Summary.CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITETMflow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38+B cells from 131 patients requiring therapy. The 92 patients (70%) with ≥ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2·30, 95% confidence interval 1·28–4·12; two‐tailedP = 0·003). Among the 64 patients with < 30% CD38+cells, OS of the 25 with ≥ 100 ABC was worse than that of the 39 with < 100 ABC (P = 0·018). OS of patients with < 30% CD38+cells and ≥ 100 ABC was actually similar to that of patients with ≥ 30% CD38+cells.BrightCD20 expression (≥ 20 000 ABC) was not associated with a worse OS (P = 0·10). The presence of ≥ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38+cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.

Iron transferrin has been found to induce a mean 10‐fold increase in the activity of protein kinase C in CCRF‐CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1–0.5 μg/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1–100 μg/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF‐CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2‐fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF‐CEM cells and in mitogen‐stimulated human lymphocytes that may occur through increased synthesis of the enzyme.

Lectins are useful probes for studying cell surface glycoconjugates. Pea (PL) and lentil (LL) lectin each requires for binding a fucosyl- and two alpha-mannosyl residues in core regions of glycopeptides, but differences in outer chain carbohydrates may alter their relative binding affinities. We used binding studies with [125I]-PL and LL and flow cytometry with fluorescein-conjugated (FITC)-PL and -LL to study their interactions with peripheral lymphocytes. Binding of both lectins to lymphocytes was saturable, reversible, and inhibited by alpha-methyl mannose. Scatchard analyses were consistent with two classes of receptors for each lectin. Flow cytometric analyses demonstrated that cell to cell receptor densities varied. Sixty-five percent of lymphocytes bound PL (mean 2 X 10(6) receptors/cell) and 45% bound LL (mean 3 X 10(6) receptors/cell). Competition studies demonstrated mutual inhibition, but flow cytometry revealed persistent FITC-PL or -LL binding depsite 20-fold molar excess of the other lectin. Distributions of receptors for PL and LL on lymphocytes were as follows: 45% of lymphocytes bound both PL and LL; 20% of lymphocytes bound PL alone; 35% of lymphocytes bound neither PL nor LL. Despite similar binding requirements for PL and LL and overlap between their receptors on lymphocytes, there appear to be subsets of receptors specific for each lectin. These results may reflect abilities of PL and LL to discriminate subtle carbohydrate differences on lymphocyte surfaces.

Flow cytometry was used to compare binding of wheat germ agglutinin (WGA) and Lens culinaris lectin (Lcl) to peripheral lymphocytes (PBL) from normal subjects and 26 patients with chronic lymphocytic leukemia (CLL). As defined by the interactions of WGA and Lcl with cell surface receptors, normal unfractionated PBL and purified B-cells were heterogeneous, containing subclasses that bound to WGA (WGA+Lcl-), Lcl (WGA-Lcl+), both (WGA+Lcl+), or neither (WGA-Lcl-). By contrast, in 25 of 26 CLL patients, PBL were homogeneous with approximately 90% of cells binding one or both lectins. Apparent monoclonal tumor cell populations were identified in these patients corresponding to the normal lectin defined cell subclasses: WGA-Lcl- in 18 patients; WGA-Lcl+ in four patients; WGA+Lcl+ in three patients. Ten patients were studied sequentially on more than one occasion at intervals of five to 15 months. Lectin binding patterns remained stable within this time frame. These findings demonstrate that lectin binding may detect several CLL subclasses corresponding to lectin-defined subsets of normal PBL. Lectins can serve as useful tools for dissecting the heterogeneity of human lymphoproliferative diseases.

I have used lectin affinity chromatography of radiolabelled components in cell extracts to examine molecular heterogeneity of membrane glycoproteins from 10 cases of chronic lymphocytic leukemia (CLL). Different complements of radiolabelled surface glycoproteins were found in extracts from cells with different lectin binding characteristics. Additional confirmation of heterogeneity was obtained by analyses of hexose and sialic acid contents of the extracts and various lectin-adherent fractions. The fact that CLL cells from different patients may carry different cell surface oligosaccharides provides the possibility that clinical variability in this disorder may relate in part to this molecular heterogeneity. The data further indicate that different lectins may bind to different saccharide structures at CLL cell surfaces. CLL cells represent excellent source material for isolation and purification of specific lectin receptor molecules.

We have used binding experiments with radiolabeled lectin and flow microfluorometry to characterize the interaction of wheat germ agglutinin (WGA) with human peripheral blood mononuclear cell preparations. Results indicated that WGA bound to these cells in a complex fashion described by a curvilinear (upwardly concave) Scatchard plot and a nonlinear dissociation curve. Binding was rapid and reversible and was inhibited 80–90% by 0.1 M N -acetylglucosamine, a specific haptenic inhibitor of WGA binding. Approximately 1 5 of the total binding was attributable to monocytes, (but phagocytic uptake was excluded), and the remainder was due to lymphoid cell variants. Kinetics of binding were only modestly altered by formalin fixation of the cells mitigating against a role for site-site interactions in the generation of the non-linear Scatchard plot. Flow microfluorometric analysis revealed that the capability to bind WGA was a characteristic of only 35% of blood mononuclear cells and further allowed identification of a small cell subclass constituting less than 10% of the total that nonetheless accounted for fully one half of the entire WGA binding potential of these cells.

When human lymphocytes are cultured in the presence of phytomitogens, 70-90% of the cells undergo blast transformation and synthesize DNA. However, less than 40% of these lymphocytes actually undergo mitosis while 35-90% of the newly synthesized DNA is excreted into the media. The release of DNA by the cells is selective since experiments with [(14)C]uridine indicate that RNA is not lost into the culture media. DNA excretion occurs under many culture conditions. The excreted DNA has an estimated molecular weight of 3 to 12 x 10(6) as determined by gel filtration on Sepharose 2B. It forms a single sharp peak at a density of 1.055 g/cm(3) when examined by sucrose density gradient centrifugation, suggesting that the DNA is complexed to protein or lipid.

We have identified two populations of human lymphocytes differing in responsiveness to the plant mitogen concanavalin A (Con-A). When peripheral blood lymphocytes are passed through a nylon column a population of lymphocytes highly responsive to Con-A adheres to the fibers while a second population of cells relatively unresponsive to Con-A emerges from the column. The untreated peripheral blood lymphocytes are termed "unfiltered" cells while the lymphocytes which pass through the column are termed "filtered" cells. Under standard assay conditions the Con-A-stimulated DNA synthesis is 6.5-fold greater, and the percentage blast formation is four-to fivefold greater in the unfiltered than in the filtered population. Mixing unfiltered with filtered cells fails to induce responsiveness in the latter indicating that a "helper" cell is not involved. The failure of filtered cells to respond to Con-A is specific for that mitogen since both populations respond nearly equally to erythroagglutinating phytohemagglutinin (E-PHA) and the poke weed mitogen (PWM). Binding studies with Con-A-(131)I demonstrate that the unfiltered population possesses approximately three times as many Con-A receptor sites per cell as the filtered cells, although both cell populations bind the mitogen with the same affinity (apparent association constant [K] of 1.67 x 10(6)m(-1)). The relationship between Con-A binding and lymphocyte activation was determined by measuring the effect on DNA synthesis of incubating the two lymphocyte populations with increasing amounts of Con-A. The concentration of Con-A required for half-maximal stimulation of DNA synthesis was 5-14 times greater for the filtered cells. However in the presence of very high Con-A concentrations the filtered cells achieved a maximal rate of DNA synthesis approaching that of the unfiltered population. These data implicate the decreased number of Con-A receptor sites on the filtered cells in their failure to respond to low concentrations of Con-A. A crucial event in the activation of lymphocytes by plant mitogens may be the binding of a critical number of the mitogen molecules to the cell surface.