Selective triggering of human T and B lymphocytes in vitro by polyclonal mitogens.

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

Infrequent normal B lymphocytes express features of B-chronic lymphocytic leukemia.

An infrequent (2-3%) B lymphocyte subpopulation was found in the normal human tonsil and lymph nodes that shows the phenotypic characteristics of B-chronic lymphocytic leukemia (B-CLL) (rosette formation with mouse erythrocytes, weak expression of membrane Ig, staining for HLA-DR, and OKT1 or Leu-1 detecting a T cell-associated p65 antigen). Preliminary evidence suggests that at least a subpopulation of these cells is found, in small proportions, within the germinal centers. These cells were not observed in the human bone marrow. B-CLL may involve this peripheral B lymphocyte subset.

The specific recognition by macrophages of CD8+,CD45RO+ T cells undergoing apoptosis: a mechanism for T cell clearance during resolution of viral infections.

During viral infections, CD8+,CD45RO+ T populations expand. These primed cells express abundant levels of cytoplasmic granules that contain perforin and TIA-1. Recent work has suggested that the majority of this CD8+ population downregulates Bcl-2 protein expression and is destined to undergo apoptosis. In this study we have investigated the elimination of these apoptotic CD8+ T cells by both human monocyte-derived and murine bone marrow macrophages. We have found that these phagocytes recognize and ingest both apoptotic CD8+ and CD4+ T cells using an alpha v beta 3 (vitronectin receptor)/CD36/thrombospondin recognition system, with the same receptors being used in the recognition of apoptotic neutrophils. These data provide new evidence for a mechanism that enables the clearance of greatly increased populations of CD8+ effector cells which are found during viral infections. This enables cellular homeostasis to occur in the host upon resolution of viral diseases in vivo.

The significance of low bcl-2 expression by CD45RO T cells in normal individuals and patients with acute viral infections. The role of apoptosis in T cell memory.

The bcl-2 gene product has been shown to prevent apoptotic cell death. We have now investigated the bcl-2 protein expression by resting and activated mature T cell populations. Freshly isolated CD45RO+ T cells within CD4+ and CD8+ subsets expressed significantly less bcl-2 than CD45RO- (CD45RA+) T cells (p < 0.001). When CD45RA+ T cells within both CD4+ and CD8+ subsets were activated in vitro, the transition to CD45RO phenotype was associated with a decrease in bcl-2 expression. Patients with acute viral infections such as infectious mononucleosis caused by Epstein-Barr virus infections or chickenpox, resulting from varicella zoster virus infection, had circulating populations of activated CD45RO+ T cells which also showed low bcl-2 expression. In these patients, a significant correlation was seen between low bcl-2 expression by activated T cells and their apoptosis in culture (r = 0.94, p < 0.001). These results suggest that the primary activation of T cells leads to the expansion of a population that is destined to perish unless rescued by some extrinsic event. Thus the suicide of CD45RO+ T cells could be prevented by the addition of interleukin 2 to the culture medium which resulted in a concomitant increase in the bcl-2 expression of these cells. Alternatively, apoptosis was also prevented by coculturing the activated T lymphocytes with fibroblasts, which maintained the viability of lymphoid cells in a restinglike state but with low bcl-2 expression. The paradox that the CD45RO+ population contains the primed/memory T cell pool yet expresses low bcl-2 and is susceptible to apoptosis can be reconciled by the observations that maintenance of T cell memory may be dependent on the continuous restimulation of T cells, which increases their bcl-2 expression. Furthermore, the propensity of CD45RO+ T cells to extravasate may facilitate encounter with fibroblast-like cells in tissue stroma and thus be an important additional factor which promotes the survival of selected primed/memory T cells in vivo.

Clinical application of a rapid lung-orientated immunoassay in individuals with possible tuberculosis.

BACKGROUND: Immunological ex vivo assays to diagnose tuberculosis (TB) have great potential but have largely been blood-based and poorly evaluated in active TB. Lung sampling enables combined microbiological and immunological testing and uses higher frequency antigen-specific responses than in blood. METHODS: A prospective evaluation was undertaken of a flow cytometric assay measuring the percentage of interferon-gamma synthetic CD4+ lymphocytes following stimulation with purified protein derivative of Mycobacterium tuberculosis (PPD) in bronchoalveolar lavage fluid from 250 sputum smear-negative individuals with possible TB. A positive assay was defined as >1.5%. RESULTS: Of those who underwent lavage and were diagnosed with active TB, 95% (106/111) had a positive immunoassay (95% CI 89% to 98%). In 139 individuals deemed not to have active TB, 105 (76%) were immunoassay negative (95% CI 68% to 82%). Of the remaining 24% (34 cases) with a positive immunoassay, a substantial proportion had evidence of untreated TB; in two of these active TB was subsequently diagnosed. Assay performance was unaffected by HIV status, disease site or BCG vaccination. In culture-positive pulmonary cases, response to PPD was more sensitive than nucleic acid amplification testing (94% vs 73%). The use of early secretory antigen target-6 (ESAT-6) responses in 71 subjects was no better than PPD, and 19% of those with culture-confirmed TB and a positive PPD immunoassay had no detectable response to ESAT-6. CONCLUSIONS: These findings suggest that lung-orientated immunological investigation is a potentially powerful tool in diagnosing individuals with sputum smear-negative active TB, regardless of HIV serostatus.

Long-term clinical and surrogate marker effects of subcutaneous intermittent interleukin-2 without antiretroviral therapy in HIV-infected patients.

OBJECTIVES: Subcutaneous administration of interleukin-2 (IL-2) has been shown to increase CD4 counts in HIV-infected patients. It remains unclear whether this effect is associated with a clinical benefit. PATIENTS AND METHODS: We conducted a long-term follow-up in the cohort of the UK-Vanguard study in which three groups of 12 antiretroviral-naive subjects with CD4 cell counts >350 cells/mm(3) received no treatment or IL-2 at either 4.5 or 7.5 MIU twice daily in 5 day cycles, respectively. RESULTS: Mean follow-up was 376 weeks. IL-2 therapy was associated with a higher area under the curve of CD4 cell count change from baseline at week 48 but not thereafter. HIV-RNA levels were unaffected. Highly active antiretroviral therapy (HAART) was initiated after a mean of 172, 175 and 152 weeks in the control group, low-dose and high-dose IL-2 treatment group, respectively, a statistically non-significant difference. There was a tendency to start HAART soon after discontinuation of IL-2 therapy which may have been triggered by the steep decay of CD4 counts. There were two serious adverse events in the control group, seven in the low-dose IL-2 group and eight in the high-dose IL-2 group. No pattern of disease was detected, making an association with IL-2 therapy unlikely. CONCLUSIONS: We could detect neither a benefit of IL-2 therapy after week 48 nor delayed initiation of HAART. This is currently the longest follow-up data comparing IL-2 therapy with no therapy in antiretroviral-naive HIV-infected patients and does not show a persistent benefit of the intervention.

Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma.

Precursors of plasma cells were studied in the bone marrow of 28 patients with multiple myeloma, plasma cell leukemia, and benign monoclonal gammopathy. Pre-B and B cell populations were analyzed with anti-B monoclonal antibodies corresponding to the clusters standardized at the Leucocyte Typing Workshops in Paris and Boston (CD9, CD10, CD19-22, CD24). In advanced forms of plasma cell malignancies, such as cases of multiple myeloma in stages II and III and of plasma cell leukemia, some cells of lymphoid morphology expressed common acute lymphoblastic leukemia antigen (CALLA, CD10) and HLA-DR, but contained no detectable terminal deoxynucleotidyl transferase enzyme. These CALLA+ cells were absent in benign monoclonal gammopathies. In multiple myeloma, the CALLA+ cells were negative for surface and cytoplasmic immunoglobulins (Ig), and, unlike CALLA+, terminal deoxynucleotidyl transferase (TdT+) pre-B cells in the normal bone marrow also failed to react with antibodies to B cell-associated antigens such as CD9, CD19, CD22, and CD24. The CALLA+, Ig- cells could be regarded as preplasmacytic since, after having been separated and stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13 acetate in vitro, they transformed into plasma cells and synthesized the same heavy and light chains as myeloma cells.

Subpopulations of normal and leukemic human thymocytes: an analysis with the use of monoclonal antibodies.

Combinations of antibodies to membrane antigens and to terminal deoxynucleotidyl transferase (TdT) were used to study human thymocyte and bone marrow subpopulations and leukemia cells. Cortical thymocytes were TdT+ and expressed T-cell antigens (HuTLA+), a thymocyte-specific antigen (HTA-1+), and a leukocyte antigen (HLe-l++) but lacked detectable HLA-A,B,C and la (HLA-D) antigens. In contrast, medullary thymocytes were TdT-, HuTLA+, HTA-1-, HLe-l++. A small subpopulation of larger, probably immature, thymocytes were strongly TdT+, HuTLA+, la-, HTA-1-, HLe-l +/-. Many blast cells from cases of thymic acute lymphoblastic leukemia (Thy-ALL) showed the phenotype of this small subset, and only a proportion of Thy-ALL blast cells exhibited HTA-1 and HLe-l antigens as strongly as was observed on normal cortical thymocytes. In contrast, TdT+ cells observed in normal juvenile bone marrow were HuTLA, HTA-1-, HLA+, la+. This phenotype corresponded to the phenotype of the common form of ALL (non-T, non-B) and indicated that further studies are necessary to analyze the differentiation of bone marrow precursors to thymic cells.

Evaluation of ricin A chain-containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B-cells as potential reagents for in vivo therapy.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases.

Current views about the origin of acute lymphoid leukemia (ALL) emphasize the importance of maturation arrest at a precursor cell level. Recently, the CD22 antigen has been identified in the cytoplasm of normal bone marrow-borne immature B lineage cells, while the CD3 antigen (epsilon chain) has been detected within normal immature thymic blasts. In the first part our study performed on 100 cases of known acute leukemias, the expression of such cytoplasmic molecules, referred to as cCD22 and cCD3, was analyzed together with their appearance in the leukemic cells' membrane (mCD22 and mCD3). The presence of cCD22 in B-lineage ALL and that of cCD3 in T-ALL has indeed fully confirmed the diagnosis reached by other markers, and mCD22 and mCD3 were expressed on only a few cases of B- and T-lineage ALL, also revealing a degree of developmental asynchrony within leukemic blasts. In the subsequent analysis both cCD22 and cCD3 have been included in a standard panel of diagnostic reagents applied on 500 consecutive cases of acute leukemia. Here the aim was to analyze both the diagnostic precision of individual markers and the heterogeneity of various leukemic types in terms of the expression of membrane and intracellular antigens and their cytochemical features (Sudan Black B and esterases). It has been found that cCD22 and cCD3 are exquisitely specific for B-precursor ALL (TdT+, CD19+) and T-ALL (TdT+, CD7+), respectively, while both markers are absent in acute myeloblastic leukemia (AML) and acute myelomonocytic and monocytic leukemia (AMML/AMoL). These observations contrast the findings which demonstrate that 31% of cases among nonlymphoid acute leukemia (including AML and AMML) express CD7 and/or TdT. The study of myeloid antigens detected by CD13, CD33, and CD14 is also informative and complementary, both in diagnosing and subdividing the AML and AMML/AMoL groups. The peculiar main observation of this study is that only with the combined use of these markers in a microplate assay for membrane antigens, followed by double staining for intracellular antigens such as terminal deoxynucleotidyl transferase, cCD3, cCD22, c mu heavy chain, and T cell receptor beta, it is possible to safely establish the lineage affiliation and subgrouping of virtually all acute leukemias. Among these cases are those with aberrant combinations of markers, including 14% of B-lineage ALL (cCD22+,CD19+,TdT+) and a single case T-ALL (cCD3+,CD7+,TdT+), which exhibit CD13 and/or CD33 antigens, cases with mixtures of ALL and AML blasts, and 1.2% of acute leukemias which lack lineage affiliation and can be regarded as acute undifferentiated leukemia.

Molecular investigation of 19p13 in standard and variant translocations: the E12 probe recognizes the 19p13 breakpoint in cases with t(1;19) and acute leukemia other than pre-B immunophenotype.

The gene E2A has recently been cloned, mapped to 19p13 and shown to be rearranged in cases of pre-B acute lymphoblastic leukemia (ALL) with t(1;19) (q23;p13). Nine cases with a 19p13 breakpoint, four having a phenotype other than pre-B, have been investigated with the E12 probe to the E2A gene. Five cases had t(1;19) (q23;p13) and C-ALL with pre-B phenotype in four out of four cases tested. Two cases had t(1;19) (q21;p13), one with Null cell phenotype, t(4;11), and 'jumping translocations' and the other with acute non-lymphocytic leukemia M5 following bone marrow transplantation for C-ALL. Variant translocations in patients with ALL were t(15;19) (q15;p13) and t(17;19) (q21;p13). Southern blotting with E12 showed rearrangement in the cases with t(1;19) (q23;p13) and t(1;19) (q21;p13), but not in other cases with variant 19p13 breakpoints. Thus rearrangement of the E2A gene is not restricted to cases with pre-B ALL but may also occur in acute leukemias with other immunological phenotypes. Failure to detect rearrangement in 19p13 variants may be due to an E2A breakpoint outside the E12 recognition region. Alternatively, there may be further genes in this location with relevance to leukemogenesis.

Leukemia-associated changes identified by quantitative flow cytometry. III. B-cell gating in CD37/kappa/lambda clonality test.

Normal and malignant B-lymphoid cells were studied for CD37 antigen expression with three-color immunofluorescence (IF) in combination with kappa/lambda light chain staining, and by quantitative immunofluorescence utilizing the QIFI test. Peripheral B cells brightly expressed CD37 antigen (median 80-114 x 10(3) molecules/cell). Moderate to high levels (> 20 x 10(3)/cell) of CD37 expression were detected in 364 in 366 cases of peripheral B-cell disorders including all cases of B-ALL, B-cell lymphomas and B-CLL as well as eight of ten cases of PLL. By contrast, slg- B-cell precursors and other cell types in normal bone marrow (BM) were CD37-/CD37dull (< 10 x 10(3) molecules/cell). The negativity for CD37 or only CD37dull expression was confirmed in 180 of 182 cases of precursor B-ALL and 196 cases of non-B malignancies. Among the CD37 cluster, the RFB7 antibody of IgM class showed the weakest binding to non-B cells. In 64 normal samples of blood and BM the CD37+ gated cells showed normal kappa/lambda ratios as expected, while in 100 cases of B malignancy striking changes such as kappa/lambda monoclonality (79%) and aberrant slg- or sigdull expression (21%) were seen among the gated CD37+ B cells. The CD37/kappa/lambda test identified as few as 0.5% kappa+ or lambda- monoclonal B cells admixed to normal BM: circulating B-lymphoma cells were seen in nine patients with morphologically normal blood count. The discrimination of the Kolmogorov-Smirnov (KS) test for kappa/lambda excess was also improved by CD37+ B gating. Thus CD37+ B-cell gating and kappa/lambda analysis is a simple and sensitive routine test, e.g. when combined with autogating on a Cytoron-Absolute cytometer, for identifying malignant B cells in minimally involved BM and blood.

Leukemia-associated changes identified by quantitative flow cytometry. II. CD5 over-expression and monitoring in B-CLL.

The CD5 antigen density on B cells was studied on fetal spleen, cord blood, and adult peripheral blood (after immunomagnetic bead purification) using an indirect immunofluorescence technique. In fetal spleen, there was a continuum in CD5 expression, whereas all cord blood and less than 20% adult peripheral blood B cells were CD5+. Mean CD5 antigen density on these normal cells was low (3-6 x 10(3) molecules/cell); eight to 20 times lower than on normal T lymphocytes. In adult blood, less than 10% B cells expressed more than 3 x 10(3) CD5 molecules/cell. In chronic malignancies, 34/35 cases had a CD5 antigen density lower than on residual T cells, but mean antigen density was higher (14.8 +/- 2.1 x 10(3) molecules/cell) than on normal B cells. Sixteen cases of chronic lymphocytic leukemia (50%) expressed a CD5 density above 10 x 10(3) molecules/cell. This aberrantly high CD5 expression was used to detect neoplastic cells after dilution in normal lymphocytes, with a limit of detection between 1:100 and 1:1000. Quantitation of the CD5 antigen allows better characterization of the B1 population and should be used for the monitoring of chronic malignancies.

Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia.

The expression of bcl-2 protein that is involved in preventing apoptosis in hemopoietic and other cells was evaluated by quantitative flow cytometry in various subpopulations in the normal fetal bone marrow (FBM) and in different types of acute myelogenous leukemias (AML). In the FBM the highest bcl-2 levels (mean antibody binding capacity 51 x 10(3) molecules/cell) were found in CD34+ intermediate sized blasts and myeloblasts, while a CD34+ subset of CD10+ lymphoblasts had low bcl-2 content (8-10 x 10(3) molecules/cell) and the CD34-, CD10+ lymphoblasts were, as expected from previous studies, bcl-2- (< 5 x 10(3) molecules/cell). Variable levels of bcl-2 (5.1-222 x 10(3)) were found in 43 tested cases of AML. The bcl-2 levels decrease with granulocytic and monocytic differentiation and, accordingly, cases of AML with M1 and M2 features showed significantly higher mean bcl-2 levels than the leukemias with promyelocytic (M3) or myelo-monocytic (M4/M5) features. Nevertheless, in seven cases of AML the bcl-2 levels were higher than seen in the normal FBM cells and none of these patients remain in remission after 2 years. Furthermore, in several AML cases intraclonal heterogeneity was observed. The undifferentiated smaller blasts with Class-IIdim display had higher bcl-2 content than the more differentiated larger blasts with more granular side scatter and Class-bright expression. In the same subsets of AML blasts the proliferative S-G2-M fractions showed a reciprocal correlation to bcl-2 content. Thus the higher bcl-2 levels may give a survival advantage and confer some degree of drug resistance to the least differentiated blast populations. The multi-parameter analysis described in this paper, including a combined bcl-2 and cytokinetic analysis of phenotypically defined subgroups of AML blasts, may detect early population shifts during relapse and also guide combination drug therapy.

Expression of the E2A-PBX1 fusion transcripts in t(1;19)(q23;p13) and der(19)t(1;19) at diagnosis and in remission of acute lymphoblastic leukemia with different B lineage immunophenotypes.

The chromosomal translocation t(1;19)(q23;p13) and its variant form der(19)t(1;19) found in 3-5% of acute lymphoblastic leukemia (ALL) results in the expression of the E2A-PBX1 fusion transcript. Although strongly associated with a pre-B immunophenotype, we report the occurrence of t(1;19) in bone marrow or peripheral blood in nine patients with ALL with the following immunophenotypes: pre-B ALL (four), c-ALL (two), c-ALL clg not tested (one), null-ALL (one) and mature B-ALL (one). The E2A-PBX1 fusion transcript investigated by reverse-transcriptase polymerase chain reaction (RT-PCR) was seen in all patients at diagnosis and/or on follow-up samples. Six patients are alive in first clinical remission. Of these patients, three were PCR+ve from between 2 and 38 months from diagnosis, and three were PCR-ve when examined at 5, 26 and 51 months from diagnosis. Two patients are in second remission. One was PCR+ve at 18 months, suffered a CNS relapse at 21 months but was PCR-ve 1 month later. The other was PCR+ve in remission at 2 and 11 months from diagnosis and in testicular relapse at 31 months, but was PCR-ve 5 months later. The remaining patient died 2 months from diagnosis and was not investigated in remission. The prognostic significance of these findings remains to be investigated.

The detection of residual acute lymphoblastic leukemia cells with immunologic methods and polymerase chain reaction: a comparative study.

In this study we applied double color immunofluorescence analysis and polymerase chain reaction (PCR) amplification of rearranged TCR delta genes for detecting residual leukemia in the posttreatment bone marrow (BM) samples taken from four patients in morphological remission. In three of these patients (nos. 1-3; T-ALL) a combination of CD3 and anti-TdT antibodies (Abs) was used to identify residual blasts while in patient 4 (B lineage ALL) the combination CD13/TdT served to detect residual disease. Two rounds of PCR primed by nested amplimers were carried out to prepare clonospecific probes from presentation DNA and to investigate the follow-up samples. In patients 1 and 2 no cCD3+/TdT+ cells were seen posttreatment, but PCR amplification of the TCR V delta 1-D-J delta 1 region revealed residual disease in both patients. Patient 1 underwent allogeneic BM transplant (BMT) 8 months after diagnosis and is well 3 months post-BMT while patient 2 relapsed 12 months after presentation. In patient 3 the remission samples investigated 2 and 3 months after diagnosis did not contain cCD3+/TdT+ cells, but in the sample collected at 4 months a few such cells (0.0001-0.001%) could be detected. In the same sample, PCR amplification of the TCR V delta 2-D-J delta 1 region indicated the presence of 10(-4)-10(-3) residual leukemic cells. These findings predicted full morphological relapse which occurred 2 months later. In patient 4 CD13/TdT double positive cells were clearly seen 2 and 3 months after presentation. PCR amplification of the V delta 2-D delta 3 recombination also revealed residual blasts when applied to one of such "remission" samples. After further remission induction treatment, no immunologic evidence of residual disease was detected. This patient received an allogeneic BMT 8 months after diagnosis and is disease free 4 months after BMT. Our data indicate that both double color immunofluorescence and PCR analysis offer powerful tools to study residual leukemia and highlight the advantages as well as the potential limitations of each technique.

Quantification of CD24 and CD45 antigens in parallel allows a precise determination of B-cell maturation stages: relevance for the study of B-cell neoplasias.

Quantitative expression, i.e. absolute number of monoclonal antibody molecules bound per cell, was evaluated for CD24 and CD45 by flow cytometry with standards of fluorescence intensity on a panel of normal and neoplastic B cells. The CD24 antigen was expressed at homogeneous high level in fetal bone marrow and liver. Its density decreased progressively in the other normal tissues in parallel with the B-cell maturation. The ratio between CD24 density measured on fetal bone marrow B cells and that seen on adult peripheral B cells was 6:1. The CD45 antigen density was lower on fetal bone marrow cells than in the more mature stages. Fetal spleen lymphocytes and all the mature B lymphocytes displayed the same CD45 density than that seen on normal adult peripheral T cells. The CD24/CD45 antigen density ratio was precisely related to the stage of B-cell maturation. The same pattern of variation of CD24 and CD45 antigen density was seen on B-cell neoplasias, with a significantly higher value of CD24 and lower value of CD45 in acute lymphoblastic leukemias than in chronic malignancies. CD24 and CD45 antigen levels were frequently out of the range observed in the corresponding normal population. Among ALL patients, a low CD24/CD45 antigen density ratio was associated with a good prognosis. These data confirm the interest of an absolute quantitative study for widely distributed antigens.

Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation.

In the present study, we explored the suitability of a new cell fixative (ORTHO PermeaFix, OPF) for the detection by flow cytometry of intracellular molecules while preserving the cell surface immunoreactivity, scatter features and morphology. The effect of OPF was investigated on whole blood of ten normal donors, and on separated blasts of 17 leukemic patients. OPF fixation for 45 min to 24 h maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity. For at least 1 week following fixation, the cells were still suitable for immunostaining with monoclonal antibodies that recognize the main lymphoid populations. These included CD3, CD4 and CD8 for T-cell subsets, CD19 and CD16 for B lymphocytes and NK cells, and CD45 for leukocyte common antigen (LCA). The OPF fixation of leukemic cells allowed the simultaneous detection of nuclear TdT in conjunction with membrane CD19, and with membrane and/or cytoplasmic CD22 in common-ALL, as well as with cytoplasmic CD3 in T-ALL cases. Our findings suggest that with the introduction of this new fixative into the routine laboratory service, a number of convenient and practical arrangements can be made which increase the efficiency of immunodiagnosis. Small laboratories with no inhouse flow-cytometric facilities can now accumulate OPF-treated whole blood samples for at least 3-4 days and send these to reference laboratories. In addition, the immunodiagnosis of acute leukemia is greatly facilitated by combination staining for membrane and intracellular antigens both at diagnosis and when the analysis of minority populations is warranted for detecting minimal disease.

Phenotypic, genotypic, cytochemical, and ultrastructural characterization of acute undifferentiated leukemia.

During the diagnostic investigation of 750 acute leukemias, nine cases were morphologically, cytochemically, and phenotypically undifferentiated. In seven of these cases the blasts were class II+, CD34+ and TdT+, in one were class II+, TdT+, CD7+ while in the remaining leukemia blasts expressed class II only. Cytoplasmic and membrane CD22, CD3, CD13, and Ig as well as membrane CD19, CD10, CD37, CD2, CD33, CD14, glycophorin C, and CD61 were absent. The further characterization of these rare leukemias yielded the following results. The TCR-beta, -gamma and -delta genes were in germline configuration in seven cases studied while IgH genes were rearranged on both alleles in two cases and germline in the other five. By ultrastructural analysis peroxidase activity was detected on unfixed cells in a minority of blasts from four of seven cases. In two of the peroxidase-positive cases a small proportion of blasts also reacted with an anti-myeloperoxidase monoclonal antibody. In one of the peroxidase-negative cases, 7% of blasts were labeled by the antibody, suggesting the presence of peroxidase in its proenzyme form. Importantly, the two cases with Ig gene rearrangements did not have cytochemically or immunologically detectable peroxidase. Three of the nine patients were treated as ALL while six received AML chemotherapy. In five patients complete remission was achieved while the other four died from infections during remission induction. Four patients are still in remission 7, 12, 24, and 30 months after diagnosis while one patient relapsed after 12 months. In conclusion, we have characterized the genotypic and ultrastructural features of subtype of acute leukemia in which blasts expressed immaturity markers and lacked lineage associated antigens. In contrast to previously reported "unclassifiable" cases, the leukemias were phenotypically homogeneous and showed a good response to chemotherapy.

Autologous bone marrow transplantation in acute lymphoblastic leukemia--preclinical immunologic studies.

Immunologic aspects of autologous bone marrow transplantation (ABMT), immunodiagnosis, patient monitoring, and the purging of bone marrow have been studied in individual patients. It was demonstrated that the most sensitive method for detecting lymphoid cells which show the phenotypes of ALLs of B or T lineage was double immunofluorescence staining for nuclear terminal transferase (TdT) and B or T lineage antigens. With the help of these sensitive tests in the presence of rabbit complement (C'), MAbs CD10 (RFAL3 of IgM class), CD19 (SB4 of IgM class), and their cocktail were capable of eliminating greater than 3 log blast cells of B lineage ALL in 84%, 75.5%, and 90% of cases, respectively. The same reagents lysed 26.8%, 0%, and 45% of blasts in the presence of human C'. CD7 (RFT2, IgG2) eliminated greater than 3 log T-ALL blast cells in 73% of cases. The proliferative fractions of leukemic blasts were also TdT+ and sensitive to lysis with MAb and C'. On the basis of these observations MABs were selected for purging in 36 patients undergoing ABMT in first remission (10 patients considered to be at a high risk of relapse), second and third remissions (23 and 2 patients), and without entering into remission (1 patient). The efficacy of eliminating the MAb-reactive cells from the bone marrow inoculum was also documented in five patients. By the use of sensitive immunologic assay (TdT/cytoplasmic CD3 double staining) in patients with T-ALL, no residual leukemia (less than 10(-4] could be detected at the time of transplantation. Following an observation period of 5-34 months, 24 of the 36 patients are alive and well with no procedure-related mortality.