Mitogen stimulation of chronic lymphocytic leukemic lymphocytes: defective phytohemagglutinin stimulation independent of immunologic cell surface markers

Peripheral blood lymphocytes (PBL) from 107 untreated patients with chronic lymphocytic leukemia (CLL) were analyzed for the presence of surface immunoglobulin (Ig) and the ability to form rosettes with sheep erythrocytes (SRBC). Four groups were identified based on the cell surface markers: (1) 81 patients' PBL expressed primarily IgM kappa or IgM lambda, 4 further patients' PBL expressed IgM with equal percentages of kappa and lambda surface markers; (2) 13 patients had equal percentages of PBL expressing lg and SRBC receptors; (3) 6 patients' PBL primarily formed rosettes with SRBCs, and (4) in 3 patients and the majority of cells had no detectable markers (null cells). Lymphocytes from all patients within each group were tested for their ability to respond to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). The maximum response in PHA-stimulated normal cell cultures appeared at 2--3 days; for PWM-stimulated cultures, maximal response was at 3--5 days. CLL cultures from all patients in each of the four groups required 5--7 days to develop a maximal PHA response. The response of CLL lymphocytes in all groups to PWM stimulation was similar to normal lymphocytes. Thus, the abnormal PHA response of CLL lymphocytes was independent of the presence or pattern of cell surface markers.     

Analysis with antiidiotype antibody of a patient with chronic lymphocytic leukemia and a large cell lymphoma (Richter's syndrome)

A murine monoclonal antibody made against an idiotypic determinant (Id) of surface IgM/IgD lambda molecules on chronic lymphocytic leukemia (CLL) cells of a 71-year-old woman was used for clonal analysis by two- color immunofluorescence. The anti-Id antibody identified IgM+/IgD+/lambda+ B cells as the predominant cell type of her CLL clone. In addition, substantial proportions of the IgG and IgA B cells and most of the IgM plasma cells in her bone marrow and blood were Id+. Six years after diagnosis, the patient died of respiratory failure due to infiltration of lungs by malignant cells. Autopsy revealed a dramatic change in the tumor cell morphology. The lungs, hilar nodes, and liver were infiltrated by a diffuse large cell lymphoma admixed with the leukemic cells. By immunohistologic staining these anaplastic lymphoma cells were IgM+/IgD-/lambda+ B cells expressing the same Id noted earlier on the CLL cells. The immunoglobulin gene rearrangement pattern on Southern blot analysis was also the same in leukemic blood cells and in the tissues involved by the lymphoma. Thus, the combination of antiidiotype and immunoglobulin gene analyses in this patient with Richter's syndrome revealed that a CLL clone, seemingly "frozen" in differentiation, was actually undergoing isotype switching, differentiation into plasma cells, and evolution into a rapidly growing and fetal lymphoma.     

High-frequency expression of a conserved kappa light-chain variable-region gene in chronic lymphocytic leukemia.

Malignant B lymphocytes from several patients with chronic lymphocytic leukemia (CLL) were examined for reactivity with murine monoclonal antibody 17.109. This antibody, prepared against the rheumatoid factor (RF) paraprotein Sie, recognizes a crossreactive idiotype on 48% of human IgM RF paraproteins, but does not react with IgM paraproteins without RF activity or substantially with normal pooled immunoglobulin. The 17.109-reactive idiotype is a marker for a kappa III variable-region gene, designated V kappa RF, that is conserved in outbred human populations. In a limited study of 31 CLL patients, the leukemic cells from 5 of 20 patients with kappa light chain-expressing CLL were recognized by the 17.109 monoclonal antibody. Despite having malignant cells specifically reactive with this antibody, patients with 17.109-positive CLL did not have elevated serum levels of circulating antibody bearing 17.109-reactive determinants. Total RNAs isolated from the CLL B lymphocytes, or from hybridomas produced by fusing the CLL cells with the WI-L2-729-HF2 cell line, were fractionated electrophoretically and examined by blot hybridization. Under stringent hybridization conditions capable of discerning a single base-pair mismatch, RNA from the 17.109-idiotype-positive CLL cells hybridized to synthetic oligonucleotide probes corresponding to framework and complementary-determining regions in the V kappa RF gene. The high frequency of the 17.109-associated idiotype and the V kappa RF gene in CLL suggests that the disease may arise from B lymphocytes that express a restricted set of inherited immunoglobulin variable-region genes with little or no somatic mutation.     

Light chain composition of serum granulocyte binding immunoglobulins

Serum granulocyte binding IgG, IgM, and the light chain composition of granulocyte binding immunoglobulins were measured in 58 adult subjects, including 8 normal individuals, 6 with Felty syndrome, 6 with chronic idiopathic neutropenia, 32 with B-cell chronic lymphocytic leukemia (CLL), and 6 with multiple myeloma. An abnormal kappa/lambda ratio of granulocyte binding immunoglobulins was detected in 12 of 32 patients with CLL. Neutropenia in patients with CLL did not correlate with an abnormal kappa/lambda ratio or excess granulocyte binding IgG, but did correlate with granulocyte binding IgM (P less than 0.02). Eight of the 12 patients (5 with chronic idiopathic neutropenia and 3 with Felty syndrome) with an immune neutropenia without underlying neoplastic disorder had light chain restricted granulocyte binding immunoglobulins. Of all patients' sera with light chain restriction, 76% were of lambda light chain isotype. Thus, the frequent detection of light chain restriction of granulocyte binding immunoglobulins is not a reflection of malignancy but is suggestive of the somatic mutation of immunoglobulin light chain genes.     

Lymphocyte surface characteristics in malignant lymphoma

The surface of lymphocytes obtained from fresh biopsy specimens from 41 patients with malignant lymphoma and from 30 normal subjects or patients with non-neoplastic lymphadenopathy were investigated. Immunoglobulin on the cell surface was used to identify B cells, whereas T cells were recognized by their reactivity with an antithymocyte antiserum and their ability to form rosettes with sheep erythrocytes. Normal and inflammatory lymph nodes were composed predominantly of T lymphocytes, as were nodes from 14 patients with Hodgkin's disease. Two thymomas were T cell proliferations, whereas a node from a patient with ataxia-telanglectasia was devoid of T lymphocytes. The presence of immunoglobulin on the cell surface indicated that 19 of 21 lymphocytic lymphomas were B cell proliferations, whereas the cells from 3 histiocytic lymphomas (reticulum cell sarcomas) and 1 mixed histiocytic and lymphocytic lymphoma were devoid of surface immunoglobulin. In immunoglobulin-positive tumors, one predominant heavy chain and one predominant light chain could usually be identified, thus establishing the clonal character of the neoplastic proliferation. Ten of 11 diffuse poorly differentiated lymphocytic lymphomas were composed of cells with large amounts of surface immunoglobulin, whereas only 1 of 5 diffuse well differentiated lymphocytic tumors contained such abundant surface immunoglobulin. The surface immunoglobulin data indicate the existence of at least two subspecies of B cell neoplasms. A small lymphocyte with sparse surface immunoglobulin proliferates as diffuse well differentiated lymphocytic lymphoma and chronic lymphocytic leukemia, whereas a larger lymphocyte with abundant surface immunoglobulin proliferates as diffuse poorly differentiated lymphocytic lymphoma and lymphosarcoma cell leukemia.     

Cellular immunologic studies of a patient with monocytic leukemia.

Cellular immunologic studies were performed on the leukemic cells of a 59 year old white man with monocytic leukemia. Morphologically, most circulating leukocytes were monocytes. They demonstrated Fc and C3 (third component of complement) receptors, phagocytized latex particles, showed In vitro cytoplasmic spreading and lysed antibody-coated chicken erythrocytes. Phagocytosis of, as well as rosetting with, C3-coated erythrocytes and very rapid cytoplasmic spreading suggested in vivo monocyte “activation.” The cells were easily maintained in primary culture for up to 13 weeks, with acquisition of typical macrophage morphology. In addition, nearly all cells demonstrated surface immunoglobulin (Slg) which was (1) trypsin-sensitive and did not regenerate in culture, (2) could be restored after trypsinization by incubation of cells In autologous or normal serum and (3) persisted in culture on 75 per cent of cells for at least seven days. Fluoresceinated monospecific antiserums showed an immunoglobulin G (IgG), kappa pattern on one occasion and an IgG, kappa, lambda pattern on another. Eluates of the cells prepared on three occasions showed only IgG and kappa reactivity using numerous antiserums. We concluded that most Slg was probably Fc-receptor bound with unusually high affinity and with greater representation of IgG (kappa) than IgG (lambda). These studies suggest that apparent “monoclonal” Slg does not necessarily indicate a truly clonal proliferation of B cell origin.     

Evidence that chronic lymphocytic leukemia B lymphocytes are frequently committed to production of natural autoantibodies

CD5+ B lymphocytes have been postulated to be primarily involved in autoantibody production. To investigate whether CD5 B lymphocytes from chronic lymphocytic leukemia (CLL) patients display the same function, we fused B lymphocytes from 23 CD5+ B-CLL with nonsecreting murine myeloma X-63 cells. Hybrids were derived from 18 of the 23 patients, but only hybrids from 13 patients were found to secrete immunoglobulin (Ig) (IgM kappa 4, IgM lambda 8, and only kappa light chain 1). Supernatants of these hybrids were tested against an extensive panel of antigens by enzyme-linked immunosorbent assay, indirect immunofluorescence, and hemagglutination. At the same time, peripheral blood mononuclear cells (PBMC) from 15 of these patients, including most patients from whom secreting hybrids had not been obtained, were stimulated with phorbol myristate acetate (PMA). Our results indicated that secreting heterohybrids from CLL B lymphocytes are frequently obtained; however, this is highly dependent on the light chain phenotype, since 8 of 9 lambda-expressing CLL produced hybrids secreting complete Igs, as compared with 4 of 12 kappa-expressing CLL. In addition, B lymphocytes of most patients from whom we failed to derive secreting hybrids also failed to secrete Ig on PMA stimulation. Secondly, CD5+ B-CLL lymphocytes were frequently committed to the production of natural autoantibodies, since in 8 of 15 cases (including hybrids and PMA stimulation) anti-Fc activity was found; three of these also displayed a multispecific binding pattern.     

Monoclonal antibodies against the chronic lymphatic leukemia antigen cCLLa: characterization and reactivity

Monoclonal antibodies (MoAbs) were developed against the cCLLa, a 69-kilodalton leukemia-associated antigen expressed on malignant cells of B-type chronic lymphatic leukemia (B-CLL) and its variants: prolymphocytic (PLL) and hairy cell leukemias (HCL). Two hybridomas yielded approximately 2 and approximately 7.5 mg/mL of IgG2a kappa and IgM kappa, respectively. Monoclonal surface immunoglobulin-bearing cells of all B-CLL patients studied (n = 30) reacted with the MoAbs (r greater than .99) regardless of stage or lymphocyte count. This suggests that the malignant clone in CLL can be identified and its size monitored by using our MoAbs. In contrast, normal B lymphocytes, a large panel of normal, reactive and neoplastic cells, and malignant cell lines failed to react with either MoAb as judged by indirect immunofluorescence and by flow cytometry. Only two patients (one with non-Hodgkin's lymphoma, the other with acute myeloblastic leukemia) exhibited a small cell subset reactive with the MoAbs. cCLLa specificity was suggested by selective target cell reactivity and competitive inhibition-absorption and confirmed by immunoprecipitation. MoAbs IgG2a kappa and IgM kappa appeared to share antigenic determinants and were moderate and avid complement binders inducing 100% and 40% target cell lysis, respectively. cCLLa density on malignant CLL and HCL cells was estimated by equilibrium binding studies using the IgG2a kappa MoAb at 1.7 and 9 X 10(6)/cell, respectively. The restricted expression of the cCLLa and the specificity and cytolytic activity of the anti-cCLLa MoAbs support these antibodies as probes for the classification of lymphoproliferative diseases and for the specific diagnosis and treatment of B-CLL and its variants.     

Central nervous system expression of a monoclonal paraprotein in a chronic lymphocytic leukemia patient.

An unusual complication of chronic lymphocytic leukemia (CLL) is reported. The patient, a 79-year-old man, had a long standing history of CLL, that had been complicated by the development of a Guillain-Barré-like syndrome and a peripheral biclonal gammopathy. The biclonal immunoglobulins identified in the serum were IgM lambda and IgG lambda. The patient's condition progressed and he eventually developed ophthalmologic complications. Cerebrospinal fluid (CSF) obtained during evaluation of his visual dysfunction contained numerous small, mature lymphocytes consistent with the presence of CLL cells in the central nervous system (CNS); immunoperoxidase staining of these cells revealed a monoclonal population. Protein electrophoretic evaluation of the patient's CSF showed a single monoclonal band and immunofixation electrophoresis of the CSF revealed that the immunoglobulin present was IgG lambda. No evidence for the monoclonal IgM paraprotein identified in serum could be appreciated in the CSF by immunofixation. Taken together, these findings strongly implied that there was CNS involvement by the leukemia and this process caused the patient's neurologic symptoms. Furthermore, this study demonstrates that chronic lymphocytic leukemia should also be considered as one of the hematopoietic malignancies associated with monoclonal gammopathies involving the CNS.     

Immune Phenotype and Prognosis in Chronic B-Lymphocytic Leukaemia and Leukaemic Immunocytoma

ABSTRACT. Fifty-nine patients with a leukaemic B-lymphocytic malignancy (“CLL”) were studied. According to the Kiel classification, 29 patients had chronic lymphocytic leukaemia (CLL) and 30 had immunocytoma (IC). Cell surface immunoglobulin staining showed μ heavy chain phenotype in 14 patients, μ? in 35, gamma in 7; in cells from 3 patients the staining was too weak to permit identification. The light chain phenotype was kappa in 39 patients, lambda in 17, and unidentified in 3. The immunoglobulin isotypes differed between the diagnoses. The gamma chain phenotype was found only in IC patients (p < 0.02), and more CLL than IC patients showed a lambda chain phenotype (p < 0.04). Blood lymphocytes from IC patients contained more T cells than CLL cell samples (p < 0.002). No prognostic difference was found between the CLL and IC group. Compared to the lambda phenotype, the kappa phenotype was associated with a poorer prognosis in the IC group, but with a better prognosis in the CLL group. IC patients with μ phenotype had a poorer prognosis than those with gamma phenotype. Low relative T cell numbers were associated with a poor survival (p < 0.01).     

T-cell subpopulations in chronic lymphocytic leukemia: abnormalities in distribtuion and in in vitro receptor maturation.

Purified human thymus-derived (T) lymphocytes were analyzed by detection of Fc receptors for either IgG or IgM in healthy controls and in patients with chronic lymphocytic leukemia (CLL). There was a significant and persistent increase in the numbers of T cells bearing receptors for IgG (Fc gamma) in CLL patients in comparison to the controls. After an in vitro culture period, there was a significantly decreased appearance of cells with IgM receptors (Fcmu) in CLL patients in comparison to the control group. These results indicate an imbalance in circulating T-cell subpopulations for CLL patients. In addition, an in vitro defect in CLL T-cell membrane receptor appearance is present.     

Detection of clonal excess in lymphoproliferative disease by kappa/lambda analysis: correlation with immunoglobulin gene DNA rearrangement

Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by "kappa/lambda" analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.     

Simultaneous manifestation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL)

We report a unique case of 83-year-old Caucasian male with the initial simultaneous manifestation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). The patient presented with absolute lymphocytosis in the blood, asymptomatic generalized lymphadenopathy, and mild splenomegaly. The diagnosis of CLL was suggested from the blood film, but subsequent flow cytometric (FC) analysis on peripheral blood mononuclear cells (PBMNC) revealed two distinct abnormal clones of mature B cells. A small subpopulation (7%) of lymphoid cells expressed CD20, CD11c, FMC-7, CD103, CD25, and kappa surface light chain, consistent with HCL. The larger subpopulation (75%) of lymphoid cells expressed CD19, CD20, CD23, CD5, and lambda light chain, consistent with CLL. The expression of different immunoglobulin light chains on the circulating CLL (lambda) and HCL (kappa) cells suggested two, independent, malignant B-cell clones. Interestingly, FC analysis of bone marrow (BM) cells done 6 months later revealed bright lambda light chain expression on the HCL cells. Despite administration of several different courses of chemotherapy, the HCL subpopulation was not eliminated from the BM but remained stable between 7% and 10% of total BM lymphoid cells. The CLL, responsible for most of clinical symptoms in our patient, responded to combination chemotherapy with fludarabine and cytoxan, and later to monotherapy with rituximab.     

Richter's syndrome with identical immunoglobulin gene rearrangements

A 75-year-old man was admitted to our hospital because of hepatosplenomegaly, generalized lymphadenopathy and lymphocytosis in February, 1989. The leukocyte counts were 93,200/microliters with 95% small lymphocytes which expressed surface membrane immunoglobulin (SmIg) M, D and kappa. Histological finding of the cervical lymph node was diffuse small cell lymphoma. A diagnosis of chronic lymphocytic leukemia (CLL) was made. He was followed up without chemotherapy. In January, 1990, he was re-admitted because of progressively enlarged lymph nodes and increased white blood cell counts, up to 183,200/microliters with 98% lymphocytes. He was treated with vincristine, cyclophosphamide, prednisolone. The leukocyte counts decreased to 5,000/microliters and lymph node swelling decreased in size. In April, 1990, generalized lymphadenopathy re-appeared. The biopsied lymph node specimen showed diffuse large cell non-Hodgkin lymphoma (NHL-DL). The lymph node cells were found to express SmIgM and kappa. The diagnosis of Richter's syndrome was made. DNA analysis using Southern blot method revealed identical immunoglobulin heavy and kappa chain gene rearrangements in the two neoplasms. These findings suggest that the CLL cells and the NHL-DL cells originate from the same clone in this case.     

Intestinal lymphoma in a patient with chronic lymphocytic leukemia of atypical phenotype: Richter's syndrome of unusual presentation

A patient who developed an intestinal large-cell pleomorphic lymphoma during the course of untreated chronic lymphocytic leukemia (CLL) with an atypical phenotype (SIgG kappa) is reported. This is an unusual presentation of Richter's syndrome since RS with primary gastrointestinal involvement has only been described in two patients. In our case, immunological studies disclosed the same immunoglobulin (IgG kappa) in the large-cell pleomorphic lymphoma and on the surface of CLL cells, suggesting that both had arisen from the same clonal proliferation.     

Characterization of malignant lymphomas in leukemic phase by multiple differentiation markers of mononuclear cells. Correlations with clinical features and conventional morphology.

Peripheral blood and bone marrow cells of 38 patients with malignant lymphoma in the leukemic phase were defined by multiple immunologic markers and determination of the enzyme terminal deoxynucleotidyl transferase (TdT). Despite shared B cell markers (surface immunoglobulins, binding of aggregated immunoglobulin G [IgG]) distinctions could be made between the cells of patients with chronic lymphocytic leukemia (CLL) and those of patients with other B-lymphoproliferative disorders on the basis of morphology, intensity of surface immunofluorescence and number of mouse rosette-forming cells (MRFC). In 15 patients with CLL, the mean value for MRFC was 47.5 per cent, differing significantly (p < 0.001) from the value found in 10 patients with non-CLL B-cell leukemic lymphomas (7.5 per cent) and in normal control subjects (4 per cent ± 3/mean ± 1 SD). Of 11 patients with leukemic diffuse poorly differentiated lymphocytic lymphoma (DPDL), four had cells that formed spontaneous rosettes with sheep erythrocytes (T cells), and seven had a predominance of null cells. TdT activity was present in six of six patients studied, including three patients each with the null and T cell proliferations. This group of 11 patients with leukemic DPDL was characterized clinically by prominent mediastinal involvement in 10, a median age of 29 years and a median survival of 12 months. Malignant cells of two patients with leukemic diffuse histiocytic lymphoma seemed to be of B cell origin, as they were characterized by monoclonal surface immunoglobulin M (IgM) of kappa light chain type. Leukemic cells in the remaining two patients were characterized by a prominent Fc receptor, which might be considered evidence of a monocytic disorder. These data further support the usefulness of cell marker analysis in delineating the heterogeneity of cellular types involved in leukemic and lymphomatous disorders, and suggest its eventual clinical usefulness in the diagnosis and prognosis of these disorders.     

Cell surface phenotype in lymphoproliferative disease

The surface phenotype of peripheral blood lymphocytes from 45 patients with chronic lymphocytic leukemia (CLL) and of lymphoid tissue from 100 patients with other lymphoproliferative disease was determined. Surface immunoglobulin (SIg) and complement receptor were employed as B cell markers, and reactivity with sheep erythrocytes (E rosettes) and antithymocyte globulin was used to identify T cells. Of these markers, only SIg and E rosettes reliably identified cells of the respective lineages. SIg identification with fluorescent antiserums had the added advantages of allowing the discrimination between different B cell subsets on the basis of staining intensity, and establishing the clonal character of a proliferation by the presence of predominant light and heavy chains.A uniform surface phenotype was observed in CLL, characterized by faintly staining SIgM with or without SIgD (one sixth of the patients were SIgG-positive instead), the presence of complement receptor, and the absence of reactivity with either sheep erythrocytes or antithymocyte globulin. Nodular (poorly differentiated lymphocytic) lymphoma was also uniformly of B cell lineage, but staining for SIgM was brighter (one third of these tumors were instead SIgG-bearing): Although cell suspensions of these neoplasms were uniformly E rosette-negative, the presence of complement receptor and reactivity with antithymocyte globulin were variable. Diffuse poorly differentiated lymphocytic lymphoma and two cases of Burkitt's lymphoma were very bright SIgM-positive, E rosette-negative neoplasms. Sixty per cent of the diffuse histiocytic lymphomas exhibited either SIgM- or SIgG-positive B cells which stained brightly, but the other 40 per cent comprised tumors either of T cell lineage or of a phenotype which could not be conclusively identified. The mixed histiocytic and lymphocytic lymphomas, which occupy an awkward place in pathology, could not be defined by surface markers. Surface markers should not replace routine pathologic criteria as the basis of classifying lymphoma, but cell phenotype is proving an increasingly useful added dimension for understanding and classifying this complex group of diseases.     

Comparison of Normal and CLL Lymphocyte Surface Ig Determinants Using Peroxidase-labeled Antibodies. I. Detection and Quantitation of Light Chain Determinants

Chronic lymphocytic leukemia (CLL) andnormal human blood lymphocytes wereinvestigated with regard to their membrane-associated light (L) chains. Peroxidase-labeled anti-kappa or anti-lambdaantibodies were used to visualize by lightmicroscopy the cells bearing L chain determinants. In addition, the activity of theenzyme coupled to the antibody was measured spectrophotometricly in order to determine the number of antigenic determinants on each positive cell. The resultsdemonstrated that 16%-25% of lymphocytes from six normal control subjects werepositive for kappa chains, and 4%-10%were positive for lambda chains. The average number of antigenic sites on the surface of each positive cell was calculated tobe 90,000. Lymphocytes of each of the 14CLL patients studied carried either kappaor lambda antigenic determinants on theirsurface, but not both; 50%-70% of thelymphocytes were stained. The averagenumber of antigenic sites per positive CLLwas calculated to be 9000. These resultsconfirm previously reported studies indicating that CLL is a monoclonal proliferative disease. In addition, the quantitativeresults demonstrate that the CLL lymphocyte surface membrane bears, on the average, only 10% of the L chain determinantspresent on the normal lymphocyte.

Submitted on August 3, 1973 Revised on November 13, 1973 Accepted on November 14, 1973     

Normal maturation sequence of immunoglobulin light and heavy chains in hematogone stages 1, 2 and 3 in acute leukemia after treatment

The cellular diversity of bone marrow samples was studied by using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on acute leukemia blast cells and regeneration of normal B-cells, hematogones. This approach should enhance the ability to study normal hematopoiesis, and to identify and monitor hematopoietic disorders. The study was performed on a homogeneous group of patients (mainly children), all of them after finishing complete therapy for AL, mostly B-ALL. In all of these patients complete pattern of all three individual Hg stages was present. Maturation spectra of surface immunoglobulin kappa (sIgkappa) and lambda (sIglambda) light chains and IgM, IgA heavy chains in all three stages of Hgs are presented as reliable reports on sIgs as their incidence on Hgs are scarse and even contradictory. The Ig expression paralles CD20 expression. SIg of light (kappa,lambda) and heavy (IgM, IgA) chains were completally absent in stage 1 Hgs and their expression increased through stage 2 to 3; IgM was expressed similarly. Light Ig chains kappa/lambda were expressed in a polytypic way. The results completed information on normal maturation sequence of bone marrow stage 1, 2 and 3 hematogone regeneration in treated acute leukemia patients.     

Spurious E rosette formation in B cell chronic lymphocytic leukemia due to monoclonal anti-sheep RBC antibody

The apparent simultaneous presence of surface markers characteristic of both B and T cells is a phenomenon being described with increasing frequency in patients with chronic lymphocytic leukemia (CLL). We describe a patient with CLL whose B lymphocytes possessed surface immunoglobulin reactive with neuraminidase-treated sheep erythrocytes (SRBCs) and produced E rosette formation. Cytofluorography using monoclonal antibodies demonstrated the B cell nature of these cells and the absence of the SRBC receptor. Further documentation that the binding of SRBCs was mediated through immunologic reaction included E rosette formation inhibition by monospecific antisera and hemagglutination of SRBCs by a paraprotein isolated from the patient's serum. Fusion of the CLL cells with a human hypoxanthine-aminopterin- thymidine-sensitive plasma cell line resulted in the production of human hybridomas that secreted the SRBC-reactive IgM antibody. An analysis of clinical histories of CLL patients whose cells exhibited this phenomenon from both immunologic and clinical perspectives is presented.