Receptors for Helix pomatia A haemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL).

Blood lymphocytes from thirteen patients with CLL were studied for surface-bound Ig (SIg), Fc receptors (EA rosettes), receptors for sheep erythrocytes (E rosettes) and receptors for Helix pomatia A haemagglutinin (HP), a carbohydrate-binding protein with specificity for N-acetyl-D-galactosamine and related sugars. Fluorescein-labelled HP binds to subpopulations of human peripheral blood lymphocytes (PBL) treated with neuraminidase. In normal peripheral blood, HP binds to the T-lymphocytes while the majority of the B cells bearing surface-bound immunoglobulin do not have receptors for HP. In untreated CLL, HP binds to 90-100 percent of the neuraminidase-treated PBL. Almost all of the SI-G-POSITIVE CELLS IN CLL patients also have receptors for HP. Two groups of patients were found: in one the total fraction of SIg+ cells was less than or equal to 50 percent and about 30 percent of these lost their Ig during incubation at 37 degrees C. No such loss of SIg was revealed in the remaining patients where total SIg+ fraction was approximately 70 percent. These patients usually had higher blood lymphocyte counts, probably reflecting a more advanced disease. CLL patients in remission with low numbers of leukaemic cells also had low numbers of blood lymphocytes carrying both SIg and HP-receptors. It is concluded that leukaemic cells carry both HP receptors and SIg. Testing of this combination therefore provides a valuable new tool for monitoring patients with CLL.     

B-cell function in patients with chronic lymphocytic leukemia

Summary Using a reverse hemolytic protein A plaque assay, spontaneous and pokeweed mitogen (PWM)-induced immunoglobulin (Ig) secretion was determined in peripheral blood from 22 patients with B₁-chronic lymphocytic leukemia (CLL), one patient with B₂-CLL, and one patient with suppressor T-CLL. Diagnoses were established by cytological and histological criteria as well as several marker analyses. Lymphocytes from B₁- and B₂-CLL patients were unable to secrete Ig either spontaneously or after PWM stimulation. In T-CLL lymphocytes, spontaneous Ig secretion was suppressed very probably by the OKT-8-positive leukemic population, since, after cultivation with PWM, a normal Ig secretion could be demonstrated which was paralleled by a decrease in the OKT-8-positive cells. Cocultivation experiments with freshly isolated, unseparated lymphocytes from normal subjects and lymphocytes from patients were of no informational value, since isolated normal B-cells alone already showed a high rate of Ig secretion. However, coculture experiments with separated subpopulations after PWM stimulation revealed an intrinsic B-cell defect in lymphocytes from B₁-CLL patients, whereas their T-lymphocytes were found to be normal helper cells.

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Abbreviations CLL Chronic lymphocytic leukemia - PWM Pokeweed mitogen - ISC Immunoglobulin-secreting cells - Ig Immunoglobulin(s) Supported by the Deutsche Forschungsgemeinschaft (Ru 215/2)     

Expression of the mid-stage differentiation B-cell antigen GP-70 in patients with early and stable form of B-CLL

The expression of B-cell surface markers SmIg and GP-70 was determined on the mononuclear cells from the peripheral blood of 25 patients with chronic lymphocytic leukemia (CLL), 10 patients with multiple myeloma (MM), 6 normal blood donors and in 3 cases of unexplained persistent lymphocytosis. GP-70 was expressed only in CLL patients whereas multiple myeloma patients, blood donors and patients with unexplained lymphocytosis were all negative to GP-70. SmIg was expressed in CLL patients and in some of the multiple myeloma patients. Subgrouping of CLL patients to "early", stable patients (10) and to more advanced CLL patients (15) revealed differential expression of GP-70 on CLL cells from patients with more advanced stage of disease rather than in the patients with the "early" stable clinical course of disease. SmIg, on the other hand, was expressed equally on both subgroups of CLL. Furthermore, in some cases (25%) where SmIg and the light chains of Ig were only weakly expressed or were absent, GP-70 was markedly expressed. These findings suggest that GP-70 can be used as a single laboratory determination to establish clonality of the B-lymphocyte proliferation in CLL. In addition, expression of GP-70 correlates with significant clinical parameters of the disease.     

Quantitation of immunoglobulin isotypes in chronic lymphocytic leukaemic cells compared with normal adult and neonatal lymphocytes

Radioimmunoassays were used to measure the total cellular content of mu, gamma, alpha, delta, kappa and lambda immunoglobulin chains in peripheral blood lymphocytes from normal adults, neonates and 11 patients with chronic lymphocytic leukaemia (CLL). Normal adult lymphocytes contained all classes of immunoglobulin, but predominantly IgG, associated with both types of light chain (kappa:lambda ratio 2:1). In contrast, mu was the major heavy chain in cells from 10 of the CLL patients, and the small amount of IgG found in CLL cells was not produced by the leukaemic clone. Approximately equimolar amounts of one type of light chain were also present, indicating monoclonality. The class distribution of the immunoglobulin in the neonatal cells was intermediate between that of CLL and normal adult cells. CLL B cells had substantially less surface IgM than normal but more cytoplasmic IgM. These data demonstrate the immaturity of neonatal B cells and suggest that CLL cells are also immature--at a stage not normally found in the adult circulation.     

Impaired recirculation of B lymphocytes in chronic lymphocytic leukemia

Lymphocytes of the peripheral blood and thoracic duct lymph were studied in 4 patients with chronic lymphocytic leukemia (CLL),1 patient with lymphosarcoma (LSA) and 3 patients with nonhematolo-gical diseases (controls). When stimulated in vitro with phytohemagglu-tinin (PHA) lymph lymphocytes of CLL patients responded markedly as determined by ¹⁴[C]thymidine incorporation, whereas blood lymphocytes showed a delayed and diminished response. The response of blood and lymph lymphocytes of the LSA and control patients was equal. Purified rabbit antisera against κ,λ, μ and γ-chains were labeled with ²⁵I and the labeled cells assessed by autoradiography. In CLL patients, the percentage of lymphocytes bearing κ and μ determinants was higher in the blood than in the lymph. Controls showed a much lower percentage of lymphocytes with immunoglobulins, which was equal in blood and lymph. Furthermore, the membrane dynamics of HL-A-anti-HL-A complexes on the surface of blood and lymph lymphocytes were studied by means of membrane fluorescence. In CLL, the percentage of lymph lymphocytes showing “cap formation” within 2 h was higher in the lymph than in the blood. Using autotransfusion of [³H-]cytidine-labeled blood lymphocytes, it is shown that the recovery of labeled cells in the lymph of CLL patients within 48 h is diminished compared to controls. It is concluded that in CLL, the leukemic cells are B cells whose capacity to recirculate from blood to lymph through the postcapillary venules is impaired. Only a minor population of PHA-responsive T cells appears to recirculate normally. Consequently, the concentration of T cells is higher in the lymph than in the blood and the leukemic B lymphocytes accumulate in the vascular pool. The impaired ability for recirculation and “cap formation” suggests a membrane abnormality of the CLL cell.     

Some chronic lymphocytic leukemia cells bearing surface immunoglobulins share determinants with T cells.

One set of antigens is common to some chronic lymphocytic leukemia (CLL) cells bearing surface immunoglobulins (Ig) and normal T cells. Proliferating cells from thirty-eight patients were studied with four antisera recognizing normal human T but not B cells. These antisera were raised in rabbits against (a) Sezary cells, (b) blood lymphocytes from a patient with sex-linked agammaglobulinemia, (c) T lymphoblasts and (d) thymus cells. In four CLL cases, the cells expressed the receptor for sheep erythrocytes and lacked surface Ig. Cells from thirty-four CLL cases bore monoclonal surface Ig and did not bind sheep erythrocytes. Twelve out of these thirty-four cases of CLL had cells which were lysed by one or, more frequently, by the four anti-human T cell xenoantisera. By absorption experiments, one set of at least three antigens common to the cells of some of these CLL and T cells was defined. Depending on the patient, the cells can either carry one, some or all of the antigens of this set. However, it was also demonstrated by absorption that these cells lacked antigens particular to the T cell lineage, while the cells from T CLL cases carried both sets of antigens.     

The immunomodulating effect of theophylline on lymphocytes from chronic lymphocytic leukemia patients

The in vitro immunomodulating effects of theophylline on E-rosette formation, phytohemagglutinin (PHA) response, and Ig surface receptors of B lymphocytes were studied on fresh as well as on preincubated lymphocytes from patients with B cell chronic lymphocytic leukemia (CLL). In 11 out of 14 CLL patients, 24 hours preincubation at 37 degrees C significantly enhanced E-rosette formation. Subsequent treatment of preincubated cells with appropriate concentrations of theophylline further enhanced E-rosette formation in 11 cases. On fresh lymphocytes the enhancing effect of theophylline on E-rosette formation was not significant. The same was true for PHA stimulation; in 5 out of 7 cases the mitogen enhanced the stimulating effect of preincubation and had no significant effect on fresh lymphocytes from CLL patients. Preincubation significantly reduced the percentage of surface immunoglobulin positive B cells from CLL patients in all cases studied, and theophylline treatment had an additional effect on this phenomenon. No such effect of theophylline on fresh B cells from CLL patients could be observed. Preincubation had no significant effect on control lymphocytes. The effect of theophylline on control lymphocytes as compared to lymphocytes from CLL patients was completely different for T as well as for B lymphocytes. E-rosette formation from control lymphocytes (fresh and preincubated) was significantly inhibited in the presence of theophylline. No significantly enhanced responsiveness to PHA could be observed after treatment of fresh or preincubated lymphocytes with theophylline. Preincubation and theophylline treatment had no significant effect on the percentage of Ig positive B cells from control lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delay and not deficiency in cap formation of peripheral blood B cells in patients with multiple myeloma

A major problem in the study of peripheral blood (PB) B cells from patients with multiple myeloma (MM) is the distinction between the cells really able to synthesize membrane (m) immunoglobulins (Ig) and those able only to absorb serum Ig passively, since the lymphocytes of such patients are bathed in very high concentrations of monoclonal Ig. In order to reappraise PB B cells (including putative pre-B cells) in MM, we have used three different criteria: (a) the capacity of PB B cells to cap mIg when triggered by an anti-Ig; (b) the presence of B-cell differentiation antigens (CD19, CD20, CD21, and CD37) as specific B-cell markers; and (c) the expression of cytoplasmic heavy chain as a marker of pre-B cells. We have found that, in active myeloma (N=13), the percentages and absolute numbers of PB B cells able to cap mIg (4.25%; 45.43 cells/mm³) were significantly lower than those in healthy donors (8.4%; 151.2 cells/mm³) and those in stable MM (7.67%; 134.39 cells/mm³). In addition, the capping formation in patients with stable or active MM was significantly delayed compared to that in healthy donors. For all the normal individuals and patients investigated, there has been found an excellent correlation between the percentages and absolute numbers of PB B cells able to cap their mIg and those of PB mononuclear cells bearing the four B cell-specific differentiation antigens: CD19, CD20, CD21, and CD37. Finally, virtually no pre-B cells bearing cytoplasmic chains have been identified in the peripheral blood from healthy donors and patients with MM.     

Electrophoresis of lymphocytes from normal human subjects and patients with chronic lymphocytic leukaemia

The electrophoretic mobility (EM) of peripheral blood lymphocytes from twelve normal subjects and four patients with chronic lymphocytic leukaemia (CLL) was studied in relation to the thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytic system. An attempt was made to correlate electrophoretic results with other methods of identifying T and B lymphocytes—T cells by the formation of sheep-cell rosettes and B cells by the formation of erythrocyte–antibody–complement (EAC') rosettes and by staining for surface immunoglobulin.

In the normal subjects the majority of cells migrated quickly with a small `tail' of slower cells. It is suggested that the faster populations are T cells and the slower, B. In CLL the majority of the cells were slow migrators. There was agreement between the percentage of fast cells as assessed by electrophoresis with that of T cells by sheep-cell rosetting; there was also some agreement between the percentage of electrophoretically slow cells to B cells by EAC' rosettes or surface immunoglobulin.

It was possible to remove some of the B cells by density centrifugation after forming EAC' rosettes. This further defined the T cell peak on electrophoresis.

It is concluded that T and B cells carry different surface charge densities which permit them to be separated by electrophoresis and that the malignant B lymphocytes of CLL migrate electrophoretically in a similar fashion to normal B cells.

     

Lymphocyte binding of aggregated IgG and surface Ig staining in chronic lymphocytic leukaemia

Eleven selected patients with chronic lymphocytic leukaemia were evaluated for lymphocyte binding of aggregated IgG and surface Ig staining in order to classify them into B and T cell types. Ten of the eleven patients bound aggregates and stained for surface Ig. In the individual ten patients the number of cells binding aggregates was high (88–100%, mean 96%) whereas the number staining for surface Ig was more variable (8–100%, mean 62%). Parallel and double labelling experiments with aggregates and sheep red blood cell rosettes, a human T cell marker, provided evidence that aggregates were binding to B cells only, even when surface Ig was not detectable. Aggregates did not bind to human thymocytes. Evidence was presented that lymphocytes from some cases of CLL have low but not absent amounts of surface Ig that may be only partially detected by fluorescence techniques. Aggregate binding appears to be a more sensitive method for the detection of B lymphocytes than surface Ig staining.In one of the eleven patients the leukaemic cells were negative in the aggregate binding test. Separate studies on this case also indicated an absence of surface Ig staining and a high percentage of cells forming sheep red blood cell rosettes. It would appear that this case represented a T cell leukaemia.     

Lymphocyte membrane receptors in human lymphoid leukemias.

Membrane-bound immunoglobulins, receptors for the Fc fragment of IgG and receptors for the third component of human or murine complement were used as B cell membrane markers to study peripheral blood lymphocytes from twenty-two patients with chronic lymphatic leukemia (CLL), five patients with acute lymphoblastic leukemia (ALL) and one patient with Sézary syndrome. The capacity of human T cells of forming "spontaneous rosettes" with sheep erythrocytes was employed as T cell membrane marker. In nineteen out of twenty-seven CLL or ALL cases tested a larger percentage of cells than that found in normal individuals expressed at least one of the three B cell membrane markers studied. In the patient with Sézary syndrome the percentage of cells forming "spontaneous rosettes" with sheep erythrocytes was larger than the normal, while cells bearing B cells markers were below the normal values.     

Quantitation of antibodies to IgM, IgD and beta2-microglobulin in antisera to chronic lymphatic leukemia lymphocytes.

Rabbit antisera were prepared to chronic lymphatic leukemia (CLL) lymphocytes and were tested for their reaction with radioiodinated, solubilized, cell surface proteins of normal and CLL lymphocytes. A pooled, hyperimmune antiserum precipitated at least 16 polypeptides from both normal and CLL lymphocytes as shown by sodium-dodecyl sulfate polyacrylamide gel electrophoresis. These polypeptides varied in molecular weight from 11,000 to 180,000. None was prominent or unique to the CLL lymphocytes, although four peptide bands in the CLL cells usually showed more radioactivity than their counterparts in normal cells. Precipitation with antisera of known specificity showed that the cell surface proteins from CLL and normal lymphocytes included HLA antigens and beta2-microglobulin. IgM and IgD were found in preparations of normal cells and in cells of 3 of 5 CLL patients. Of cells from the other 2 patients, one showed only IgD and the other no Ig. An antigen-binding capacity test was employed to quantitate the antibody content of a pooled anti-CLL lymphocyte serum. The antiserum reacted with all Ig classes; however, after absorption with light chains and F(ab')2 fragments, the serum retained activity only for IgM and IgD. The absorbed antiserum bound 45 microgram IgM, 1.5 microgram IgD and 4.5 microgram beta2-microglobulin per milliliter. These data indicate that rabbit anti-CLL lymphocyte sera fail to detect a qualitatively unique tumor-specific polypeptide on the surfaces of CLL cells. However, such antisera contain antibodies to many cell surface proteins including IgM, IgD, HLA antigens and beta2-microglobulin.     

Modulation and High Frequency Expression of Autoantibody-Associated Cross-Reactive Idiotypes linked to the VhI Subgroup in CD5-Expressing B Lymphocytes from Patients with Chronic Lymphocytic Leukaemia (B-CLL)

Leukaemic B cells from patients with chronic lymphocytic leukaemia (B-CLL) are known to express the pan T-cell marker CD5 and a restricted set of immunoglobulin (Ig) variable region heavy (V_h) and light (V_L) chains encoded by germline or minimally mutated germline genes. We have studied surface expression of certain V_H and V_K gene products on peripheral blood B lymphocytes from 23 patients with B-CLL, using a panel of monoclonal antibodies (MoAbs) recognizing germline encoded cross-reactive idiotypes (CRI) associated with V_HI (G6, G8), V_HIII (B6, D12), V_KIIIb (17–109) and an epitope linked to the V_KIII light chain subgroup (C7). While only 1.7–3.2% of peripheral blood B lymphocytes from normal individuals expressed the V_HI-associated CRI (V_hI-CRI), these CRI were expressed on virtually all the leukaemic B cells from 17–22% of the CLL patients. The V_HIII-associated CRI (V_HIII-CRI), however, were found in 8.5–13% of the CLL B cells. Fifty per cent of the IgMK-expressing CLL cells (7/14) expressed the V_RIII light chain subgroup of which only one expressed the V_KIIIb-associated CRI (V_KIIIb-CRI), 17–109. The anti-V_HI-associated CRI antibodies were used to study their regulatory effect on in vitro Ig synthesis by the leukaemic cells. A significant suppression of spontaneous and mitogen-driven Ig production was observed in all cases studied. These results demonstrate an over-expression of V_HI and V_KIII gene products in B-CLL and suggest that B cells expressing these CRI are particularly susceptible to lymphoproliferative stimuli. The anti-CRI antibodies can be used to modulate Ig production by the leukaemic cells and may be of potential value for selective immunotherapy.     

Beta-glucuronidase activity of lymph node imprints from malignant lymphomas and chronic lymphocytic leukaemia.

beta-Glucuronidase activity was semiquantitatively estimated in the cells of lymph node (LN) imprints from patients with Hodgkin's disease (HD), diffuse non-Hodgkin's lymphomas, chronic lymphocytic leukaemia (CLL), normal lymph nodes, and benign lymphadenopathies. In addition, in some of these cases beta-glucuronidase activity was semiquantitatively determined in peripheral blood smear lymphocytes. The beta-glucuronidase score (betaGS) was very low in the cells of the LN imprints from patients with diffuse non-Hodgkin's lymphomas. The LN lymphocytes of HD had a normal betaGS independently of the histological subtype of the disease, while in the LN imprint of CLL the enzyme activity was low, normal, or high. The betaGS of the lymphocytes in LN imprints of normal controls and HD were in general significantly lower compared with he lymphocytes of the peripheral blood smears in the same cases. The relation of our findings to the B and T cell origin of malignant lymphomas and chronic lymphocytic leukaemia is discussed.     

Differentiation of human lymphocytes by pokeweed mitogen in vitro: Studies in normal and in immunodeficient subjects

Summary Immunoglobulin (Ig) synthesis and secretion by peripheral blood lymphocytes activated by pokeweed mitogen (PWM), and by phytohemagglutinin (PHA) was measured in normal individuals and in subjects with primary immunodeficiency. Unstimulated cultures demonstrated stable, low Ig synthesis of all major Ig classes; PWM showed a peak of Ig synthesis at day 5; PHA cultures demonstrated a late peak of Ig production at day 9. Three cases of common variable immunodeficiency showed different patterns of data when the percentage of B cells in blood and Ig production in vitro were used as parameters. Two persons with immunodeficiency and thymoma showed decreased Ig production in vitro, and had suppressor cells capable of blocking Ig production by normal lymphocytes in co-cultivation experiments.

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Abbreviations used PWM pokeweed mitogen - PHA phytohemagglutinin - Ig immunoglobulin - IDD immunodeficiency disease - GARIg goat-anti-rabbit immunoglobulin serum - PBS phosphate buffered saline Supported by: United States Public Health Service Grants AI 09239, AM 20122, AM 11796     

Reactivity of neoplastic cells of hairy cell leukemia with antisera to S-100 protein

Rabbit antibodies to bovine S-100 protein were tested by immunoperoxidase technics against fresh hairy cell leukemia (HCL) cells obtained from nine patients (peripheral blood in six and spleen in three), as well as lymphoblastoid cell lines derived from three patients with HCL. Peripheral mononuclear cells from three normal persons and two patients with chronic lymphocytic leukemia (CLL) and cells from two melanoma lines were used as controls. The melanoma cell lines, cell lines derived from patients with HCL, and fresh HCL cells displayed cytoplasmic and nuclear positivity after exposure to anti-S-100 protein sera. By contrast, normal peripheral blood lymphocytes and CLL cells were negative for S-100 protein. Additional studies were performed by immunoperoxidase technics on representative sections of formalin-fixed splenic tissues from eight patients who had splenectomies. The cause of splenectomy was HCL in three, traumatic rupture in one, CLL in one, Hodgkin's disease in one, and hypersplenism in two patients. Sections from all three HCL patients showed moderate to marked positivity with antisera to S-100 protein. These results strongly suggest the presence of S-100 protein in HCL cells.     

Carbohydrate composition of peripheral, cultured and leukaemic human lymphocyte plasma membranes.

Plasma membranes isolated from peripheral blood lymphocytes of normal donors, lymphocytes from patients with chronic lymphatic leukaemia (CLL), a T cell and B cell line (MOLT-3 and RPMI-1788) were analysed and compared for total carbohydrate contents. T cells and peripheral blood lymphocytes contained the highest relative amounts of sialic acid and fucose, whereas chronic lymphatic leukaemic cells possessed the highest amounts of N-acetylgalactosamine and also more total cell surface carbohydrate. The Thomsen-Friedenreich antigen (TF) was detected serologically on membrane fractions by the use of anti-TF containing sera and specific lectins from Arachis hypogaea, Agaricus bisporus and Vicia graminea. The disaccharide beta-D-galactosyl(1-3)-N-acetyl-D-galactosamine is the immunogdominant carbohydrate group of the FT antigen and was detected as its reduced form, by gas chromatography, in all cells, thus correlating serological and analytical evidence. The haemagglutinating activity of the lectins and sera used was only inhibited by plasma membranes after the removal of sialic acid showong that the native form of this antigen is normally masked by sialic acid in CLL cells as well as normal lymphocytes.     

Cell volumes of normal and malignant mononuclear cells.

The cell volumes of mononuclear cells, T lymphocytes, B lymphocytes, and monocytes from the peripheral blood of 20 normal individuals were compared to neoplastic lymphoid cells from 14 patients with chronic lymphocytic leukaemia (CLL), 20 individuals with acute lymphocytic leukaemia (ALL), and 18 cases of non-Hodgkin''s lymphoma (NHL). Normal T cells were obtained by rosetting mononuclear cells with sheep erythrocytes followed by centrifugation on a gradient composed of Ficoll and diatrizoate salts. Monocyte populations were prepared by adhering mononuclear cells to plastic dishes and B cells were obtained by the depletion of T lymphocytes and monocytes from a mononuclear cell population. Cell volumes were determined on a Coulter Counter Model H4 Channelyzer. In normals, the average mean cell volume (MCV) of T lymphocytes was smaller than B lymphocytes and the average MCV of B lymphocytes was smaller than the average MCV of monocytes (p less than 0.05). The average MCV of lymphocytes from patients with CLL was smaller than the average MCV of normal B cells (p less than 0.01). The average MCV of lymphoblasts from cases of ALL was larger than the average MCV of normal peripheral blood lymphocytes (p less than 0.01). In addition, the size of lymphoblasts showed great variation within and among cases of ALL. The MCV of lymphocytes from most cases of NHL was larger than the MCV of lymphocytes from reactive lymph nodes and from the peripheral blood of normal individuals. An association was observed between the MCV of neoplastic cells and the classification according to Rappaport. We believe that the measurement of lymphoid cell volumes may be helpful in the diagnosis and prognosis of patients with a variety of lymphoproliferative disorders.     

Rosette formation with mouse erythrocytes by lymphocytes from normal donors and patients with various diseases.

Rosette formation of human lymphoid cells with mouse erythrocytes has recently been proposed as a marker for a subpopulation of B lymphocytes. In this work we studied the percentage of mouse rosette forming cells (MRFC) in normal and pathological conditions and compared them to the percentage of sheep rosette forming cells (SRFC) a marker for T lymphocytes. Peripheral blood lymphocytes (PBL) from normal donors contained 6.2 +/- 1.1% (mean +/- 1 S.D.) MRFC. High percentages of MRFC were found in CLL patients, and a slight increase was observed in patients with systemic lupus erythematosus. MRFC were absent in Bruton's type agammaglobulinaemia, but were normally present in patients with T cell defects. Cryopreservation of lymphocytes in 10% DMSO did not significantly affect the mean percentages of SRFC and MRFC, though a slight increase of the former and a small reduction of the latter was observed. Double binding experiments on peripheral blood lymphocytes showed a predominant association of MRFC with cells staining for surface IgM and/or IgD. In all samples tested, we also observed a small population of MRFC negative for sIgM or sIgD and a few sIgM or sIgD positive cells that did not rosette with mouse erythrocytes.     

Surface and cytoplasmic immunoglobulin expression in B-cell chronic lymphocytic leukemia (CLL)

Flow cytometric analysis of abnormal lymphocyte populations in chronic lymphocytic leukemia (CLL) has been widely reported to show weak expression of surface immunoglobulin (sIg). The international scoring system to help discriminate between CLL and other B-cell lymphoproliferative disorders lists this as the first of 5 criteria worth 1 point each. In the present study, 30 cases of CLL were studied for surface and cytoplasmic Ig expression. 23 of these 30 (76.7%) cases were positive for sIg. Of these 23, 14 cases (60.9%) showed moderate to bright sIg expression. All of these 23 cases were positive for CD5 and CD23; all were negative for CD10 and only 6 (26.1%) were positive for FMC7. 27 of 30 (90.0%) cases expressed cytoplasmic immunoglobulin (cIg) compared to 5% reported by others. This shows that cytoplasmic Ig occurs in a much greater percentage of cases than reported previously. 23 of 30 (76.7%) cases showed positivity for both surface and cytoplasmic Ig, with 15 showing kappa light chain restriction and 8 showing lambda light chain restriction. Six expressed cytoplasmic Ig only, with 4 showing kappa light chain restriction and 2 showing lambda light chain restriction. One case expressed neither cytoplasmic nor surface Ig. CD38 positivity portends a worse prognosis. Of the 29 cases tested for CD38, 13 (44.8%) were positive. Of these 13 cases, 12 were in the surface Ig/cytoplasmic Ig+ group and 1 in the cytoplasmic Ig+ group only. Also, of the 23 cases tested for CD22 expression, 16 (69.6%) were positive. These data question the use of both "weak" surface Ig expression and lack of CD22 expression as valid scoring criteria for CLL.