follicular lymphoma (FL) and large B cell lymphomas, while the opposite occurred for marginal-zone and lymphoplasmacytic lymphomas. FCM enhances the diagnostic ability of FNA cytology, playing a crucial role in a rapid and accurate differential diagnosis between RP, B-NHL and T-NHL. In addition, immunophenotyping of FNA samples contributes to a more precise subclassification of B-NHL when combined with histopathology and genetic/molecular data.

Chicken anemia virus infection in broiler chickens in Shahrekord, Iran: serological, hematological, and histopathological findings

Chicken infectious anemia caused by a single-strand DNA circovirus is a disease in young chickens that is characterized by aplastic anemia, generalized lymphoid atrophy, and immunosuppression. In the present study, the presence of chicken anemia virus (CAV) infection and the hematologic and histopathologic changes in CAV seropositive broiler chickens in Shahrekord region, center of Iran, were investigated. Blood and lymphoid tissue samples were obtained from 271, 2–6-week-old chicks of 23 commercial broiler chicken flocks. Measurement of CAV antibody titers by enzyme-linked immunosorbent assay (ELISA) and differential leukocyte counts were carried out on serum and whole blood, respectively. The results of ELISA showed that all flocks of different ages were positive for anti CAV antibodies. Histological examination of lymphoid organs revealed lymphocytic depletion in 72 chicks, of which 66 cases were seropositive in ELISA. Twenty chicks had lymphocytic depletion in both thymus and bursa of Fabricius and 46 cases had only bursal lymphoid depletion. Macroscopic appearance of bone marrows and spleen were normal. The mean antibody titers, packed cell volumes (PCVs), and number of lymphocytes and heterophils of the chickens with both thymic and bursal lymphoid depletion were significantly different than antibody titers and PCVs of the other group. These results have demonstrated CAV infection and immunosuppressive effect of this virus in broiler chickens and the need for vaccination of breeder flocks in center of Iran.

Reactivation and Mutation of Newly Discovered WU, KI, and Merkel Cell Carcinoma Polyomaviruses in Immunosuppressed Individuals

Infection with the human polyomaviruses BK (BKV) and JC (JCV) is almost ubiquitous, asymptomatic, and lifelong. However, reactivation during immunosuppression, associated with mutations in the transcriptional control region (TCR) that up-regulates viral replication, can cause life-threatening disease. In this study, we investigated whether the recently discovered WU and KI polyomaviruses (WUPyV and KIPyV) and Merkel cell polyomavirus (MCPyV) could, like BKV and JCV, persist, mutate, and reactivate in immunodeficient subjects. Autopsy samples of lymphoid tissue from 42 AIDS-immunosuppressed subjects and 55 control samples were screened by polymerase chain reaction for all 5 polyomaviruses. TCR sequences from KIPyV and WUPyV recovered from both immunosuppressed and nonimmunosuppressed subjects were compared. Combined polyomavirus detection frequencies were much higher for the immunosuppressed group, compared with the nonimmunosuppressed group (35.7% vs. 3.6%), with viral loads in lymphoid tissues ranging from < or = 8.4 x 10(5) to > 1.5 x 10(5) viral genome copies per 10(6) cells. MCPyV was recovered from only 1 HIV-negative study subject. TCR sequences from reactivated WUPyV and KIPyV variants showed a number of point mutations and insertions that were absent in viruses recovered from respiratory tract specimens obtained from nonimmunosuppressed subjects. KIPyV and WUPyV show reactivation frequencies comparable to those of BKV and JCV during immunosuppression. TCR changes that potentially lead to transcriptional dysregulation may have pathogenic consequences equivalent in severity to those observed for JCV and BKV.

Evaluation of the Risk of PRRSV Transmission via Ingestion of Muscle from Persistently Infected Pigs

The objectives of this experiment were to determine how long porcine reproductive and respiratory syndrome virus (PRRSV) could be detected in muscle tissues of experimentally infected pigs and to evaluate the transmissibility of PRRSV to pigs via ingestion of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)-positive muscle tissues. Serum, lymphoid tissues, and muscle (M. longissimus dorsi) samples were collected from 135 pigs (89 PRRSV-inoculated pigs and 46 negative control). Between 28 and 202 days post-inoculation, 13 of 89 (14.6%) muscle samples were positive by qRT-PCR. Among these 13, PRRSV was isolated from four of the 13 corresponding serum samples and three of 13 lymphoid tissue samples. In addition, infectious virus was detected in lymphoid tissue homogenates of six of 13 pigs by intramuscular bioassay. Swine transmissibility studies were performed by feeding thirteen 3-week-old PRRSV-naive pigs (recipient pigs) qRT-PCR-positive muscle and then monitoring recipients for evidence of PRRSV viremia by qRT-PCR. No transmission of PRRSV to recipient pigs via consumption of muscle samples was observed. These data suggested that qRT-PCR detected non-infectious PRRSV in pig meat and/or PRRSV is not highly transmissible to susceptible pigs via consumption of PRRSV-contaminated meat.

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis-specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1.2% (2/162) and 17.2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27.7% (45/162) of blood and 29.0% (47/162) of lymphoid tissue samples. The species-specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0.01 in blood PCR, P < 0.001 in tissue PCR) and serologically negative (P < 0.001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.

The Reliability of Lymphoma Diagnosis in Small Tissue Samples Is Heavily Influenced by Lymphoma Subtype

A specific pathologic diagnosis is important in malignant lymphoma because the diverse disease subtypes require tailored approaches to clinical management. Reliance on small samples obtained with cutting needles has been advocated as a less invasive alternative to using larger, excised samples. Although published studies have demonstrated the safety and apparent sufficiency of this approach in informing clinical care, none have systematically determined the accuracy of pathologic lymphoma subtyping based on very small samples. We used a tissue microarray representing 67 cases of malignant lymphoma and 17 samples of nonneoplastic lymphoid tissue to model lymphoma diagnosis in small samples. Overall, 73.8% of the cases were diagnosed with a level of confidence deemed sufficient for directing clinical management; 85.9% of these diagnoses were accurate. Small cell lymphomas with highly distinctive immunophenotypes, including small lymphocytic, mantle cell, and T-lymphoblastic lymphoma, were recognized most consistently and accurately in the small samples. In contrast, follicular lymphoma and marginal zone lymphoma were especially difficult. Our results indicate that the reliability of lymphoma diagnoses based on small samples is heavily influenced by lymphoma subtype.

Expression of mucosal addressin cell adhesion molecule-1 during small bowel graft rejection in rat

To investigate the expression of mucosal addressin cell adhesion molecule-1(MAdCAM-1) during small bowel graft rejection and the effects of MAdCAM-1 on the development of acute rejection. Rat heterotopic small bowel transplantation (SBT) was performed in F344/N rats with syngeneic and allogeneic (BN-F344/N) grafts. Bowel and gut-associated lymphoid tissue(GALT) samples were collected from small bowel transplants on postoperative day(POD) 1, 3, 5 and 7. Histopathology assessment of the grafts was conducted to identify the rejection. MAdCAM-1 was detected by immunohistochemistry and Western blot. During acute rejection, MAdCAM-1 was highly-expressed on gut lamina propia and GALTs, particularly on vascular endothelial cells in the gut lamina propia. There were no change of MAdCAM-1 expression in syngeneic grafts from POD1 to POD7. In allogeneic grafts, MAdCAM-1 expression in mesenteric lymph nodes was down-regulated, while up-regulated on the vascular endothelial cells in the lamina propria during acute rejection. Alteration of MAdCAM-1 expression may be associated with the development of SBT graft rejection.

Survey for transmissible spongiform encephalopathies in Irish pigs fed meat and bone meal

Samples of brain and lymphoid tissues from 1107 meat and bone meal-fed, culled adult pigs from 24 Irish farms were examined for evidence of transmissible spongiform encephalopathy (tse) by histopathological,...

Loss of CD19 expression in B‐cell neoplasms

To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas. A polyclonal antibody to the C terminus of CD19 was used to immunostain paraffin-embedded samples of normal and neoplastic lymphoid tissues. CD19 was widely expressed in normal B cells and in extramedullary plasma cells. It was found in most B-cell neoplasms, but expression in follicular lymphoma was weak (33/69) or negative (four cases). Similarly, CD19 expression in diffuse large B-cell lymphomas was weak (28/56) or negative (eight cases). In T-cell-rich B-cell lymphomas, CD19 was also weak (4/10) or negative (three cases). CD19 was often absent in post-transplant B lymphoproliferative disease, classical Hodgkin's disease and plasma cell neoplasms. An unexpected finding was the frequent absence of CD19 in the neoplastic cells in lymphocyte predominant Hodgkin's disease. CD19 can now be detected in routine biopsy specimens. In contrast to the classical pan-B marker CD20, CD19 is not always strongly expressed in B-cell neoplasms. Furthermore, the lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin's disease (which express most B-cell-associated markers) commonly lack CD19.

Pathogenesis and prevalence of variant Creutzfeldt–Jakob disease

In the late 1980s and early 1990s, there was widespread exposure of the UK population to bovine spongiform encephalopathy (BSE)-contaminated food products, which has led to over 150 deaths from variant Creutzfeldt-Jakob disease (vCJD). Although the pathogenesis in humans is not fully understood, data from animal models and, to a lesser extent, patients with vCJD suggest that oral exposure to BSE is rapidly followed by accumulation of PrP(res) in gut-associated lymphoid tissue, then, after haematogenous spread, throughout the lymphoreticular system. Spread to the central nervous system may not occur for several years, but blood from individuals in the pre-clinical phase appears to be able to transmit disease. The incidence of vCJD has remained low and is in decline, but it is known from iatrogenic CJD and kuru that human prion disease can have incubation periods of up to 40 years. Cases of vCJD are therefore likely to occur for many more years and alternative phenotypes may develop in individuals with different PRNP genotypes to those seen to date. Studies in transgenic mice have shown that sub-clinical infection is frequent following oral exposure to BSE and a study looking at the accumulation of PrP in anonymized human lymphoid tissue samples found positive cases. There are likely to be a number of asymptomatic 'carriers' of disease within the UK and although it is unclear whether these individuals will develop clinical disease, there is a potential for iatrogenic spread to others. These uncertainties highlight the importance of developing a reliable blood test for vCJD and the continued need for surveillance.

Evaluation of CD52 Expression in Hematopoietic Neoplasms by Standard Immunohistochemistry: Implications for the Expanded Use of Alemtuzumab (CAMPATH-1H) in the Treatment of Hematological Malignancies.

CD52 is a GPI-linked glycoprotein expressed by B cells, T cells, monocytes and macrophages. The humanized monoclonal antibody alemtuzumab (CAMPATH-1H) is specific for CD52 and is FDA-approved for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL). The utility of alemtuzumab in the treatment of other lymphoid and non-lymphoid malignancies has been recently explored; however, a comprehensive survey of CD52 expression among the various classes of hematopoietic neoplasms has not been completed. In addition, most methods of detecting CD52 rely on flow cytometric techniques that cannot be performed in a retrospective manner. Here we present methods for the reliable detection of CD52 on fixed, paraffin-embedded human bone marrow and lymphoid tissue samples using standard immunohistochemical staining methods and a non-humanized form of CAMPATH (CAMPATH-1G; Serotec, UK). Over 200 cases of B and T cell lymphomas and leukemias were evaluated. As summarized below (see table), the vast majority of low-grade B cell lymphoproliferative disorders (CLL/SLL, follicular lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma) express CD52. Interestingly, some heterogeneity was noted within certain of these tumor types. For example, plasma cells were largely negative in lymphoplamsmacytic lymphomas, and proliferation centers were more intensely positive in CLL/SLL. In addition, we found that the majority of precursor B-cell acute lymphoblastic leukemia/lymphomas express this antigen. In contrast, there is some heterogeneity in CD52 expression among diffuse large B cell lymphomas, with approximately 22% of these tumors expressing undetectable or extremely low levels of CD52. Neoplasms of the T cell lineage also show marked heterogeneity in CD52 expression. The majority of precursor T cell acute leukemia/lymphomas and anaplastic large cell lymphomas showed no detectable CD52 expression; however, among the remaining T cell tumors, there was more pronounced tumor to tumor variation in CD52 expression. Among acute myeloid leukemias, we found CD52 expression only in AML-M4Eo. Finally, all cases of Hodgkin lymphoma and multiple myeloma tested were negative for CD52 expression. Together, these findings expand the subclasses of hematopoietic neoplasms that are rational candidates for alemtuzumab (CAMPATH-1H) therapy, and, in addition, reveal that CD52 is not universally expressed by lymphoid malignancies. Thus, target validation using this standard immunohistochemical procedure can now be readily performed on a case-by-case basis and is likely to be essential to guide alemtuzumab (CAMPATH-1H) therapy for hematological tumors.Expression of CD52 by Hematopoietic NeoplasmsDiagnosis #Cases Tested# Cases CD52 Positive% Cases CD52 PositiveCLL/SLL2525100Follicular Lymphoma77100Mantle Cell Lymphoma2020100Lymphoplasmacytic Lymphoma2727100Burkitt Lymphoma11100Diffuse Large B Cell Lymphoma272178Pre-B ALL181689Pre-T ALL1417AML232*9Peripheral T cell Lymphoma18739Anaplastic Large Cell Lymphoma600Angioimmunoblastic Lymphoma3133ATLL22100Hepatosplenic T cell Lymphoma100NK/T Cell Lymphoma2150Multiple Myeloma1800Hodgkin Lymphoma1100* 2 of 2 Cases of AML-M4Eo positive

Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance.

The prognostic significance of IgH/Bcl2 rearrangement in follicular lymphoma (FL) remains contentious; polymerase chain reaction (PCR) methodology and tissue source variability may account for some inconsistencies. As IgH/Bcl2 major breakpoint region (MBR) sequences may be found in normal blood, an MBR+ result by conventional PCR in blood/bone marrow may not indicate FL. To establish tumour MBR status, 190 lymphoid tissue samples with histologically evident FL (and therefore >1% tumour cells) were examined by real-time quantifiable PCR; 50% (95/190) had clonal MBR IgH/Bcl2 (MBR was considered clonal when >1%). Overall survival (median = 11.5 years) of MBR+ and MBR- patients was not significantly different.

Gizzerosine-induced histopathological lesions in broiler chicks

1. The aim of this study was to investigate pathomorphological changes in broiler chicks fed with different doses of gizzerosine, a substance produced during the heat treatments of fish meal. 2. The experiment was carried out in Ross broiler chicks which were divided into three groups: group A received 100% of non-medicated commercial mash for broiler chicks. During an experimental 5-d period, 50% of commercial mash was replaced with unheated fish meal (0.65 ppm gizzerosine) in group B and in group C with heated fish meal (1.15 ppm gizzerosine). Fourteen chicks from each group were killed every day. Samples of gastrointestinal and lymphoid organs, lung, pancreas, liver, brain and kidney tissue were sampled for histopathological analysis. Organs were embedded in paraffin and stained with hematoxylin-eosin stain and using periodic acid-Schiff reagent (PAS) and Sudan III (frozen sections). 3. Necropsy did not reveal notable differences between treated groups. There were no significant histopathological changes in immunocompetent organs nor in the lungs, the pancreas, the kidney or the brain. Sharply demarcated multiple vacuoles were observed in the myocardium in group C toward the end of the experiment. In group C, the prevalent changes in the gizzard and the proventriculus were slight to severe cuticle erosions and oedema of the lamina propria with or without multiple vacuoles, respectively, towards the end of the experiment. The most prominent changes toward the end of the experiment were dispersed cell vacuolisation in duodenal, jejunual, ileal and caecal lamina propria in group C. 4. In conclusion, it should be emphasised that extra-gizzard gizzerosine-induced lesions are probably not mediated by H 2 -receptor stimulation, but could be a consequence of cellular hypoxia.

Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome

The aim of this work was to characterise the lesions and agents present in clinically normal and clinically affected pigs on a farm during an outbreak of postweaning multisystemic wasting...

PCR Analysis of Immunoglobulin Heavy Chain (IgH) and TcR-γ Chain Gene Rearrangements in the Diagnosis of Lymphoproliferative Disorders: Results of a Study of 525 Cases

This report summarizes a cumulative 4-year experience in polymerase chain reaction (PCR) analysis of immunoglobin heavy chain (IgH) and TcR-γ chain gene rearrangements in 525 cases of lymphoproliferative disorders. Because the sensitivity of the PCR methodology was found to be tissue dependent, in the study of the presence of clonal cell population in tissues containing a small number of polyclonal lymphocytes, such as skin and gastrointestinal biopsy specimens, we used the multiple–PCR run approach. In this latter methodology, we repeat the PCR reaction from the same sample at least three times to confirm the reproducibility of the results. In the study of 273 cases of B- or T-cell lymphomas with characteristic immunomorphological and clinical features, a clonal IgH or TcR-γ chain gene rearrangement was detected in approximately 80% of cases. A clonal rearrangement involving both IgH and TcR-γ chain genes was found in 10% of cases of both B-cell and T-cell lymphomas. The study of 167 cases of nonneoplastic lymphoid tissue samples showed the presence of clonally rearranged cell populations for IgH or TcR-γ genes in 3 and 9% of cases, respectively. We also applied PCR for the study of 85 cases of lymphoproliferations with no definite diagnosis (i.e., benign versus malignant) after immunomorphological analysis. In 65 cases (76%), the correlation of immunomorphological features with the presence (48 cases) or the absence (17 cases) of clonal lymphoid cell populations led to a definite diagnosis. In almost all these cases, the final diagnosis was found to be in agreement with the clinical course. In the 20 remaining cases (24%), no definite diagnosis could be made. We also assessed the value of PCR in detecting bcl-2/JH gene rearrangement as an additional clonal marker in the diagnosis of follicular lymphoma. Bcl-2/JH rearrangement and/or IgH gene rearrangement was found in approximately 85% (71/85) of follicular lymphoma cases studied.

Clonality assessment of lymphoproliferativedisorders by multiparameter flow cytometry of paraffin-embedded tissue: an additional diagnostic tool in surgical pathology

A major drawback of immunohistochemical detection of monoclonalityin B-cell lymphoproliferative disorders is the lack of contrast between surface-immunoglobulin staining and extracellular immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell suspensions by flow cytometry is commonly used. Although the expression of immunoglobulin light chain subtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin-embedded material to measure clonality in B-cell lymphoproliferative disorders (lymphoid tissue ( n = 10) and non-Hodgkin's B-cell lymphoma ( n = 10). Immunocytochemistry indicated that common cell surface markers as well as the immunoglobulin light chains could be detected in the cell suspensions derived from archival material. In addition, the technique also allowed combined high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to lymphocytes, a double-immunostaining experiment for both light chain immunoglobulins (κ and λ was performed. This experiment showed that this cross-linking was minimal (less than 2%). All cases of reactive lymphoid hyperplasia were DNA diploid and showed a polyclonal expression of immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA aneuploid nature of the Bell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA-diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DNA-aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of malignant lymphoma in routinely processed archival samples of lymphoid tissues.

A lysis, storage, and transportation buffer for long-term, room-temperature preservation of human clinical lymphoid tissue samples yielding high molecular weight genomic DNA suitable for molecular diagnosis.

Molecular diagnosis of malignant lymphoma depends on the ability to extract high molecular weight genomic DNA. However, collection, storage, and transportation of frozen tissue is time consuming and expensive. We used a simple, low-cost lysis, storage, and transportation buffer (LST) to maintain clinical tissue samples at room temperature for up to 4 weeks before molecular analysis. Immersion of lymphoid tissue in LST at room temperature for 2 to 4 weeks was compared with snap-freezing in liquid nitrogen followed by storage at -75 degrees C. Southern blot analysis using an immunoglobulin heavy chain JH probe yielded identical results in 5 clonal and 6 nonclonal samples. The DNA recovered from the LST of a 12th sample was too degraded to be analyzed; however, the tissue had large zones of geographic necrosis. We also demonstrated that DNA extracted from tissue stored in LST is suitable for amplification by the polymerase chain reaction. Results from 4 of the snap-frozen and LST samples analyzed for rearrangements at the immunoglobulin heavy chain VDJ locus were identical. LST can be used in a clinical laboratory for storing tissue samples at room temperature up to 4 weeks before molecular analysis.