Leon W. M. M. Terstappen

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.

Donor-Derived Long-Term Multilineage Hematopoiesis in a Liver-Transplant Recipient

Graft-versus-host disease (GVHD) has been well documented in several recipients of liver transplants1–8. In this syndrome alloreactive cells from the donor attack the recipients skin, gastrointestinal tissue, and hematopoietic tissue. Severe myelosuppression commonly results, but there has been a degree of recovery of hematopoiesis in some patients after immunosuppressive therapy. The recovery of hematopoiesis resulted from the recovery of the recipients bone marrow in some cases,1,3 but it also could be due to the proliferation of hematopoietic precursor cells in the donors liver, since the liver is a site of hematopoiesis in fetuses and, under certain circumstances, .xa0.xa0.

Automatic lineage assignment of acute leukemias by flow cytometry

A method for automatic lineage assignment of acute leukemias was developed. Input are eight list mode data files acquired with a FACScan flow cytometer. For each cell, four parameters are measured: forward light scatter, orthogonal light scatter, fluorescein fluorescence, and phycoerythrin fluorescence. Eight data files are acquired in the following sequence: unstained, isotype controls, CD10/CD19, CD20/CD5, CD3/CD22, CD7/CD33, HLADR/CD13, and CD34/CD38. First, each of the data files 3 to 8 are clustered independently employing an algorithm based on nearest neighbors. Next, the clusters are associated across the data files to form cell populations, using the assumption of light scatter invariance across tubes for each population. The mean positions of each cell population are fed into a decision tree. The decision tree first identifies normal cell populations, i.e., monocytes, neutrophils, eosinophils, basophils, NK cells, T-lymphocytes, and B-lymphocytes. After elimination of the normal cell populations from the data space, the residual cell populations are classified as B-lineage ALL, T-lineage ALL, AML, AUL, B-CLL, or unknown. The effectiveness of this novel approach is shown with case studies of B-lymphoid, T-lymphoid, and Myeloid acute leukemias.

Impaired growth and elevated Fas receptor expression in PIGA + stem cells in primary paroxysmal nocturnal hemoglobinuria

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

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.

Expression of the DAF (CD55) and CD59 antigens during normal hematopoietic cell differentiation

Expression of decay‐accelerating factor (DAF or CD55) and of CD59 during hematopoietic cell development in normal bone marrow and on peripheral blood leukocytes were characterized by three‐color immunofluorescence experiments. With this technique cell subsets were identified by forward light scatter, orthogonal light scatter, and two cell‐surface antigens. For each cell lineage, specific combinations of two monoclonal antibodies labeled with different fluorochromes were used. DAF or GD59 were then quantitated on the defined cell subsets from the fluorescence signal of the respective antibody conjugated with a third fluorochrome. Early uncommitted hematopoietic progenitor cells (CD34+, CD38+) all expressed both proteins homogeneously. Initial commitment to the eryth‐ roid (CD71+, CD45dim), myeloid (CD33+), or B lymphocyte (CD10+) lineages was not associated with changes in DAF or CD59 levels. With erythroid development, i.e., after loss of CD45 and decrease of CD71, expression of both proteins decreased. With myeloid maturation, expression of GD59 remained constant and expression of DAF varied. During neutrophil maturation, DAF decreased initially and then reemeiged on maturing neutrophils concurrently with the appearance of GD16 (FcγRIII), whereas during monocyte maturation, DAF increased concurrently with up‐ regulation of CD14. With B cell development, expression of DAF increased concurrently with down‐regulation of GD10 and up‐regulation of CD20, whereas expression of CD59 diminished slightly late in B cell maturation. Analysis of peripheral blood elements showed that monocytes, neutrophils, and B lymphocytes expressed both proteins homogeneously, but that in contrast to other cell subsets, which all expressed CD59, only a subset of (CD3+) T lymphocytes and (GDI 6+) Natural killer cells expressed DAF. The absence of DAF was not related to CD4 or CD8 expression or to the presence of activation markers (CD25+, CD38+), memory cell markers (CD58+, CD45RO+), or virgin T cell markers (CD45RA+), but was correlated with expression of CDllb (CR3) and CDllc (gpl50/95). Although CD21” (CR2) and CD35* (CR1) cells all expressed DAF, CDlla (LFA‐1) levels correlated inversely with those of DAF. Although the presence of CD55 and CD59 on early progenitor cells and throughout hematopoietic cell development is consistent with the requirements for both proteins in protection of host cells from complement‐mediated injury, the physiological relevance of the unique patterns of variation for each cell lineage is unclear. Nevertheless, the availability of a detailed DAF and CD59 expression map in normal marrow will facilitate analyses of alterations during hematopoietic development that may occur in hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH).

Defective and normal haematopoietic stem cells in paroxysmal nocturnal haemoglobinuria

Summary. The expression of decay‐accelerating factor (DAF or CD55) and CD59 during haematopoietic cell development in bone marrow aspirates of two patients with paroxysmal nocturnal haemoglobulinuria (PNH) was compared with that in normal bone marrow by five‐dimensional flow cytometry. In contrast to early uncommitted haematopoietic progenitor cells (CD34+, CD38‐) in normal bone marrow which uniformly express DAF and CD59, the majority of CD34+, CD38‐ cells in both patients marrow exhibited the absence of the two proteins. In both specimens, however, subpopulations of CD34+, CD38‐ cells expressing DAF and CD59 were detectable, indicative of the presence of two lines of haematopoiesis, one abnormal and the other normal. Concurrent abnormal and normal haematopoietic development was further evident by the presence of subpopulations of DAF‐, CD59‐ and DAF+, CD59+ cells along the differentiation and maturation pathways of the myeloid (CD33+, CD15‐ → CD33+→++, CD15+), the erythroid (CD45dim, CD71dim→ CD45‐, CD71++), and the B‐lymphoid cell lineages (CD10++, CD20‐ → CD10‐, CD20++). While the majority of cells differentiating into and maturing along each cell lineage lacked DAF and CD59, the majority of mature B (CD20++, CD10‐) and T‐lymphocytes (CD3+) expressed both proteins suggestive of the presence of lymphocytes with a long life span which were generated from normal haematopoietic progenitors before the onset of the disease. The detection of distinct sets of CD34+, CD38‐ →+ progenitor cells which are DAF+, CD59+ or DAF‐, CD59‐ in marrow of PNH patients has relevance for the treatment of PNH. Cells with the phenotype CD34+, CD38‐, DAF+, CD59+ are capable of self renewal and represent potential candidates for autologous bone marrow transplantation following depletion of CD34+, CD38‐, DAF‐, CD59‐ cells.

Detection of Aberrant Antigen Expression in Acute Myeloid Leukemia by Multiparameter Flow Cytometry

Acute leukemia’s are conventionally diagnosed by light microscopical evaluation of bone marrow aspirates or biopsies. Distinction between acute lymphoblastic (ALL) and acute myeloid leukemia (AML) is supported by cytochemistry and is well reproducible between experienced hematologists and/or pathologists (Bennett et al. 1976; MIC Classification 1988). Disagreement can arise within the myeloid lineage, e.g. in the distinction of “de novo” and “secondary” leukemia’s, in the distinction of myelodysplastic syndromes (MDS) in advanced stages (RAEB-T), and in the distinction of benign myeloproliferative disease in infants. All these exceptions comprise far less than 5% of all cases. The largest discrepancies arise in the sub classification of AML using the FAB classification (Argyle et al. 1989). The usefulness of additional diagnostic tools in the routine of leukemia diagnosis must be judged based on their ability to provide the correct diagnosis in disputed cases, to identify clinically relevant subpopulations for early treatment stratification, and to detect residual leukemic cells in hematological complete remission for postremission therapy monitoring. Since today, only patients within one subset of AML, the acute promyelocytic leukemia APL, FAB M3, may receive different induction therapy (Castaigne et al. 1990; Warreil et al. 1991), the lack of concordance between different hematocytologists has little significance for the patients. This may change, when more subpopulations with distinctly different prognosis are identified.

Distribution of Cells with a “Stem Cell Like” Immunophenotype in Acute Leukemia

The differentiation of human pluripotent progenitor cells to the functional effector cells in the peripheral blood is accompanied by the sequential acquisition and loss of characteristic cell surface molecules. Stem cells are identified by function [1, 2] and by immunophenotype. They express a 110–115 kD cell surface molecule, classified as CD34 [3; 4]. The compartment of CD34 positive progenitor cells can be further subdivided based on coexpression of other, lineage-restricted and lineage-nonrestricted cell surface antigens such as CD38, HLA-DR [5], stem cell factor receptor (c-kit) [6], CD45 RA [7], CD19 [8], CD33 [9] and cytoplasmatic myeloperoxidase [10].