Stefan Serke

Multiparameter flow‐cytometrical quantitation of circulating CD34+‐cells: correlation to the quantitation of circulating haemopoietic progenitor cells by in vitro colony‐assay

Summary. A multiparameter flow‐cytometrical method for the quantitation of CD34+‐cells present in adult human peripheral blood cells (PBMC) has been developed. PBMC from 13 healthy adult subjects were analysed for CD34+‐cells by flow‐cytometry, with only the lymphoid population of cells negative for anti‐CD3‐moAb included in the analysis. At the same time mononuclear cells from the same individuals were depleted of CD3+‐ and CDl4+‐cells by immunomagnetic purging. These cells were cultured for haemopoietic colonies. A correlation coefficient of 0·92 was calculated by regression analysis of CD34+‐cells number versus the numbers of colonies (CFU‐GM, BFU‐E).

Identification of CD71 (transferrin receptor) expressing erythrocytes by multiparameter-flow-cytometry (MP-FCM): correlation to the quantitation of reticulocytes as determined by conventional microscopy and by MP-FCM using a RNA-staining dye

Reticulocytes express the CD71‐defined antigen, the transferrin receptor. This report describes how by means of dual‐colour immunofluorescence using MP‐FCM (multiparameter‐flow‐cytometry) CD71+ erythrocytes can be detected regularly in blood of healthy adults. Percentages of these CD71+ erythrocytes were compared to the percentages of reticulocytes as determined by conventional microscopy using brilliant cressyl blue, and to the percentages of erythrocytes with high RNA content, as detected by MP‐FCM using a RNA‐staining dye (thiazole‐orange). Only about two‐thirds of the percentages determined by the two latter methods were detected by MP‐FCM using the CD71 expression for definition of reticulocytes. Studying clinical samples, however, including both specimens with very low and very high numbers of reticulocytes. almost identical percentages were determined by all the three methods described.

Analysis of CD34-positive hemopoietic progenitor cells from normal human adult peripheral blood : flow-cytometrical studies and in-vitro colony (CFU-GM, BFU-E) assays

SummaryHemopoietic progenitor cells are present in minute numbers in the peripheral blood of healthy adults. By in vitro colony-assays evaluating BFU-E-and CFU-GM-growth, their numbers have been estimated to be about 1,000 per 1.0 ml of whole blood. Employing a CD34-moAb, detecting an antigen present on virtually all hemopoietic progenitor cells, and by using multiparameter flow-cytometry, we have designed a flow-cytometric method for the quantitation of CD34+-cells in blood. Comparative studies on bone marrow and blood have shown that CD34+-cells from both sources display almost identical light-scatter characteristics. They differ, however, with regard to the coexpression of the CD33-and the CD19-antigens. In vitro colony-assays for BFU-E and CFU-GM in single cultures have shown that about 25% of the CD34+-cells from blood were clonogenic in vitro. Our data indicate that the CD34+-cells from peripheral blood differ substantially from bone marrow CD34+-cells.

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization.

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19‐infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3‐ and CD14‐poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhll‐3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19‐containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow‐cytometry, demonstrating a selective inhibition of GlyA‐positive cells following B19‐inoculation. Typical morphological changes were observed on cytocentrifuge‐spots, and typical giant‐cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19‐DNA increased by about x 100 after 72 h of culture.

Lymphocyte activation by phytohaemagglutinin and pokeweed mitogen: Identification of proliferating cells by monoclonal antibodies

Using a two-colour immunofluorescence technique, we have investigated the mitogenic effects of phytohaemagglutinin-M (PHA-M) and of pokeweed nitrogen (PWM) on human lymphocyte subsets. These were identified by CD1, CD3, CD4, CD8, and CD16 monoclonal antibodies, and proliferation was demonstrated by a polyclonal anti-transferrin antibody. Evidence has been obtained for the generation of a population expressing both the CD4 and CD8 antigens simultaneously, in short-term cultures of peripheral blood mononuclear cells in the presence of PWM and of PHA-M.

Heat stability of parvovirus B19 : kinetics of inactivation

Heat inactivation of parvovirus B19 (B19) was studied in a culture of hematopoietic progenitor cells generated in vitro from peripheral human blood. After inoculating cell cultures with identical volumes of plasma (MII) containing B19 (B19-MII) heat-treated (60 degrees C) for various periods of time, a time-dependent inactivation of the input virus was determined by a decrease of viral DNA replication. No B19 DNA was detected after infection with B19-MII heat-treated for 20 min or more by Southern blot. Viral B19 protein production decreased time-dependently and was not detected after infection with samples treated for 12 min at 60 degrees C or more determined by the enzyme immunoassay. This study indicates that infectivity of B19 virus in plasma can be reduced in vitro by heat-treatment (60 degrees C). However, this does not mean that the heat treatment completely inactivated B19 virus.

Effects of various recombinant human hemopoietic growth factors (rhEpo, rhG-CSF, rhGM-CSF, rhIl-3) on the growth of peripheral blood progenitor cells (BFU-E, CFU-GM)

SummaryThe effects of rhEpo 1, rhG-CSF, rhGM-CSF and rhIl-3 on the growth of both CFU-GM and BFU-E from normal human adult peripheral blood have been studied in plasma clot cultures. Using optimal concentrations of all growth factors, alone and in combination with all other factors, rhIl-3 showed the highest activity in regard to growth of both CFU-GM and BFU-E, whereas rhGM-CSF treatment resulted only in half-maximal colony growth compared to rhIl-3. No synergism or additive effect was seen with the combination of rhIl-3 and rhGM-CSF. Treatment with rh-G-CSF had no additional effect with optimal concentrations of rhIl-3 and/or rhGM-CSF. When suboptimal concentrations of rhGM-CSF and rhIl-3 were applied, however, they showed a marked synergism on both BFU-E and CFU-GM. RhGCSF, added to a suboptimal concentration of rhGM-CSF, resulted in a marked growth increase of CFU-GM but had no effect on BFU-E.

A liquid culture method for the in vitro growth of hemopoietic progenitor cells from normal human adult peripheral blood allowing for analysis by multiparameter flow-cytometry.

A liquid culture method has been developed allowing for the in vitro growth of peripheral blood‐derived hemopoietic progenitor cells of myeloid, erythroid, monocytic and megakaryocytic lineages. Adherent cell‐ and CD21‐positive cell‐depleted PBMC from normal subjects have been cultured in the presence of rhEPO, rhGM‐CSF or rhII‐3. Culturing cells in liquid cultures and in plasma clots, a similar dose‐response was observed for granulocytic cells/liquid culture and granulocytic colonies/plasma clot with rhGM‐CSF, and also for erythroid cells/liquid culture and erythroid colonies/plasma clot with rhEPO. Comparing serum‐ liquid cultures to serum+ liquid cultures, the ratio of CD13+ cells to CD15+ cells was higher in serum‐ cultures, indicating a maturation arrest of myeloid cells with serum deprivation. Using dual‐colour flow‐cytometry, cell‐cycle analysis of CD13+ cells, comparing the effects of rhGM‐CSF to those of rhII‐3, have been performed. The liquid culture method promises to be a useful tool for the study of in vitro differentiation and proliferation of hemopoietic progenitor cells.

Overexpression of the Raf-1 proto-oncogene in human myeloid leukemia

The Raf-1 protein, a cytoplasmic serine/threonine kinase, plays an important role in signal transduction pathways. In order to examine the role of Raf-1 in human myeloid leukemia, we determined raf-1 mRNA expression by Northern blot analysis in blast cell samples from 27 acute myeloid leukemia (AML) cases and peripheral blood mononuclear cells from six healthy donors. A normal raf-1 transcript size was detected in all cases investigated. However, overexpression of raf-1 mRNA was found in 2 of 27 AML cases, both of which were erythroleukemias (AML, FAB M6).

Binding of mitogenic plant lectins to human lymphocytes: Flow cytometric analysis

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.