Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.     

HLA-DR expression in B-cell non-Hodgkin's malignant lymphomas: a multiparameter flow cytometry study

Ten cases of reactive follicular hyperplasia and 31 cases of B-cell non-Hodgkin's malignant lymphoma were studied using multiparameter flow cytometry. A bimodal distribution for HLA-DR expression, but not for surface immunoglobulin or B cell-specific antigens CD19 and CD20, was observed commonly in mixed cell type and infrequently in non-mixed cell type B-cell malignant lymphomas. On the basis of HLA-DR distribution alone, 31 cases of B-cell malignant lymphomas of low, intermediate, and high grades could be separated into mixed and non-mixed cell types, with only two misclassifications (P = 0.0001). Exceptionally, one case of malignant lymphoma, follicular and diffuse, mixed-cell type had a unimodal HLA-DR distribution, and one case of malignant lymphoma, diffuse, large noncleaved cell type had a bimodal HLA-DR distribution. In all cases of malignant lymphoma, follicular, mixed-cell type studied, low HLA-DR was correlated with small cells, and high HLA-DR was correlated with large cells. In contrast, HLA-DR expression and cell size were not as directly correlated in cases of malignant lymphoma, diffuse, mixed-cell type. These observations suggest that most, but not all, cases of B-cell malignant lymphomas of the mixed cell type can be separated from other B-cell lymphomas on the basis of HLA-DR distribution.     

Critical analysis and diagnostic usefulness of limited immunophenotyping of B-cell non-Hodgkin lymphomas by flow cytometry

We report the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of various immunophenotypes characteristic of each class of B-cell non-Hodgkin lymphoma (NHL) based on analysis of 352 morphologically well-characterized B-cell NHLs and 175 benign lymph nodes (LNs) using 2-color flow cytometry. All B-cell NHLs that exhibited a characteristic immunophenotype (except diffuse large B-cell lymphoma) had a high NPV. The immunophenotypes of small lymphocytic lymphoma and mantle cell lymphoma showed high specificity, but only small lymphocytic lymphoma also showed a high PPV. One third of follicular lymphomas coexpressed CD23 and CD10. Diffuse large B-cell NHL showed no consistent immunophenotype. About 90% of all benign LNs expressed no substantial amounts of CD5, CD10, or CD23. Most benign LNs also failed to express substantial amounts of immunoglobulin heavy chains. In contrast, about 90% of NHLs showed expression of 1 or 2 heavy chains. The expression pattern of immunoglobulin light chains was not found helpful in favoring one lymphoma type over another. The usefulness of each immunophenotype for each lymphoma group is of particular diagnostic importance in limited specimens, such as fine-needle aspiration biopsies, small core biopsies, body effusions, extranodal sites, and nodal tissues with various artifacts.     

Clinical utility of bone marrow flow cytometry in B-cell non-Hodgkin lymphomas (B-NHL)

AIM: To determine the efficacy of flow cytometry (FC) in the assessment of bone marrow (BM) in B-cell non-Hodgkin lymphoma (B-NHL). FC is a common practice, but is far from being validated. METHODS AND RESULTS: Morphological analysis and FC immunophenotyping were performed on 421 samples. T-cell lymphomas, Hodgkin's disease, chronic lymphocytic leukaemia and hairy cell leukaemia were not included in the study. Clonality was assessed by the standard kappa/lambda/CD19 test. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. A double-step procedure was employed in all cases to increase the sensitivity of the FC procedure. Of 380 evaluable samples, 188 corresponded to follicular lymphoma (FL), 58 to diffuse large B-cell lymphoma (DLBCL), 57 to mantle cell lymphoma (MCL), seven to Burkitt's lymphoma and the remaining 70 samples to other low-grade lymphomas. Morphological marrow infiltration was found in 148 cases, and flow immunophenotyping identified 138 cases with BM involvement. A concordance between the two methods was detected in 298 cases (79%). There was a discordance in 82 cases (21%): morphology positive/FC negative in 46 cases and morphology negative/FC positive in 36 (61% of all cases with discordance were from FL). There was no difference in outcome when patients with discordances were compared with patients without discordances. CONCLUSIONS: Most samples showed concordance between morphological and FC results. FC identified BM involvement in the absence of morphological infiltration. Morphology/FC discordance seems to have no influence on the outcome of FL patients.     

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.     

Two B-cell subpopulations identified by flow cytometry.

Ig+ spleen cells were analysed by light scatter analysis on a flow cytometer and two distinct subpopulations were identified. The large Ig+ cells (10-11 micron in diameter) were found in spleen and bone marrow, whereas the small Ig+ cells (7-8 micron in diameter) were found in all lymphoid tissues. Of the total Ig+ splenic lymphocytes, 40-60% were large Ig+ cells and had surface IgM, Ia and Fc gamma receptors. The large Ig+ cells were highly enriched for responsiveness to the B-cell mitogens, anti-Ig, Nocardia water-soluble mitogen and LPS, whereas the small Ig+ splenic cells had little or no responsiveness to these mitogens.     

Immunophenotyping of angioimmunoblastic T-cell lymphomas by multiparameter flow cytometry

Angioimmunoblastic T-cell lymphoma (AITL) is a distinct form of peripheral T-cell lymphoma (PTCL) frequently involving lymph nodes, spleen and bone marrow, and is associated with systemic symptoms. Its histologic features may be subtle at an early phase and difficult to diagnose. Despite the success of flow cytometry (FCM) in diagnosing B-cell neoplasm, FCM has not been widely accepted as a useful method for establishing the diagnosis of PTCL. Recently, the neoplastic T-cells in AITL have been shown to express CD10. We prospectively applied multiparameter FCM immunophenotyping to three cases of histologically confirmed AITL and identified a small (5-7%) population of CD4+/CD10+ T-cells in two cases. In one case, the CD4+/CD10+ population lacked surface signals of CD3 and CD7, but strongly expressed CD2, whereas CD45 expression was very weak; partial loss of surface CD3 was observed in the other. None of the lymph nodes with reactive hyperplasia, B-cell lymphomas, or Hodgkin's lymphoma studied during the same time period contained the CD4+/CD10+ population. These findings suggest that addition of CD4/CD10 and CD3/CD10 to FCM immunophenotyping panels is useful in the diagnosis of AITL. To the best of our knowledge, this is the first report to demonstrate CD10-expressing T-cells in AITL by FCM.     

In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry

The process of sperm chromatin decondensation occurs when a spermatozoon enters an ovum. Protamine disulphide bonds are reduced to SH and the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA which then combines with histones. Defective chromatin decondensation will thus prevent further development of the male pronucleus. In this study human sperm samples were incubated in vitro at 28 degrees C (using a medium in which the polyanion, heparin, substitutes for nucleoplasmin and beta- mercaptoethanol for egg glutathione) for 10, 20 and 30 min before stopping the reaction with formalin (to 3.6%). The DNA of the fixed cells was stained with Acridine Orange by a one-step method and subjected to flow cytometry and data analysis, in which a zone characteristic of condensed chromatin is outlined on red-green fluorescence contour plots. After 20 min of incubation 97% of the control spermatozoa that were in the mature window (WIN M) had decondensed and moved out of this region. Defects in sperm decondensation were seen in four semen samples of the 20 that were tested. In cases where spermatozoa fail to produce a fertilized egg the cause may lie with defective chromatin quality, including failure of the sperm chromatin to decondense. The method described here is a simple procedure for detecting sperm samples containing such defective cells.      

Diffuse large B-cell lymphoma arising in a composite lymphoma with biclonality by flow cytometry and one monoclonal band by PCR

Composite lymphoma (CL) refers to the presence of two or more distinct types of lymphomas in a single organ or tissue. CL is an infrequent finding and may be due to the existence of two genetically related neoplasms, i.e. transformation of a single lymphoma into another lymphoma, or be due to the presence of two clonally unrelated lymphomas. CL composed of more than two lymphomas is even rare. Herein we describe a case of diffuse large B-cell lymphoma (DLBCL) arising in a CL of follicular lymphoma (FL) and small lymphocytic lymphoma (SLL) in an inguinal lymph node of an 85 year old woman. The three lymphomas were morphologically and immunophenotypically distinct while flow cytometry detected two monoclonal B-cell populations. Karyotyping and Polymerase Chain Reaction (PCR) for B-cell clonality each detected a single monoclonal B-cell population. The morphology findings may suggest DLBCL being transformed from FL while Richter transformation from SLL appears to be less likely in our case. Due to the single clone by chromosome study and PCR study, the precise relationships of the three lymphomas are unknown.     

Clinical significance of an anti-Dib assessed by flow cytometry

Although antibodies to the Dib antigen are generally considered to be of potential clinical significance, we know of no reports assessing the clinical significance of anti-Dib (in vivo or in vitro). We report on an 88-year-old Japanese male gastrectomy patient who had alloanti-Dib. After transfusion of two Di(b-) units, three Di(b+) units had to be transfused, and there were no clinical signs of acute hemolysis. Di(b+) RBC survival was followed retrospectively by flow cytometry. On days 1, 7, and 10, the percent of circulating Di(b+) RBCs was determined to be 39, 30, and 11 percent, respectively, compared to an expected 49, 43, and 41 percent based on calculations. The Di(b+) RBCs appear to have been tolerated for about 6 days, then were removed from the circulation. Direct anti-IgG tests were 1-2+ mixed field with all posttransfusion samples. Monocyte monolayer assays (MMAs), which have been reported to predict the clinical significance of alloantibodies, gave borderline positive results. MMA results using sera from days 0, 3, and 9 were 2.7 and 5.5, 0.8 and 4.8, and 3.0 and 3.7 percent, respectively, without and with added fresh normal serum as a source of complement (clinical significance = > 3% reactivity). The subclass of the anti-Dib was IgG1. This is the first documentation of the clinical significance of an anti-Dib.     

Non-endemic Burkitts's lymphoma. A B-cell tumor related to germinal centers.

To investigate the nature of non-endemic Burkitt's lymphoma, we examined neoplastic cells from eight American patients for receptors for sheep erythrocytes (E), complement (EAC), and Fc fragment of lgG (igGEA), and for surface immunoglobulins (Slg) and hydrolytic enzymes. In addition, we reviewed 47 biopsies and 17 autopsies from American patients to ascertain patterns of involvement by tumor in lymph nodes, spleens and Peyer's patches. Neoplastic cells in all cases studies bore monoclonal surface immunoglobulins of the igM class. Receptors for EAC and igGEA were identified on a minority of the cells. Little or no hydrolytic enzyme activity was demonstrable. These results indicate that, like Burkitt's lymphoma in Africans, this histologically identical tumor in American patients consists of B lymphocytes. In 10 biopsies and two autopsies, germinal centers were selectively involved by tumor, suggesting that these neoplastic cells may be related to some B lymphocytes of normal germinal centers.     

Large B-cell lymphomas: fine-needle aspiration plays an important role in initial diagnosis of cases which are falsely negative by flow cytometry

Large B-cell lymphomas (LBCLs) have significant false-negative results when immunophenotyped by flow cytometry (FC). To clarify the role fine-needle aspiration (FNA) in reducing this false-negative rate, 28 cases ultimately diagnosed as LBCL that had FNA as part of the workup and a negative FC were identified. We examined their clinical and cytologic features, in comparison with cases of LBCL with FNAs that were positive by FC. In 24/28 FC-negative cases (86%) a cytologic diagnosis of suspicious or positive for malignancy was rendered. We conclude that cytologic analysis is more sensitive than FC in the diagnosis of malignancy in FNA of LBCL, particularly in aspirates with low cellularity and/or low viability. Examination of cytospin preparations of the actual material analyzed by FC may provide an indication that an FC result is falsely negative. It is important to recognize the potential of false-negativity by FC of LBCLs when interpreting FNAs with features suggesting lymphoma.     

Prognostic importance of DNA flow cytometry in non-Hodgkin''s lymphomas.

Eighty one cases of non-Hodgkin's lymphoma were examined by DNA flow cytometry, using fixed embedded histological tissue. The frequency of detection of DNA aneuploidy and the values for S phase fractions depended on the histological subtype and grade of lymphoma. Twenty two of the patients with low grade centroblastic/centrocytic non-Hodgkin's lymphoma had repeat biopsies. Eleven of these patients remained histologically and cytometrically stable, but the remaining eleven transformed into high grade non-Hodgkin's lymphoma. The mean value for the S phase fraction in the initial biopsy specimens from patients which transformed was higher than that for patients whose lymphomas remained stable (p less than 0.001). It is proposed that estimates of S phase fraction prospectively identify patients with low grade non-Hodgkin's lymphoma at risk from transformation.     

Comparison between image and flow DNA cytometry in non-Hodgkin's lymphomas

To assess the reliability of DNA estimation in cytological material, Feulgen lymph node imprints from 22 cases of malignant non-Hodgkin's lymphomas were examined by image cytometry (ICM) for both ploidy and cell kinetics, and the results obtained were compared with flow cytometry (FCM). The DNA distribution pattern was less accurate with ICM than with FCM; however, a high correlation was found between proliferative indices (r = 0.91) and between aneuploidy rates (agreement in about 86% of the cases) determined by FCM and ICM. Moreover, DNA tetraploid (or near-tetraploid) stem lines were more easily detected by ICM, due to the morphological selection of lymphomatous cells. The proliferation rate and the aneuploidy frequency according to morphological classification were in agreement with larger, previously reported studies with FCM. Therefore, ICM appears to supply additional complementary information to that obtained with FCM, particularly for the study of heterogeneous cell populations, which may be usefully applied to refine the large cell lymphoma subclassification.     

Functional heterogeneity and pathogenic significance of interleukin-6 in B-cell lymphomas.

A possible autocrine effect of interleukin-6 (IL-6) on the growth and differentiation of the tumor cells of 55 B-cell lymphomas was examined. Interleukin-6 was detected in a few types of B-cell lymphomas, including polymorphic immunocytoma (PI), small lymphocytic lymphoma (SLL), and immunoblastic lymphoma (IBL) with or without plasmacytoid differentiation. In PI and in IBL with plasmacytoid differentiation (IBL-P), IL-6 was detected only in immunoglobulin-containing plasmacytoid cells, and it was absent from most proliferating (Ki-67/PCNA-positive) lymphoma cells. In SLL, IL-6 was not observed in lymphoplasmacytoid cells; instead, IL-6 was observed in transformed (Ki-67/PCNA-positive) tumor cells in proliferation centers. The lymphoplasmacytoid cells in SLL exhibited a phenotype (IL-6/glutathione-S-transferase-pi [GST-pi]-negative), different from that of normal plasma cells (IL-6-negative/GST-pi-positive) and from the plasmacytoid cells (IL-6/GST-pi-positive) in PI and IBL-P. In IBL without obvious plasmacytoid differentiation, IL-6 was detected in most tumor cells that were highly proliferative (Ki-67/PCNA-positive). In this study, IL-6 was undetectable in most lymphomas related to follicular centers, in lymphoblastic lymphoma, in small noncleaved cell lymphomas of the Burkitt and non-Burkitt types, and in diffuse large cell lymphoma. This finding is compatible with a previous finding that IL-6 mRNA was absent from follicular center cells in reactive lymphoid tissues. The functions of IL-6 in these lymphomas may be quite diverse. It appears that IL-6, as an autocrine factor, is responsible for the plasmacytoid differentiation of lymphoma cells in IP and some IBL (IBL-P). The differentiation of lymphoplasmacytoid lymphoma cells in SLL, however, may not be mediated by an autocrine IL-6 mechanism. Interleukin-6 may provide a growth signal, rather than acting as a differentiation factor, for some IBL cells and for some transformed tumor cells in proliferation centers in SLL.     

Morphometric nuclear analysis of lymphoid cells in center cell lymphomas and in reactive germinal centers

A comparison between neoplastic and nonneoplastic lymphoid cell nuclei within germinal centers of nodular lymphomas and of reactive follicular hyperplasias has been carried out with the help of a semiautomated image analyzer by measuring nuclear area, by assessing the presence or absence of invaginations, and by defining the form of the invagination. Nuclear areas are larger in lymphomas, where the invagination, if present, decreases in depth and increases in angle as the value of nuclear area becomes greater. Nuclei with such a large area and shallow invaginations are not present within the nonneoplastic germinal centers. Moreover, no correspondence has been found between neoplastic and nonneoplastic nuclei with regard to the form of the invagination, that is, its symmetry and angle. Therefore, it is possible to argue that the neoplastic nuclei in nodular lymphomas are different from the nuclei found in the sequential pathway of reactive germinal centers.     

Acrosomal integrity assessed by flow cytometry in men with variable sperm quality.

The binding of a specific, fluorescent acrosomal marker (FITC-labelled peanut lectin) to spermatozoa from men exhibiting differences in sperm quality by conventional criteria was quantitated by flow cytometry. When a standard number of cells was analysed, binding of the lectin diminished and became more variable as the degree of sperm pathology increased. In general, ejaculates with total sperm counts exceeding 120 x 10(6) cells exhibited a stable level of binding within relatively narrow limits. In two normozoospermic men, low levels of acrosomal fluorescence were demonstrated. The significance of these observations with regard to prognosis in assisted fertilization programmes is discussed.