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.

Use of monoclonal antibodies to detect conformational alterations in lactate dehydrogenase isoenzyme 5 on heat denaturation and on adsorption to polystyrene plates.

A monoclonal antibody (mAb) MF30 (IgGl) against human lactate dehydrogenase isoenzyme 5 (HLDH5) was prepared. MF30 was found to bind with high specificity to HLDH5 when the enzyme was adsorbed onto a polystyrene plate but did not recognize the isoenzyme when in solution. The isoenzymes HLDH1, HLDH2 and HLDH3 adsorbed onto polystyrene were not recognized by mAb MF30. Heat-treated HLDH5 (heated at 70 degrees C, pH 7.5 for 45 sec) behaved towards MF30 in the same way as the untreated isoenzyme, i.e. interaction between them took place only after the denatured isoenzyme had been adsorbed onto an ELISA plate. A second mAb, designated 2/66, prepared against porcine lactate dehydrogenase isoenzyme 5 (PLDH5), was found to interact with the porcine isoenzyme when in solution as well as when adsorbed onto polystyrene. However, no such interaction occurred after the isoenzyme had been subjected to heat treatment as above. The mAb 2/66 was found to cross-react fully with the human isoenzyme HLDH5 both in solution and when adsorbed onto polystyrene; however, as in the case of the porcine isoenzyme, all such recognition was lost upon heat denaturation. The above findings suggest that the adsorption of HLDH5 onto a polystyrene surface is accompanied by a conformational change. Denaturation of the enzyme by heat seems to lead to the appearance of a conformation differing in its antigenic pattern from that of the adsorbed enzyme. The data suggest that the mAbs MF30 and 2/66 recognize two different antigenic sites of HLDH5. The antigenic site which is recognized by 2/66 and is present in the native enzyme both when in solution and when adsorbed onto polystyrene disappears on heating. The other antigenic determinant is recognized by mAb MF30 when the enzyme is adsorbed onto a polystyrene surface either before or after heat treatment. This study illustrates the way in which appropriate mAbs might possibly be used as probes for the detection of conformational alterations occurring in proteins under various conditions.

Simultaneous analysis of DNA content and surface antigens in human bone marrow.

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.

Flow cytometric analysis of normal B lymphoid development.

Identification of the antigens expressed on marrow B lineage cells can be used to develop a model for the sequential acquisition of cell surface antigens during B lymphocyte development. The data suggest that the surface antigen expression is highly controlled during the development of B cells with the coordinated acquisition of multiple cell surface antigens during the maturational process. The developmental scheme in figure 6 is inferred from the expression of cell surface antigens on single samples. Confirmation of the progression from one stage to the next requires the isolation of a particular stage with subsequent induction to the next stage in-vitro. These data suggest that the development of B lymphoid cells may be discrete rather than continuous. The most immature cells identifiable in the bone marrow express CD34+ as well as HLA-DR. The earliest recognizable B lineage cells (CD19+, bright CD10+) also express CD34+. These cells are smaller by forward light scattering when compared to the cells which express only CD34+ (precursor of myeloid cells). Cells within stage I also express TdT in the nucleus and are proliferating. As the cells progress from stage I to stage II, the B lineage cells lose cell surface CD34 and nuclear TdT. At this time the density of HLA-DR and CD45 increases while the amount of CD10 decreases. These changes occur with no detectable change in cell size as assessed by forward light scattering. HLA-DP is first detected on the cells at this time. The progression of cells from stage II to stage III is marked by the acquisition of CD20, HLA-DQ, and sIgM. The amount of CD45 increases further in the transition between stage II and stage III. The acquisition CD21 and CD22 as well as the loss of CD10 distinguishes stage IV from stage III. Once the cellular composition of normal marrow has been defined, perturbations from homeostasis can be identified. Since marrow is the tissue most sensitive to injury by most antineoplastic chemotherapy and radiotherapy regimens, a means of quantifying the changes from the normal state can provide an assessment of the cytotoxic injury produced in individual patients. By monitoring the return to normal, it may be possible to more precisely individualize therapy for each patient. With a clear understanding of normal hematopoiesis, it should also be possible to identify maturational blocks which occur in hypoplastic marrow states. This may provide a means of identifying the regulatory points for each lineage and provide strategies for overcoming the inhibition of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Assessment of proliferation during maturation of the B lymphoid lineage in normal human bone marrow.

Different stages of B lymphoid maturation were identified in normal bone marrow using multiple cell surface markers. The proliferation status of each of these maturational stages was determined by simultaneous quantitative DNA analysis on a flow cytometer. The technique used to quantify these parameters preserved the cell surface immunofluorescence, the light scattering properties and the stoichiometric binding to DNA. The proliferating cells were confined to a distinct population of cells expressing CD10. The number of proliferating cells in these populations was relatively constant among 12 separate bone marrow samples. The data suggest that the timing and rate of proliferation of cells within a single lineage may be a preprogrammed aspect of normal maturation.