The usefulness of CD71 expression by flow cytometry for differentiating indolent from aggressive CD10+ B-cell lymphomas

We studied 34 low- and 30 high-grade CD10+ B-cell lymphomas. Forward light scatter (FSC) and CD71 fluorescence intensity (CD71i) of tumor cells were measured and normalized by corresponding values for resting T cells. Significant differences in CD71i values between low- and high-grade lymphomas were observed by the Mann-Whitney U test (P < .001) and receiver operating characteristic (ROC) curve analysis (P < .001). FSC was not significantly different between low- and high-grade lymphomas; the area under the ROC curve was less than that for CD71i. Neither FSC nor CD71i significantly differentiated follicular lymphoma (FL) grades. A comparison of all FLs (grades 1-3) and non-FL high-grade lymphomas (Burkitt lymphoma [BL] and large B-cell lymphoma [LBCL]) showed significant differences in CD71i (P < .001) and FSC (P = .021). Differences were significant in CD71i and FSC between FL and LBCL (P < .001) but not between FL and BL. CD71i is more potent than FSC for distinguishing CD10+ low- from high-grade lymphomas and FL from non-FL high-grade lymphomas. Sensitivity and specificity are limited owing to inability to identify FL3. In ROC analysis, a high value for CD71i can identify BL and LBCL with a high degree of certainty.     

Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry

AIMS: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). METHODS: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1-120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. RESULTS: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. CONCLUSION: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.     

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

A major drawback of immunohistochemical detection of monoclonality in 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 (kappa and lambda) 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 B-cell 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.     

The expression of CD22 (Leu 14) and CD11c (LeuM5) in chronic lymphoproliferative disorders using two-color flow cytometric analysis

The monoclonal antibodies (MoAbs) CD22 and CD11c recognize B-lymphocyte- and monocyte-associated antigens, respectively. Reports indicate that when these two MoAbs co-express, they represent a unique marker for hairy cell leukemia (HCL) although neither is specific for that disease. The authors evaluated the expression and diagnostic utility of CD22 and CD11C in specimens from 26 normal subjects, 29 patients, with various nonlymphoproliferative disorders (NLPDs), and 75 patients with different types of chronic lymphoproliferative disorders (CLDs) using two-color flow cytometric analysis of peripheral blood lymphocytes. Lymphocytes co-expressed CD22 and CD11c in less than or equal to 3% of the normal subjects and in less than or equal to 6% of the patients with NLPDs. These markers were expressed in greater than 10% of the lymphocytes of 46% (32/69) of the patients with B-cell CLDs: B-cell chronic-lymphocytic leukemia, 9/41; B-cell non-Hodgkin's lymphoma, 8/14; HCL, 11/11; B-cell lymphoproliferative disorder (NOS), 1/2; and B-cell prolymphocytic leukemia, 1/1. None (0/6) of the lymphocytes of patients with T-cell CLDs expressed greater than 10% CD22-positive (CD22+) or CD11c-positive (CD11c+) cells. The HCL cases demonstrated a unique CD22+CD11c+ fluorescence histogram pattern, distinct from other lymphoproliferative disorders, that was characterized by uniformly intense CD11c and CD22 fluorescence. Differences in the expression of the CD22+CD11C- and CD22+CD11C+ phenotypes between diagnostic groups were found, most notable was a paucity of CD22+CD11c+ cells in lymphocytes of patients with HCL. CD22 also had more variable expression than CD19 and HLA-DR in the cases of B-cell CLD. This study demonstrates that the CD22+CD11c+ phenotype is not unique to HCL but is a consistent feature of that disorder and that the immunofluorescence pattern of co-expression in HCL is diagnostically useful.     

Comparison of multiparameter flow cytometry with cluster analysis and immunohistochemistry for the detection of CD10 in diffuse large B-Cell lymphomas.

CD10 is a critical antigen for the distinction of follicle-center lymphoma from other B-cell lymphomas composed of small cells in fine-needle aspiration specimens, tissue core biopsies, and bone marrow. In addition, CD10 is expressed in a subset of diffuse large B-cell lymphomas (DLBCLs), where it may be an adverse prognostic indicator. We have previously demonstrated that CD10 expression detected by multiparameter flow cytometry (FC) with cluster analysis is highly sensitive and specific for follicle-center lymphoma in the differential diagnosis of small B-cell lymphomas. In this study, we assessed the utility of paraffin section immunohistochemistry (IHC) for CD10 compared with FC in a cohort of 50 DLBCLs. IHC for CD10 was technically successful in 47 of the 50 (94%) DLBCLs; 3 failed based on lack of internal CD10 reactivity. CD10 was expressed by FC in 20 of 47 DLBCLs (43%); CD10 was positive by IHC in 15 of these (75%). All 27 cases that were CD10(-) by FC were negative by IHC. The level of CD10 expression by FC in the 5 FC(+)/IHC(-) cases ranged from relatively dim to bright. Our results indicate 75% sensitivity and 100% specificity of CD10 expression by IHC compared with multiparameter FC with cluster analysis and a 6% technical failure rate.     

Usefulness of CD79b expression in the diagnosis of B-cell chronic lymphoproliferative disorders

We evaluated anti-CD79b for its usefulness in the diagnosis of B-cell chronic lymphoproliferative disorders (BCLPDs), particularly chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). We analyzed 100 BCLPDs for CD5, CD19, CD20, CD23, CD79b, and surface immunoglobulin light chain (sIg) expression by 4-color flow cytometry. CD20, CD79b, and sIg expression were quantified. Correlational analysis and univariable and multivariable logistic regression models were used to determine the best combination of antigens for the immunophenotypic classification of CLL vs other BCLPDs. Positive and statistically significant Spearman pairwise correlations between CD20, CD79b, and sIg fluorescence intensity were demonstrated. In the simplest models in which a single variable was considered, cutoff points were chosen that gave misclassification rates for CLL of 16% for CD79b, 19% for sIg, and 18% for CD20. Low-intensity CD79b, CD20, and sIg are associated highly with CLL. A panel containing CD5, CD19, CD23, and sIg allowed correct classification of most cases. Addition of CD20 or CD79b improved diagnostic accuracy; CD79b was slightly better than CD20. CD79b seems to be a useful addition to a standard flow cytometry panel for the evaluation of BCLPDs.     

Use of flow cytometry in the diagnosis of lymphoproliferative disorders in fluid specimens

The diagnostic evaluation of fluid specimens, including serous effusions and cerebrospinal fluids (CSFs), can be challenging for a number of reasons. The evaluation of lymphoid proliferations in these specimens can be particularly problematic, given the frequent presence of coexisting inflammatory conditions and the manner in which these specimens are processed. As a result, immunophenotypic analysis of hematopoietic cell populations by flow cytometry has emerged as a useful ancillary study in the diagnosis of these specimens, both in patients with and without a previous history of a lymphoproliferative disorder. In this study, we review our experience with flow cytometry in fluid specimens over a four‐year period. Flow cytometry was performed in 184 of 6,925 total cases (2.7% of all fluids). Flow cytometry was performed in 4.8% of pleural fluids (positive findings in 38%, negative in 40%, and atypical in 18%), 1.1% of peritoneal fluids (positive in 40%, negative in 50%, and atypical in 10%), 1.9% of pericardial fluids (positive in 67%, negative in 33%), and 1.9% of CSFs (positive in 23%, negative in 55%, atypical in 3%). The specimen submitted was inadequate for analysis in 9.2% of cases, most commonly with CSF specimens, but was not related to the volume of fluid submitted. Atypical flow cytometry findings and atypical morphologic findings in the context of negative flow cytometry results led to the definitive diagnosis of a lymphoproliferative disorder in a significant number of cases when repeat procedures and ancillary studies were performed. Diagn. Cytopathol. 2014;42:664–670. © 2014 Wiley Periodicals, Inc.     

Intracellular detection of cytokine expression with four-color flow cytometry in pregnant NOD/SCID mice

Bosma et al [1] originally reported that C.B-17mice homozygousforthe severely combinedimmu-nodeficiency (scid/scid) mutation lack functionalTand Blymphocytes . Although there are defectsin cellular and humoral immune function,normalNKcell function can be detected in these mice[2] . On the other hand,the NOD/Lt strain ischaracterized by a functional deficit in NK cells[3] and proneto developa highincidence of auto-immune insulin-dependent diabetes mellitus medi-ated by Tcells [4] .Shultz e…     

Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry

A major limitation to the detection of rare cell types in the peripheral blood using monoclonal antibodies is nonspecific binding of the antibody reagent to normal cells. Detection of rare common acute lymphoblastic leukemia antigen (CALLA)-positive cells in peripheral blood is significantly improved by using multiple flow cytometric parameters to exclude a variety of mature blood cells which may nonspecifically bind the antibody reagent. Monocytes and granulocytes are excluded by gating out cells with high 90 degrees light scatter. By gating on red fluorescence, a variety of mature cell types binding to phycoerythrin (PE)-conjugated Leu 3, Leu 2, and M3 monoclonal antibodies are also excluded. CALLA-positive lymphoblasts from 6 consecutive patients were not excluded on the basis of these parameters. Gating on log 90 degrees light scatter and log red fluorescence in this fashion reduced the incidence of nonspecific binding to peripheral blood mononuclear cells of a fluorescein-conjugated irrelevant monoclonal antibody by 98% from 308 cells per million to 5 cells per million. One CALLA-positive lymphoblast per 100,000 peripheral blood mononuclear cells could be detected in mixture experiments using this method. The normal range of CALLA-positive cells in adults is less than 16 cells per million peripheral blood mononuclear cells. This low background of CALLA-positive peripheral blood cells may permit the detection of early leukemic relapse in acute lymphoblastic leukemia by analysis of the peripheral blood. This methodology can be applied to the detection of any rare cell type by using phycoerythrin-conjugated antibodies to markers that the cell type does not possess.     

Primary cutaneous CD30 positive T-cell lymphoproliferative disorders with aberrant expression of PAX5: report of three cases

Accurate diagnosis of lymphoma includes the assessment of lineage-specific markers. Hematopoietic and lymphoid tissues express PAX5 exclusively in pro-B-cell to mature B-cell stages. However, some mature PAX5+ T-cell lymphomas have been reported. We report three cases of primary cutaneous CD30+ T-cell lymphoproliferative disorders (LPDs) with PAX5 expression: one cutaneous anaplastic large cell lymphoma (ALCL) and two cases of lymphomatoid papulosis (LyP). The three patients were 26 years old and female, 75 years old and female, and 65 years old and male. In all cases, Hodgkin's and Reed-Sternberg-like large lymphoid cells were present, positive for CD30, fascin, and PAX5, and negative for CD3, CD4, CD8, CD20, CD45RO, CD56, cytotoxic markers, and Epstein-Barr virus. The ALCL was accompanied by lymphadenopathy; the patient died of progressive disease 5 months after diagnosis. The LyP cases were localized in the skin with spontaneous regression. One case was diagnosed during pregnancy, transformed to ALCL, and ended in death 32 months after diagnosis despite multi-agent chemotherapy. This study is the first to address the clinical significance of PAX5+ primary cutaneous CD30+ T-cell LPDs. These cases were distinct regarding PAX5 expression and a relatively aggressive clinical course versus conventional primary cutaneous CD30+ T-cell LPDs.     

Value of CD23 determination by flow cytometry in differentiating mantle cell lymphoma from chronic lymphocytic leukemia/small lymphocytic lymphoma.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many morphologic and immunophenotypic features. In addition to histomorphologic examination, it is customary to use the absence of CD23 to differentiate MCL from CLL/SLL, based primarily on reported comparisons of immunohistochemical staining of tissue sections. These findings are widely extrapolated to flow cytometric analysis, although available data are contradictory and not sufficiently detailed. We compared expression of CD23 by flow cytometry in 22 cases of MCL and 25 cases of CLL/SLL. Lymphoma cells in 12 of 22 MCLs were negative for CD23, and 10 showed dim expression. In contrast, none of 25 CLL/SLLs were negative for CD23, 4 were dimly positive, and 21 were moderately or brightly positive. Thus, a significant proportion of MCL exhibited overlap of CD23 expression in the low-intensity range with CLL/SLL. Clinically, there was no correlation between the intensity of CD23 expression and clinical stage at diagnosis or survival. These findings emphasize that by flow cytometry, MCL can be differentiated reliably from CLL/SLL using CD23 if negative expression is observed. However, with dimly positive expression, interpretation should be cautious.     

Role of fine-needle aspirate immunophenotyping by flow cytometry in rapid diagnosis of lymphoproliferative disorders

Immunophenotyping is an essential component in the diagnostic work-up of lymphoproliferative disorders (LPD). As compared to immunohistochemistry, flow cytometric immunophenotyping (FCMI) is rapid, quantitative and a more objective technique. This study was designed to evaluate the utility of FCMI on fine needle aspirates (FNA) in rapid diagnosis of LPD in routine clinical practice. FNA from 31 consecutive cases clinically suggestive of LPD were subjected to FCMI. Representative material for FCMI was obtained in 28 (90%) cases and a definite diagnosis established in 27 cases. Histopathogical correlation was available in 22 cases and concordance with FCMI results was observed in 19 (86.4%) cases. FCMI analysis was inconclusive in 4 cases. The results of FCMI were available the same day and were crucial for therapeutic purpose in 3 patients with superior vena cava syndrome. FCMI combined with cytological examination of aspirate smears permits rapid diagnosis with high level of accuracy resulting in efficient treatment planning for critically ill patients and those from far-off rural areas.     

CD4 mAb induced apoptosis of peripheral T cells: multiparameter subpopulation analysis by flow cytometry using Attractors.

Studies describing the induction of apoptosis for CD4 mAbs do not delineate between epitope-dependent and Fc-driven epitope cross-linking induced cell death. Keliximab and clenoliximab are two CD4 mAbs that differ only in their heavy chain isotypes, being an IgG1 and a modified IgG4, respectively. These antibodies suppress CD4 T cell responses in vitro and in vivo and have been in human clinical trials for the treatment of RA and asthma. Here we compared the apoptotic activity of these mAbs to differentiate between the contributions of epitope-dependent vs. Fc-driven epitope cross-linking induced cell death in vitro as a link to differential CD4 cell depletion in vivo. We developed a simple flow cytometry procedure that measures apoptosis within intact and compromised subpopulations of PBMCs within a few hours of culture. Attractors software was used to quantitate the percentage of apoptotic CD4 T cells, which generate reactive oxygen species (ROS), express external phosphatidyl serine (PS) and cleaved fluorescein diacetate (FDA), within the intact and compromised lymphocyte populations. Treatment of freshly isolated PBMCs with keliximab resulted in the appearance of characteristic apoptotic condensed CD4 T cells that contained reactive oxygen species, were annexin V positive and had intact esterase activity. Apoptosis was evident within 3 h and continued throughout the 72-h culture period. In contrast, clenoliximab alone did not induce apoptosis. The use of multiparameter flow cytometry and Attractors to analyze subpopulations based on scatter properties and biochemical processes during apoptosis provides a sensitive assay in which to quantitate and characterize the induction of cell death. Depletion of CD4 T cells in vivo by keliximab may reflect, in part, antibody-mediated apoptosis of these cells that is dependent on Fcgamma receptors.     

The CD45 isoform B220 identifies select subsets of human B cells and B-cell lymphoproliferative disorders

The B220 isoform of CD45, a pan B-cell marker in mice, is expressed by only a subset of human B cells that do not express the memory B-cell marker CD27, suggesting that it is a differentiation-specific isoform of CD45. We examined normal human peripheral blood B cells, secondary lymphoid tissue, and a range of human B-cell lymphoproliferative disorders for the expression of B220 by flow cytometric immunophenotyping and immunohistochemical staining. We found that a subset of human B cells in peripheral blood is positive for B220 by flow cytometric immunophenotypic analysis. In reactive lymphoid tissues, B220 is expressed by B cells occupying the mantle zones and by a subpopulation of germinal center cells, but, in contrast, marginal zone B cells in the spleen do not express B220. Of 94 cases of B-cell lymphoproliferative disorders, 33 (35%) were positive for B220 by flow cytometric immunophenotypic analysis, including most cases of marginal zone lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. In contrast, all cases of precursor B lymphoblastic leukemia/lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma were negative for B220. Immunohistochemical staining for B220 correlated with flow cytometric analysis for all cases studied by both methods. Our data demonstrate that B220 is expressed in a select subset of normal, reactive B cells in a pattern that is consistent with a marker of naive B cells. However, this restricted expression pattern is not seen in B-cell lymphoproliferative disorders. Discordance between the B220 expression patterns of normal mantle and marginal zone B cells and their respective neoplastic counterparts may aid in the distinction between normal and neoplastic proliferations at these anatomical sites.     

Characteristic CD103 and CD123 expression pattern defines hairy cell leukemia: usefulness of CD123 and CD103 in the diagnosis of mature B-cell lymphoproliferative disorders

By using flow cytometry, we studied CD103 and CD123 expression by the malignant cells in 300 B-cell lymphoproliferative disorder (BC-LPD) cases, including 114 hairy cell leukemia (HCL), 20 HCL variant (HCLv), 9 splenic marginal zone lymphoma (SMZL; in 5, only CD103 was evaluated), 133 chronic lymphocytic leukemia (CLL), 3 follicular lymphoma (FL), and 21 mantle cell lymphoma (MCL). All HCLs expressed uniform CD103 and bright CD123. Among the 20 HCLv cases, 20 (100%) were CD103+ and 8 (40%) were CD123+ (partial or dim). CD103 was negative in all MCL, FL, CLL, and SMZL cases. CD123 was positive in 1 (25%) of 4 SMZL, 3.8% of CLL (5/133), 7 (33%) of 21 MCL, and 1 (33%) of 3 FL cases. CD103 is specific for HCL and HCLv. CD123 expression is more widespread in BC-LPDs but is useful in conjunction with CD25 to differentiate HCLv from HCL. These findings support the usefulness of CD123 and CD103 to aid in the differential diagnosis of BC-LPDs.     

B细胞特异性激活蛋白与CD20在淋巴组织增生性疾病中的表达

目的探讨B细胞特异性激活蛋白(B cell-specific activator protein, BSAP)在淋巴组织增生性疾病中表达情况及在淋巴瘤鉴别诊断中的意义.方法观察63例淋巴组织增生性疾病的组织病理学形态,按WHO新分类方案进行淋巴瘤分型,采用免疫组织化学S-P法同步检测比较BSAP和CD20的表达.结果 9例淋巴结反应性增生的B淋巴细胞,34例B细胞性淋巴瘤的瘤细胞均表达BSAP和CD20,但BSAP的中等到强阳性表达率(69.8%)高于CD20(53.5%),二者差异无显著性(P>0.05);6例霍奇金淋巴瘤H/RS瘤细胞表达阳性率为83.2%;14例T细胞淋巴瘤的肿瘤细胞均不表达BSAP.结论 BSAP表达定位于B淋巴细胞的核内,清晰易见,在B细胞性淋巴瘤表达多呈强阳性,可用于淋巴瘤的分型与鉴别诊断,是判断肿瘤为B细胞来源的又一重要辅助指标.     

Analysis of heavy metal immunotoxicity by multiparameter flow cytometry: correlation of flow cytometry and immune function data in B6CF1 mice

Bone marrow and spleen cells obtained from female B6C3F1 mice given a single i.p. exposure to cadmium acetate (0.9 mg/kg), lead acetate (12 mg/kg), or sodium acetate (12 mg/kg), were studied using flow cytometry, immunologic, and hematologic assays. Significant changes were detected in subpopulations of bone marrow cells using multiparameter flow cytometry within 1 day following treatment with cadmium or lead. Bone marrow cells obtained from B6C3F1 mice 5 days after treatment with cadmium or lead were found to have a decreased number of cells expressing Mac-1, 55-7.2, 14.8, and Lyt-1 antigens, suggesting a shift to immature cell types. An increase in the number of progenitor cells (CFU-C) obtained from the bone marrow of mice treated with heavy metals was also noted 5 days after exposure to cadmium or lead. A time-dependent suppression of the in vitro primary humoral immune response of spleen cells to SRBCs, TNP-Ficoll and TNP-LPS was produced by cadmium or lead treatment. Suppression of the mitogenic response of spleen cells to Con A, PHA, and LPS was also found to be time-dependent. Spleen cell surface marker expression (Mac-1, Lyt-1, Lyt-2 and 14.8) was altered in response to cadmium or lead treatments, but these changes did not appear to correlate with the humoral immunity or mitogen-induced proliferation data. These studies demonstrate that changes in cell surface markers on discrete subpopulations of lymphoid cells present in the spleens of heavy metal exposed mice may not correlate with alterations in the functional activity of these cells. However, changes in murine bone marrow surface markers in response to cadmium or lead treatment predicts a shift to immature cell types, which appeared to correlate with the increase in CFU-C activity.