D. Huhn

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).

PROPAGATION OF LARGE NUMBERS OF T CELLS WITH NATURAL KILLER CELL MARKERS

Summary. Previously, a subset of T cells co‐expressing the NK cell antigen CD56 has been described. These CD3+CD56+ cells are rare in peripheral blood collections and have been poorly characterized. We have developed culture conditions which allow for the rapid expansion of CD3+CD56+ cells. The protocol for cellular expansion includes the addition of interferon‐gamma on day O, interleukin‐1, interleukin‐2 and a monoclonal antibody against CD3 on day 1 to peripheral blood lymphocytes. Cells of the CD3+CD56+ phenotype increased up to 6000‐fold using this protocol after 16 d in culture. These cells have been characterized by flow cytometry and have been found to express the alpha, beta T cell receptor, co‐express the CD5 and CD8 antigens and do not express the CD16 antigen. Morphologically, these cells cannot be distinguished from NK cells. CD3+CD56+ killer cells lyse a variety of tumour cells with intermediate activity between CD3−CD56+ NK cells and CD3+CD56− T cells.

Deep venous thrombosis and pulmonary artery embolism in high‐grade non Hodgkin's lymphoma: incidence, causes and prognostic relevance

Abstract: To analyse incidence, risk factors, causes and prognostic significance of venous thromboembolism (VTE) in high‐grade non‐Hodgkins lymphoma (HG‐NHL) a prospective clinical trial (N = 593), also undertaken to analyse other aspects of HG‐NHL, a study of haemostasis (N = 25) and a post‐mortem analysis (N = 70) were performed. Clinical analysis documented a 6.6% incidence of VTE, and 77% of all cases occurred before or within the first 3 months of chemotherapy. Ann Arbor stage IV and B‐mediastinal clear cell histology were risk factors for VTE, while rapid changes in tumour load or application of consolidation chemotherapy were not. Vessel compression by HG‐NHL was the leading cause of VTE, whereas a significant (paraneoplastic or chemotherapy‐induced) thrombophilic state was not disclosed by haemostatic tests. While VTE‐related fatality was found to be low in the clinical trial (1.7%) and at necropsy (8.5%), the occurrence of VTE was associated with an unsatisfactory response of HG‐NHL to chemotherapy and a high incidence of treatment‐related mortality due to diffuse alveolitis. Thus, fatal VTE in HG‐NHL is rare, but VTE is associated with an unfavourable clinical course of HG‐NHL.

The value of immunocytochemical staining of lymph node aspirates in diagnostic cytology

This study investigates the applicability of immunocytochemical techniques towards improving the cytological diagnosis of lymph node disorders. Cytocentrifuge preparations of fine needle aspirates were examined using an indirect immunoperoxidase method and the alkaline phosphatase–antialkaline phosphatase method.

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.

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.

Improvement of pneumonia and arthritis in Felty's syndrome by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF).

SummaryA 63-year old man with Feltys syndrome and pneumonia of unknown origin was treated with GM-CSF. Granulocyte counts increased and arthritis-related symptoms improved under GM-CSF. Pneumonia was treated effectively with antibiotics only during or after GM-CSF application. This suggests, that antibiotic-resistent infections can be treated effectively in patients with Feltys syndrome when granulocyte counts are raised by GM-CSF.