Somatic mutations in the HLA genes of patients with hematological malignancy.

Somatic mutations and genomic alterations are frequent events in the clonal evolution of hematologic malignancies. Recent studies have reported copy neutral loss of heterozygosity (LOH) for the mismatched human leukocyte antigen (HLA) haplotype in patients relapsed after haploidentical hematopoietic cell transplantation (HCT) for a hematologic malignancy. Herein, we report 15 cases of somatic mutations in the HLA genes of patients with a variety of hematologic diseases, including acute myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and non-Hodgkin's lymphoma, encountered at our institute over the past decade. While two of the cases were identified in patient relapse specimens collected post-HCT, 13 cases were found in peripheral blood specimens submitted for HLA typing prior to transplantation. Ten patients exhibited acquired LOH for all or part of one HLA haplotype. Five other cases involved somatic mutations in the nucleotide sequences of common HLA-A or HLA-B alleles. Since they are not systematically evaluated prior to HCT, acquired mutations in HLA genes are likely under reported. Beyond the implications for accurate HLA typing and donor selection, alternations that result in the loss of HLA expression may allow escape from immune surveillance and adversely impact transplant outcome.

A clone-specific monoclonal antibody that inhibits cytolysis of a cytolytic T cell clone.

Monoclonal antibody 384.5 specifically inhibited cytolysis of P-815 target cells by cloned L3 cytotoxic T lymphocyte (CTL) effector cells. The lytic activity of other cloned CTL that have other distinct specificities was not affected. Antibody 384.5 did not inhibit the cytolytic activity of bulk populations of C57BL/6 mixed lymphocyte culture (MLC) cells. Concanavalin A-facilitated cytolysis by T cell clone L3 but not T cell clone B18 was inhibited by antibody 384.5, whereas phytohemagglutinin-facilitated cytolysis by L3 cells was not strongly inhibited. Antibody 384.5 binds specifically to L3 cells but not to several other T lymphocytes clones, or to a detectable portion of populations of primary MLC cells, normal spleen, thymus, lymph node, or bone marrow cells. In contrast, C57BL/6 anti-B10.A(5R) secondary MLC cells (genetically enriched for reactivity against the H-2Dd region gene products) contained a small population which reacted with the antibody 384.5. The determinant detected by antibody 384.5 was susceptible to trypsin treatment, and was reexpressed after overnight incubation. These results suggest that the monoclonal antibody 384.5 detects an endogenously synthesized clone-specific determinant associated with the cytolytic activity of the L3 CTL clone. These properties make antibody 384.5 an attractive candidate for an antibody that reacts with the antigen-recognition site of a cytolytic T cell antigen receptor.

Lyt-2-/Lyt 3- variants of a cloned cytolytic T cell line lack an antigen receptor functional in cytolysis.

To investigate the role of Lyt-2 and Thy-1 in cytolysis, we have generated, by ethyl methanesulfonate mutagenesis and selection, variants of the cloned cytolytic T lymphocyte line L3 that specifically lack either Lyt-2 or Thy-1. An analysis of these variants indicates that neither Lyt-2 nor Lyt-3 is responsible for the lethal hit, but suggests that Lyt-2 and/or Lyt-3 are required for an antigen receptor functional in cytolysis. The data also suggest that the expression of Lyt-3 on the cell surface is not independent of the expression of Lyt-2. Finally the data indicate the Thy-1 plays no role in cytolysis.

Expression of IgD by murine lymphocytes. Loss of surface IgD indicates maturation of memory B cells.

B lymphocytes capable of generating primary IgM and IgG plaque-forming cells (PFC) responses to burro erythrocytes have surface IgD, as do primary IgM PFC. IgG memroy cells arising after one injection of antigen are divided into two groups, one of which expresses surface IgD while the other has no detectable membrane IgD. PFC generated from the IgG memory cells lacking surface IgD show a higher average avidity than those arising from IgD-positive IgG memory cells, indicating that mature IgG memory cells do not have surface IgD. After more than one injection of antigen, few, if any, IgG memory cells have surface IgD. IgG PFC arising in primary or secondary immune response lack membrane-bound IgD. These data provide the outlines for a B-cell maturation pathway in which IgD marks unprimed and early memory B cells and is lost in mature memory cells. Studies presented here were conducted by isolating IgD+ and IgD- cells with the fluorescence-activated cell sorter and functional testing of the isolated populations in adoptive transfer experiments.

Intensive postgrafting immune suppression combined with nonmyeloablative conditioning for transplantation of HLA-identical hematopoietic cell grafts: results of a pilot study for treatment of primary immunodeficiency disorders.

This study was designed to determine the safety of a nonmyeloablative regimen in patients with primary immunodeficiency disorders (PID) who had infections, organ dysfunction or other risk factors that precluded conventional hematopoietic cell (HC) transplant. Fourteen patients received HLA-matched related (n=6) or unrelated (n=8) HC grafts from marrow (n=8), peripheral blood mononuclear cells (n=5) or umbilical cord blood (n=1), either without conditioning (n=1), or after 200 cGy total body irradiation alone (n=3) or with 90 mg/m2 fludarabine (n=10). All patients were given postgrafting immunosuppression with mycophenolate mofetil and cyclosporine. Mixed (n=5) or full (n=8) donor chimerism was established in 13 patients, and one patient rejected the graft. Eight patients developed acute grade III (n=1) and/or extensive chronic GVHD (n=8). With a median follow-up of 4.9 (range, 0.7-8.1) years, the 3-year overall survival, event-free survival and transplant-related mortality were 62, 62 and 23%, respectively. Correction of immune dysfunction was documented in 8 of 10 patients with stable donor engraftment. These preliminary results indicated that this approach was associated with stable donor engraftment and a low incidence of early mortality and, thus, can be considered for certain high-risk patients with PID. However, there was a risk of GVHD, which is an undesirable outcome for this group of patients.

T-cell lineage of a Friend virus-induced lymphatic leukemia in athymic rats.

Athymic (rnu/rnu) and euthymic rats inoculated with the Friend virus-associated lymphatic leukemia virus developed lymphocytic leukemia. Neoplastic cells from these animals were evaluated by means of indirect immunofluorescence and flow cytofluorometry with monoclonal antibodies Ox-1, Ox-7, and W3/25, which react with surface antigens present on normal rat lymphoid cell populations. Lymphoid cells from leukemic animals revealed characteristic alterations in cell surface fluorescence profiles when compared to normal, healthy controls. Athymic and euthymic leukemic rats were similar in that many cells from both the spleen and bone marrow had markers on the cell surface normally found on thymocytes but not on mature peripheral lymphocytes. These studies provided evidence supporting the presence of T-lineage lymphocytes in the athymic rat. Further, this population of early or "pre"-T-lymphocytes included the predominant leukemia cell type induced by the Friend virus-associated lymphatic leukemia virus.

Prediction of relapse of pediatric acute myeloid leukemia by use of multidimensional flow cytometry.

BACKGROUND: Most patients receiving therapy for acute myeloid leukemia (AML) enter an interval in which leukemic blast cells cannot be detected by light microscopy (i.e., morphologic remission). However, many of these patients experience a subsequent relapse. Multidimensional flow cytometry, which allows the discrimination of antigens expressed on normal and malignant cells, can detect small numbers of cancer cells in bone marrow or peripheral blood specimens. This technique enables the detection of one leukemic blast cell among 10(3) to 10(2) normal regenerating hematopoietic cells. PURPOSE: We determined whether the presence of residual leukemic blast cells, identified in the bone marrow of pediatric patients with AML by use of multidimensional flow cytometry, would be predictive of subsequent leukemic relapse. METHODS: Multidimensional flow cytometry was performed on 205 marrow specimens collected throughout the course of treatment from 39 patients who had achieved morphologic remission. The analyses employed monoclonal antibodies directed against CD45 in combination with mixed pairs of monoclonal antibodies directed against 10 other antigens. A time-varying Cox regression analysis that controlled for sample time intervals, age, sex, morphologic classification of disease, and white blood cell count at diagnosis was used to relate the multidimensional flow cytometric results to the risk of relapse after achieving remission. Reported P values are two-sided. RESULTS: Thirty-five of the 39 patients had bone marrow specimens available from the time that first morphologic remission was achieved. Leukemic blast cells were detected in the specimens from 19 (54%) of these 35 patients. Twenty-five of the 35 patients did not receive an allogeneic (i.e., from a different genetic background) bone marrow transplant during first morphologic remission, and 13 of 14 with residual leukemic cells experienced a relapse at a median time of 153 days after diagnosis (range, 48-863 days). Nine of the 11 patients who did not receive an allogeneic bone marrow transplant and lacked evidence of leukemic blast cells at first morphologic remission relapsed at a median time of 413 days after diagnosis (range, 321-794 days). Among the 10 individuals who received an allogeneic bone marrow transplant during first morphologic remission, five were positive for leukemic blast cells and five were negative; one of these patients (positive for leukemic blast cells) experienced a relapse 265 days after diagnosis, and three others died of transplant-related complications. The estimated risk of relapse during intervals of multidimensional flow cytometric positivity (i.e., intervals of remission for which the immediately preceding cytometry measurement was positive) was 2.8 times greater than that during negative intervals (95% confidence interval = 1.1-7.0; P = .02). CONCLUSIONS AND IMPLICATIONS: Multidimensional flow cytometry identifies residual leukemia in more than half of the patients with AML who are in morphologic remission. The detection of leukemic blast cells in these patients by multidimensional flow cytometry is predictive of a more rapid relapse.

Changes in susceptibility to cytotoxic antibody among tumor cells surviving exposure to chemotherapeutic agents.

We determined that myeloma cells which survived drug treatment had an altered sensitivity to cytotoxic antibody. The effects of several chemotherapeutic agents differing in drug action were compared. The antiserum was directed against viral determinants on the surface of S107 myeloma cells. This antiserum was then used to inhibit the colony formation of drug-treated myeloma cells. Tumor cells were treated with melphalan (200 ng/ml), methotrexate (40 ng/ml), actinomycin D (5 ng/ml), or 0.5 mM thymidine for 24 hr and then washed and resuspended in fresh medium. On various days after drug treatment, aliquots of these cells were exposed to complement and dilutions of antiserum and then cloned in soft agar in order to quantify the degree of antibody-mediated inhibition. Melphalan, methotrexate, and thymidine caused a severalfold increased resistance of the tumor cells to the antiserum during the first 1 or 2 days after drug treatment. During the following days, however, myeloma cells showed markedly increased susceptibility to the antiserum. The biphasic effect of methotrexate or thymidine treatment was similar to that previously observed after melphalan treatment and differed from the effect of actinomycin D. Actinomycin D caused only an increased susceptibility of the tumor cells to the antibody for a period of 4 to 5 days immediately following drug treatment. Our studies indicate that several chemotherapeutic drugs at concentrations comparable to those used in humans have long-lasting antagonistic as well as synergistic effects on the sensitivity of tumor cells to antibody and complement, depending on the particular drug used and on the time interval between drug exposure and subsequent treatment with antibody. These results suggest a model for evaluating the use of antibody in the elimination of malignant cells which, despite exposure to chemotherapy, remain clonogenic and proliferative.

Nonrandom escape of tumor cells from immune lysis due to intraclonal fluctuations in antigen expression.

Considerable heterogeneity in the amount of surface antigen can regularly be demonstrated by cytofluorometric analysis among genetically identical cells in a tumor clone. We have used monoclonal idiotype-specific antibodies to investigate the patterns of change in amounts of an idiotypic tumor antigen and how such changes affect the immune escape of malignant B cells expressing this antigen. By the use of fluorescence-activated cell sorting, we separated cells expressing either very large or very small amounts of the idiotypic target antigen and then analyzed these subpopulations of tumor cells at various times after isolation for expression of idiotype. We found that the differences in amount of antigen expression were not heritable, and that over a period of about 7 days of continuous growth in vitro, the fluorescence-activated cell sorted populations gradually came to express normal amounts of idiotype. The mechanisms regulating the quantity of surface idiotype were independent of those affecting the amounts of other membrane-associated molecules such as H-2 antigen. Furthermore, these nonheritable intraclonal differences in amounts of antigen expression were unrelated to stages of the cell cycle but clearly did affect the susceptibility of the cells from immune lysis. Thus, tumor cells expressing the lowest amount of surface idiotype were much more resistant to the lytic effects of antiidiotypic antibody and complement but had the same cell cycle distribution as did unseparated cells. These results demonstrate that nonheritable, non-cell cycle-related heterogeneity in amount of tumor antigen expression can significantly determine which cells in a cloned malignant cell population preferentially escape monoclonal tumor-specific antibody therapy.

Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse.

PURPOSE: Three open-label, multicenter trials were conducted to evaluate the efficacy and safety of single-agent Mylotarg (gemtuzumab ozogamicin; CMA-676; Wyeth Laboratories, Philadelphia, PA), an antibody-targeted chemotherapy agent, in patients with CD33-positive acute myeloid leukemia (AML) in untreated first relapse. PATIENTS AND METHODS: The study population comprised 142 patients with AML in first relapse with no history of an antecedent hematologic disorder and a median age of 61 years. All patients received Mylotarg as a 2-hour intravenous infusion, at a dose of 9 mg/m(2), at 2-week intervals for two doses. Patients were evaluated for remission, survival, and treatment-emergent adverse events. RESULTS: Thirty percent of patients treated with Mylotarg obtained remission as characterized by 5% or less blasts in the marrow, recovery of neutrophils to at least 1,500/microL, and RBC and platelet transfusion independence. Although patients treated with Mylotarg had relatively high incidences of myelosuppression, grade 3 or 4 hyperbilirubinemia (23%), and elevated hepatic transaminase levels (17%), the incidences of grade 3 or 4 mucositis (4%) and infections (28%) were relatively low. There was a low incidence of severe nausea and vomiting (11%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Many patients received Mylotarg on an outpatient basis (38% and 41% of patients for the first and second doses, respectively). Among the 142 patients, the median total duration of hospitalization was 24 days; 16% of patients required 7 days of hospitalization or less. CONCLUSION: Administration of the antibody-targeted chemotherapy agent Mylotarg to patients with CD33-positive AML in first relapse induces complete remissions with what appears to be a favorable safety profile.

Flow cytometric analysis of human bone marrow. III. Neutrophil maturation.

Neutrophil maturation was studied in normal human bone marrow aspirates using multidimensional flow cytometry in comparison with morphology. The combination of the monoclonal antibodies, CD11b, CD15, and CD16, in addition to the forward and orthogonal light scattering signals permitted the isolation of neutrophilic cells from cells of other cell lineages with a purity of greater than 99%. An unexpectedly close relationship was found between the identification of neutrophil maturation by flow cytometry and morphological classification of cells sorted based on cell surface antigen expression and light scattering properties. The neutrophils could be divided into six distinct maturational stages, i.e., stage N I contained predominantly myeloblasts; stage N II, predominantly promyelocytes; stage N III, predominantly early myelocytes; stage N IV, predominantly myelocytes and metamyelocytes; stage N V, predominantly metamyelocytes and bands; and stage VI, predominantly segmented neutrophils. These data suggest that the morphologic changes during neutrophil maturation can be identified by flow cytometry using simultaneous quantitative assessment of multiple antigens in concordance with the light scattering properties of the human bone marrow cells.

Occult B cell malignancies can be detected by three-color flow cytometry in patients with cytopenias.

Patients with unexplained cytopenias often present a diagnostic dilemma with minimal morphologic or cytogenetic changes to identify the underlying disease process. We have used multidimensional flow cytometry in a study of patients with cytopenias and found that this technology established, changed, or refined the diagnosis in 17/121 patients. Using the flow cytometric technique of CD45 and right angle light scatter (SSC) gating with two additional markers in a three-color analysis, eight of 121 patients were found to have hairy cell leukemia (HCL), in the absence of definitive morphologic findings of HCL. Two additional patients were found to have non-Hodgkin's lymphoma (NHL). Myeloid abnormalities, myelodysplasia (MDS) or acute leukemia was detected in seven of 56 patients with unexplained pancytopenia. Six of 65 patients identified with cytopenias resulting from lymphoid neoplasms had been referred for bone marrow transplantation (BMT) with a presumptive diagnosis of MDS, with subsequent deferral of BMT upon correct diagnosis. The screening technique is incorporated into an extensive immunophenotyping scheme to identify hematopoietic abnormalities using multidimensional flow cytometry (MDF). HCL cells (detected as low as 1.3%) reside in the same position as normal monocytes in the CD45 and SSC plots but could be distinguished from monocytes based on the expression of HLA-DR without CD11b, and expression of CD19. Further phenotyping of the abnormal population confirmed immunoglobulin light chain restriction, CD11c, and CD25 expression. Non-Hodgkin's lymphoma was detected as aberrant mature lymphocytes expressing B lymphoid markers, CD5 and light chain restriction. Myeloid abnormalities were identified in the myeloblast or maturing myeloid cell fractions. The flow cytometric scheme described can be used in primary diagnosis. The technique is definitive, sensitive, and stresses the importance of distinguishing lymphoid from myeloid etiology of cytopenias.

Flow cytometric characterization of acute myeloid leukemia. Part 1. Significance of light scattering properties.

Acute leukemias are classified using the morphological and cytochemical criteria set forward by the French, American and British (FAB) group. Immunophenotyping is helpful for the differential diagnosis but is secondary to the morphological criteria. Immunophenotyping performed by flow cytometry, however, can yield valuable information on cell morphology in addition to cell surface antigen expression. To provide a basis of a combined evaluation of both morphology, i.e. light scattering, and immunophenotype by flow cytometry we have compared the light scattering profiles of 70 patients newly diagnosed with acute leukemia with normal bone marrow and related the findings to the FAB classification. Three main light scattering profiles were observed in the bone marrow aspirates of the 70 patients (A1,2; B1,2,3; C1,2,3,4). A1,2, characterized by a predominant cell cluster with low forward and orthogonal light scattering, contained only and all patients diagnosed as acute lymphoblastic leukemia, acute undifferentiated leukemia, and acute non-lymphocytic leukemia M6 and M1. B1,2,3 is characterized by a predominant cell cluster with large forward and low to high orthogonal light scattering. Category B1 contained the majority of patients classified as M5; the M3 leukemias were categorized as B2. C1,2,3,4 is characterized by a predominant cell cluster with low forward and orthogonal light scattering that branches towards regions with larger light scattering. Categories C1 and C2 contained the majority of the patients classified as M2. Category C3 was specific for M4 and M4eo leukemias. The patients diagnosed as M4 were heterogeneous and equally distributed over the B and C categories. The clear relationship found between the FAB classification and classification by the light scattering profile of the acute leukemias enhances the importance of the flow cytometric classification of leukemias. In contrast with light microscopy, flow cytometry can now provide the hematologist with an objective technique to classify leukemias by the simultaneous assessment of cell surface antigen expression and cell morphology, i.e. light scattering.

Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis.

The frequency and distribution of aberrant antigen expression are analyzed on bone marrow aspirates from 80 patients with newly diagnosed acute myeloid leukemia (AML) by multidimensional flow cytometry. Parameters examined are the light scatter profile of the leukemic cells and the correlative expression of different combinations of the CD2, 4, 5, 7, 11b, 11c, 13, 14, 15, 16, 33, 34, 38, and HLA-DR antigens. Antigen expression on leukemic cells in bone marrow is described by characteristic antigen expression patterns describing: (i) the percentage of cells expressing the antigen; (ii) the antigen density; and (iii) the distribution of the antigen on the leukemic cells. Typically the non-myeloid antigens are homogeneously expressed by the leukemic cells, whereas the myeloid associated antigen CD11b, CD11c, CD14, and CD15 are heterogeneously expressed. Comparison of the antigenic profiles of 80 bone marrow aspirates revealed an extreme interclonal heterogeneity. Comparison of the antigen expression patterns found in AML patients with the antigen expression in normal bone marrow revealed four patterns of aberrant antigen expression in AML: (i) expression of nonmyeloid antigens (i.e. CD2, CD5, and CD7 were present in 57, 60, and 37% of the patients, respectively); (ii) asynchronous expression of myeloid associated antigens (i.e. co-expression of CD34 and CD15 in 25% of the patients and expression of CD16 on immature myeloid cells in 15% of the cases); (iii) over-expression of myeloid associated antigens (e.g. CD34 in 16% of the cases and CD14 on neutrophilic cells in 19% of all patients); and (iv) absence of expression of myeloid associated antigens (e.g. lack of CD33 in 21% of the cases and lack of both CD11b and CD15 in 6% of all patients. Multidimensional flow cytometric analysis of bone marrow aspirates of AML patients disclosed that the leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on aberrant antigen expression and typical light scatter profiles. The ability to distinguish leukemic cells from normal cells allows the detection of residual leukemic cells during and after chemotherapy.

Multiparameter flow cytometric analysis of human fetal bone marrow B cells.

Maturation of adult human bone marrow (BM) B cells is accompanied by the sequential acquisition and loss of characteristic cell surface antigens (Loken et al., Blood 70:1316). Little is known about these changes in fetal BM B cells. In order to compare fetal with adult B cell development, we performed three-color, flow cytometric analyses of cell surface antigens, as well as nuclear TdT staining, on lymphoid cells from fetal BM. Mononuclear cells isolated from fetal BM (18-22 weeks) were stained with combinations of antibodies against CD3, CD10, CD19, CD20, CD21, CD22, CD34, CD45, PCA-1, IgM, and HLA-DR. Analysis of six separate fetal BM specimens indicated that combinations of cell surface antigens were expressed on analogous populations in fetal and adult BM. Consistent with adult BM, greater than 95% of TdT+ cells within the CD10+ population were CD34+, whereas less than 5% were CD34-. This CD10+/CD34+/TdT+ population constituted 30-40% of the total B cell compartment, compared with 10% in adults. Quantitative changes in CD45 expression on fetal BM B cells defined three clear populations, as has been observed in adults. In striking contrast to adult BM, greater than 95% of CD19+ and greater than 95% of surface IgM+ cells were CD10+, indicating that CD10 is a pan-B cell antigen in fetal BM. Virtually no mature B cells expressing CD21, CD22, or PCA-1 were detected in fetal BM. Our results indicate a preponderance of immature phenotypes exist in the fetal BM B cell compartment. These immature cells can be grouped into three distinct populations, and probably correspond to expanded populations found less frequently in adult BM. This striking increase in the earliest identifiable stages of B cell ontogeny is consistent with an active expansion of cells destined to constitute the humoral immune system during fetal development.

Antibody-targeted chemotherapy of older patients with acute myeloid leukemia in first relapse using Mylotarg (gemtuzumab ozogamicin).

We analyzed the safety and efficacy of Mylotarg (gemtuzumab ozogamicin, an antibody-targeted chemotherapy consisting of a humanized anti-CD33 antibody linked to calicheamicin, a potent antitumor antibiotic) in the treatment of 101 patients > or =60 years of age with acute myeloid leukemia (AML) in untreated first relapse in three open-label trials. Mylotarg is administered as a 2-h intravenous infusion at 9 mg/m(2) for two doses with 14 days between doses. The overall remission rate was 28%, with complete remission (CR) in 13% of patients and complete remission with incomplete platelet recovery (CRp) in 15%. Median survival was 5.4 months for all patients and 14.5 months and 11.8 months for patients achieving CR and CRp, respectively. CD33 antigen is present on normal hematopoietic progenitor cells; thus, an expected high incidence of grade 3 or 4 neutropenia (99%) and thrombocytopenia (99%) was observed. The incidences of grade 3 or 4 elevations of bilirubin and hepatic transaminases were 24% and 15%, respectively. There was a low incidence of grade 3 or 4 mucositis (4%) and infections (27%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Mylotarg is an effective treatment for older patients with CD33-positive AML in first relapse and has acceptable toxicity.

Quantitative comparison of myeloid antigens on five lineages of mature peripheral blood cells.

Five-dimensional flow cytometry was used to identify the 5 lineages of peripheral blood leukocytes simultaneously in a single cell preparation. This technique was then used to compare quantitatively the distribution of cell surface antigens on each of these lineages of cells. Neutrophils, eosinophils, basophils, lymphocytes, and monocytes were uniquely identified by correlating their forward and orthogonal light scattering signals with the amount of cell surface-bound IgE. These three cellular characteristics were combined with two additional immunofluorescence labels to create a 5-dimensional space in which each leukocyte population occupied a unique position. The relative quantities of antigens on each cell type were determined for the monoclonal antibodies CD11b, CD13, CD14, CD15, CD16, CD33, CD38, CD45, CD45R, anti-HLA-DR, and anti-Leu-8 labeled with either fluorescein or phycoerythrin. The amount of antigen was described by the mean fluorescence intensity in comparison with the background fluorescence of each cell type. The distribution of the different cell surface antigens on the 5 major leukocyte populations as well as their interdonor variation were then correlated for 10 normal donors. Since none of the antigens studied was lineage specific, it was shown that the different lineages of blood cells could clearly be identified by quantitative comparison of the antigens. This study provides the basis for discrimination between mature cells and immature stages of differentiation of leukocytes and for distinction between normal and leukemic cells.

Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10-positive progenitor cells in normal human bone marrow.

We have previously shown [1] that the anti-My-10 murine monoclonal antibody detected an epitope of a 115-kDa glycoprotein expressed specifically on KG-1a leukemia cells and a small subset of normal human bone marrow cells. This My-10+ marrow cell subset was shown to contain a highly enriched population of morphologic blast cells and hematopoietic colony-forming cells [1]. In this report, My-10+ cells were characterized, by flow cytometry, as an approximately 1% subpopulation of normal human bone marrow cells. My-10+ cells were slightly larger than lymphocytes and agranular, as determined by their fluorescence-activating cell sorting (-er) (FACS) light-scattering properties. In two-color immunofluorescence experiments, My-10+ cells coexpressed the HLA-DR antigen. However, there was no detectable cellular coexpression of My-10 with either the Leu 1-5, 7, 9, 11, 15, M3, or My-18 antigens. There was an average of approximately 50,000 My-10 molecules per My-10+ marrow cell. This provides further evidence that the My-10 molecule is expressed, at relatively low levels, selectively on early human marrow cells but not on mature lymphohematopoietic cells.

Antigenic analysis of hematopoiesis. IV. The My-11 hematopoietic cell surface antigen is expressed by myelomonocytic and lymphoid, but not erythroid, progenitor cells.

The anti-My-11 murine monoclonal antibody reported on in this work identifies a KG-1a cell surface glycoprotein with apparent molecular mass of 210,000 daltons. Peripheral blood B-lymphocytes, and a novel subset of T-lymphocytes (not coinciding with helper or cytotoxic subsets) express My-11 antigen; granulocytes, red cells, and platelets are antigen negative. In normal bone marrow, lymphoid progenitors (TdT positive) and most granulocyte-monocyte progenitors express My-11, but erythroid and multilineage progenitors are My-11 negative. Approximately half of acute leukemia blast cell specimens are My-11 positive. The My-11 antigen distinguishes between lymphohematopoietic cells on the basis of lineage, and assists in the purification of hematopoietic progenitor cells and the subclassification of leukemias and normal lymphocytes.

A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations.

A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucleic acid stain LDS-751. Distilled water was added following incubation to induce erythrocyte lysis by hypotonic shock. After lysis for 30 s the tonicity of the sample was increased followed by measurement on the flow cytometer. The leukocyte populations were clearly resolved in the correlation of forward and orthogonal light scattering. The immunofluorescence resolution of the labeled leukocytes was equivalent to NH4Cl and a commercial lysing preparation. Absolute number of leukocytes and percentage of leukocyte subpopulations determined with this procedure correlated well with the results obtained with a clinical hematology analyzer. Cell recovery and preservation of cellular characteristics of three different procedures for lysing the human erythrocytes were compared. The LDS-751 permitted the discrimination of intact cells from residual erythrocyte ghosts, platelets and damaged nucleated cells. A considerable loss of cells was found for both NH4Cl and commercial lysing solution; the samples prepared by NH4Cl lysing had a selective loss of lymphocyte subpopulations as compared with the other two techniques. In contrast to the two procedures in which multiple washing steps are involved, the no wash, hypotonic lysis procedure provided a means of obtaining absolute numbers of leukocyte subpopulations identified by combining light scattering and immunofluorescence characteristics with no centrifugation steps required.