Hamatologie und Bluttransfusion最新文献

Peroxidase conjugated antibodies were applied to cell suspensions in order to detect surface associated immunoglobulins. Cell suspensions were fixed prior to incubation with reagents, a procedure avoiding membrane alterations induced by antibodies to surface component. By immunoelectron microscopy an identification of B lymphocytes could be made with simultaneous observation of their surface architecture. Basic findings were that normal circulating human B lymphocytes had a villous surface. This relationship was not confirmed however by examinating samples from various B and T cell proliferations establishing that surface morphology is not sufficient to categorize cells in disease. Specimens from hairy cell leukemia were also examined. Despite salient surface characteristics as revealed by the present method, the categorization of cells remains unclear.

Standardized culture of bone marrow in soft agar permits the detection of a population of granulocyte-macrophage progenitor cells (CFU-c). A spectrum of qualitative abnormalities serves to distinguish myeloid leukemic CFU-c from normal and remission populations. These abnormalities in maturation and proliferation are diagnostic of a myeloid leukemic state and serve to functionally reclassify acute myeloid leukemia at diagnosis into a number of categories based on in vitro growth pattern. The virtue of this classification is that it permits detection of a substantial number of patients who are refractory to conventional remission induction protocols. The clear distinction between normal and leukemic growth in vitro permits early detection of emerging remission CFU-c during induction therapy and of early onset of relapse in patients who are otherwise in complete remission. In patients with leukemia undergoing allogeneic bone marrow engraftment, marrow culture has proved of value in documenting the reconstitution of the patient and in detecting re-emergence of the original leukemic stem line prior to its detection by cytogenetic and hematological techniques. Serial studies on patients with chronic myeloid leukemia have allowed early diagnosis of blastic transformation and classification of blastic phase disease on the basis of in vitro growth pattern has revealed a similar spectrum of in vitro abnormalities as seen in AML. The cloning of normal or leukemic human myeloid progenitor cells (CFU-c) in agar or methylcellulose has permitted analysis of both quantitative and qualitative changes in this cell compartment in leukemia and other myelodysplastic states (1-7). Among these changes are abnormalities in maturation of leukemic cells in vitro (4, 5, 6), defective proliferation as measured by colony size or cluster to colony ratio (5, 6), abnormalities in biophysical characteristics of leukemic CFU-c (4, 5), regulatory defects in responsiveness to positive and negative feedback control mechanisms (8, 9) and the existence of cytogenetic abnormalities in vitro (10, 11). Detection of this spectrum of abnormalities has proved of clinical utility in diagnosis of leukemia and preleukemic states (5, 6, 12), in classification of leukemias and myeloproliferative diseases (5, 6), in predicting remission prognosis and response to therapy (5, 13), in predicting onset of remission or relapse in AML (13) and in monitoring the progression of chronic myeloid leukemia or preleukemic disease (4, 14). The present communication serves to illustrate the clinical applications of bone marrow culture in these various areas.

The results of T- and B-cell determinations are described in 105 cases of lymphoreticular and lymphoepithelial neoplasia, and are compared to similar investigations of 582 cases as published in the literature. In addition, T- and B-cell values are determined in blood of 35 healthy individuals, in 12 normal lymph nodes, as well as in hyperplastic conditions of lymph nodes from 30 patients and of tonsils from 85 patients. Cell characterizations are done by immunofluorescence and use of monospecific anti-immunoglobulin antisera (H chain specific), anti-thymus antiserum, as well as by the E-rosette test. While normal blood and normal and hyperplastic tissues show a polyclonal distribution or proliferation of lymphoreticular cells, neoplastic conditions are often characterized by an exuberant, possibly monoclonal proliferation of one cell type. According to this, lymphoreticular neoplasias are immunologically grouped into four main classes: B-cell neoplasias comprising most of the chronic lymphocytic leukemias, well differentiated lymphocytic lymphomas, BURKITT's tumor, follicular lymphoma BRILL-SYMMERS, and hairy cell leukemia. T-cell lymphomas represent a large part of poorly or undifferentiated leukemias of children, poorly differentiated lymphocytic lymphomas, prolymphocytic leukemia, and Sézary's syndrome. Monocytic neoplasias are malignant histiocytoses and leukemic reticuloendothelioses. A fourth group, which probably is not homogeneous and might be further classified in the future by use of more sophisticated methods, consists of tumors with T- and B-cell lack. Such tumors are histologically classified as Hodgkin's lymphomas, a certain number of histiocytic lymphomas, and mycosis fungoides. The prognostic and pathogenetic implications of a combined morphological and immunological classification of lymphoreticular neoplasias are briefly outlined.

Colony formation in vitro by marrow cells from patients with untreated acute myelogenous leukemia (AML) and from patients in AML relapse is infrequent using the standard Robinson assay. A newly developed culture system has been described in which marrow from AML patients in these disease stages form leukemic cell colonies. In this in vitro system, phytohaemagglutinin is the essential stimulator for colony formation. The leukemic origin of the colonies has been proven by ultrastructural morphology and cytogenetics. It appears that colony formation by leukemic cells in this system is predominantly independent from the leukocyte factor which is the main stimulator in the Robinson assay for growing colonies of marrow cells from haematologically normal individuals. Bone marrow cells in untreated acute myelogenous leukemia (AML) demonstrate abnormal growth in vitro in the Robinson assay (Robinson et al., 1971; and Bull et al., 1973). Characteristically, there is a near total failure of colony formation; predominantly clusters are formed containing 20 cells or less (Bull et al., 1973; Greenberg et al., 1971; Moore et al., 1973 and 1974, and van Bekkum et al., in press). The absence of colonies has been shown to be due to a marked decrease of the normal myeloid precursor cell population in untreated AML. The small agregate formation of AML cells has been attributed to the suboptimal response of leukemic cells to the leukocyte stimulation factor. Because this poor proliferation in vitro might not represent the maximal in vitro and in vivo proliferation potential of the leukemic cells, we studied a number of modifications of the in vitro culture system. A number of factors were studied which may have some influence on cell proliferation in general, notably phytohaemagglutinin (PHA), which induces lymphocyte colonies in vitro (Rozenszajn et al., 1974), and endotoxin which has been demonstrated to increase the labelling index of leukemic cells in vivo (Golde et al.). In this paper an in vitro system is described in which marrow cells from untreated AML and AML in relapse were stimulated by phytohaemagglutinin (PHA) to form leukemic cell colonies in soft agar. These (similar) cells predominantly formed small aggregates (20 cells or less) in the presence of the normal leukocyte feeder layer alone. Moreover, in the course of the experiments, it appeared that by adding low concentrations of endotoxin to the cultures, the stimulating effect of PHA could be amplified.