A new approach to the morphological and cytochemical evaluation of human bone marrow CFUC in agar cultures.

A simple procedure is described for the routine morphological examination of different types of colonies and clusters in agar cultures. The following cytochemical methods are used for the identification of cells: naphthol-ASD-chloroacetate esterase, naphthol-ASD-acetate esterase with and without addition of sodium fluoride, Biebrich scarlet and acid phosphatase. Based on their cytochemical staining properties neutrophils, eosinophils, monocytes and macrophages can be readily identified in their different stages of maturation and an assessment made of the cellular composition of colonies and clusters.

A micro agar culture system for cloning human erythropoietic progenitors in vitro.

A simple and reproducible micro agar culture method for cloning erythropoietic progenitor cells from human bone marrow is described. Mononuclear cells (MNC) were immobilized in an agar layer and stimulated by erythropoietin (Ep), which was added to a liquid overlayer. The cultures were routinely incubated, fixed, transferred to microscopic slides, dried, and stained, and erythroid colonies were morphologically examined. The dynamics of growth observed from days 2 to 26 of incubation in the presence of 2.4 U Ep/ml showed basically three kinds of aggregates, which reached maximum growth on different days of incubation. A close Ep dose-response relationship was found for CFUE and BFUE at a concentration of 7.5 x 10(4) seeded cells. By varying the plated cell concentration between 2.5 and 10 x 10(4) cells a linear increase in the aggregates formed was found. On the basis of their growth dynamics and morphologic composition, the existence of three populations of erythropoietic progenitor cells in human bone marrow is tentatively proposed.

Epidermal Langerhans cells in myelodysplastic syndromes are abnormal.

The myelodysplastic syndromes (MDS) represent clonal disorders of the hematopoietic stem cell that are associated with quantitative and qualitative disturbances of the peripheral blood cells and a high risk for the transition to overt leukemia. As epidermal Langerhans cells (LC) are bone-marrow-derived cells, we were interested to see whether they are altered in patients with MDS. Epidermal sheets were prepared from biopsies taken from the thighs of nine patients with MDS and five control persons and processed for immunoperoxidase staining of CD1a antigens. The density and morphology of CD1a+ cells (i.e., LC) was evaluated by visual assessment as well as automatic image analysis. The density of LC was reduced in seven of nine patients (range, 30-75% of normal), whereas the morphology of LC appeared to be altered in all MDS patients in that the LC displayed large and bizarre cell bodies with only a few and often abnormally long dendrites. The HLA-DR expression by LC was not altered, as shown by double immunofluorescence staining of CD1a and HLA-DR antigens. Ultrastructurally, LC again appeared enlarged and often presented with bizarre nuclei, yet displayed no other abnormalities. Our findings suggest that LC are abnormal in MDS and might even indicate a more wide-spread involvement of the dendritic cell lineage in this syndrome.

Stimulation of colony formation of various human carcinoma cell lines by rhGM-CSF and rhIL-3.

We studied the effects human recombinant granulocyte-macrophage colony-stimulating factor and human recombinant interleukin-3 on the colony formation of three human solid tumor cell lines. Using a modified double-layer soft agar clonogenic assay rhGM-CSF enhanced colony formation of all cell lines tested in a dose dependent manner (up to twofold for the breast cancer cell line BT-20, up to 163% of the control for the hypernephroma cell line C 94 and up to 147% for the non-small cell lung cancer cell line CCL 185 at a concentration of 100 ng/ml). RhIL-3 stimulated colony formation of the cell lines C 94 and BT-20, whereas on the cell line CCL 185 rhIL-3 had no effect even at the highest dose level tested (100 ng/ml). Combinations of growth factors showed subadditive stimulation on two cell lines tested (BT-20, C 94). These data indicate that haematopoietic growth factors exert a growth promoting activity on certain solid tumor cells in vitro at physiological concentrations. Therefore our results suggest that the application of these factors in immuno- and myelosuppressed cancer patients after high dose chemotherapy should be seen in light of a possible co-stimulation of the malignant cells.

Increased serum interleukin 6 levels in patients with myelodysplastic syndromes.

Serum concentration of interleukin 6 (IL-6) was measured in 45 patients with myelodysplastic syndromes (MDS). A commercially available enzyme-linked immunosorbent assay (ELISA) with a sensitivity of 3 pg/ml was used. While IL-6 was undetectable in healthy volunteers, 32 of the patients with MDS showed IL-6 concentrations higher than 3 pg/ml. In MDS we found serum concentrations of IL-6 between 0 and 150 pg/ml with a median of 9 pg/ml, mean and standard deviation (SD) were 15 and 26 pg/ml respectively. In refractory anemia with excess of blasts in transformation (RAEB-t) the serum IL-6 concentrations were significantly higher than in refractory anemia (RA; p = 0.0025), in refractory anemia with excess of blasts (RAEB; p = 0.0050) and in chronic myelomonocytic leukemia (CMML; 0.0449). No significant difference was detected between RA and RAEB or between CMML and the other types of MDS, while s significant negative correlation was found between the concentrations of IL-6 and hemoglobin (p = 0.0228).

Delayed maturation of skin window macrophages in myelodysplastic syndromes.

Blood monocyte differentiation to macrophages was examined in nine patients with primary myelodysplastic syndromes using the skin window technique. Emigrated cells were stained cytochemically for acid phosphatase reaction after 1, 2, 4, 7, 9, 12 and 23 h. Compared to age-matched controls, seven patients showed a significant delay in lysosomal enzyme acquisition, which is associated with macrophage differentiation. Our results with this in-vivo assay demonstrate an involvement of the monocyte/macrophage system in primary myelodysplastic syndromes and show that patients often have a disturbance in macrophage differentiation.

Endogenous serum levels and surface receptor expression of GM-CSF and IL-3 in patients with myelodysplastic syndromes.

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a functional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

Macrophage infiltration in non-Hodgkin's lymphomas: a quantitative in situ study.

The frequency and distribution pattern of macrophages within 93 non-Hodgkin's lymphomas (NHL) were evaluated in situ by immunomorphometry using stereological methods. For the identification of macrophages (M phi), several antibodies (Mono 1, Mono 2, OKM 1) reactive with surface antigens on cells of the monocyte-macrophage series and cytochemical staining for acid phosphatase were applied. The average number of macrophages within lymph node tissue of NHL was 6,299 +/- 760 cells/microliter (similar to reactive lymphatic tissue: 6,559 +/- 1,027). The highest number of infiltrating macrophages was detected in immunoblastic NHL (17,306 +/- 2,773), differing significantly from other histological subtypes and reactive lymphatic tissue (p less than 0.005). The possible impact of tumor-infiltrating macrophages on lymphoma cell proliferation and differentiation is discussed.

[Significance of quantitative enzyme-cytochemical studies for hematologic cytology]. / Zur Bedeutung quantitativ-enzymzytochemischer Untersuchungen für die hämatologische Zytologie.

Enzyme-cytophotometrical investigations allow evaluation of enzyme activity within single, morphologically classified cells. Special problems like development of blood cells, bone marrow cells or of macrophages may thus be studied. Knowledge about the kinetics of granulopoiesis allows a better understanding of altered functional characteristics of abnormally developed blood cells. Enzyme cytochemical microdensitometry promises to be a useful tool in haematological cytology.

Acute monocytic leukemias.

Normal monocytes and macrophages are characterized by peculiar ultrastructural and cytochemical features and, in addition, show characteristic membrane properties, a variety of special functional capacities, and important secretory activities. Almost all these cytological features can also be detected in leukemic monocytic cells, and it is quite conceivable that these peculiar features may influence or determine the clinical syndrome associated with the leukemic accumulation of monocytic cells. The morphological identification of monocytic leukemias is a very intriguing diagnostic problem and some controversies still exist concerning their cytological classification. For clinical as well as scientific purposes the diagnosis of monocytic leukemias should rely on the demonstration of specific monocytic features of the leukemic cells. Clinical findings frequently associated with acute monocytic leukemia include increased frequency of leukemic tissue infiltrations as well as increased tendency to hypokalemia and - especially in the "immature" variants - to disorders of hemostasis.

[Cytochemical aspects in bone marrow cells in macrocytic anemias]. / Zytochemische Befunde an den Knochenmarkzellen bei makrozytären Anämien.

Cytochemical investigations are useful for the characterization of different kinds of macrocytic anemias. Vitamin B12 and folate defects or chronic alcoholic myelopathy, induce in the erythroblasts cytochemical patterns which can be distinguished from those seen in preleukemia, erythroleukemia, or in drug induced toxic anemia. Tests for alpha-naphthol-acetate-esterase, for acid phosphatase, for iron, and for polysaccharides (PAS-stain), are especially valuable for these diagnostic procedures.

[Clinical and cytological differences in adult acute lymphatic and acute undifferentiated leukemia]. / Klinische und zytologische Unterschiede bei akuten lymphatischen und akuten undifferenzierten Leukämien des Erwachsenen

The usefulness for clinical purposes of the distinction of acute undifferentiated (AUL) and acute lymphocytic leukemia (ALL) is suggested by the following observations: 1. Maturation from AUL to ALL has not been observed. Transformation of ALL to AUL has been reported i.e. less of cytoplasmic polysaccharides; however this seems rather to be the effect of cytotoxic therapy and not a real change of the cytological type. 2. Significant differences among ALL and AUL can be noted as far as the therapeutic response is concerned: All of the 9 patients with ALL but only 2 out of 9 patients with AUL went into remission. The mean survival of the cases with ALL amounts to 34, that of AUL only to 4 months. Out of the patients with ALL 4 patients are still alive in persistant first remission after 77, 57, 36 and 28 months. 3. ALL occurs most frequently in young adults (mean age of 21 patients: 31.7 years): AUL is more frequent in elderly patients (Mean age of 18 patients: 57.6 years). 4. In our material ALL did never occur consequent to a typical preluekemic stage, which was followed either by myeloblastic, monocytic, erythroleukemic or undifferentiated leukemias.

[In vitro effects and possible clinical relevance of interleukin 3]. / In-vitro-Wirkungen und mögliche klinische Relevanz von Interleukin 3.

Cytokines are mainly produced by monocytes and lymphocytes and influence a broad spectrum of physiological processes within the hematopoietic and the immune system. Clinical trials have mainly been initiated with a subgroup of cytokines namely the hematopoietic growth factors. Interleukin 3 (IL-3) was recently introduced into phase I and II trials. This factor was also termed "multipoietin" because of its ability to stimulate in vitro the hematopoietic stem cell and thus all three hematopoietic cell lines. The latest in vivo data, however, demonstrate that IL-3 stimulates leukopoiesis and megakaryopoieis with only marginal effects on erythropoiesis. In large clinical trials the therapeutic effect of this factor is investigated in patients with myelodysplastic syndromes. Further indications will be other primary bone marrow failures and aplasia caused by bone marrow transplantation, aggressive chemotherapy and radiation. Potential applications offered by combinations with other cytokines can hardly be foreseen.

[Subacute and chronic monocytic leukemia: diagnostic and clinical problems]. / Subakute und chronische monozytäre Leukämie: Diagnostische und klinische Problematik.

The authors report on 16 cases of either subacute (SMML) or chronic (CMML) myelomonocytic leukemia as well as chronic monocytic leukemia (CMoL). All these cases were oligoblastic and, according to their clinical course, they could be termed as smouldering leukemias. The chronic types affected mainly males. The diagnostic cytomorphological and cytochemical criteria are discussed. Erythro- and thrombocytopoiesis were distinctly less impaired than in acute leukemias (AL). The leucocyte count in the peripheral blood of the SMML cases was within the normal range. Hepato- and splenomegaly were markedly increased as compared to AL. According to our materials leukemic skin infiltrations were less frequent in CMoL, CMML and SMML than in acute monocytic leukemias. In each of the three types of leukemia discussed monocytic leukemic cells could be readily identified by cytochemical tests and usually showed fairly normal maturation. In accordance with these observations lysozyme levels in urine and serum usually were strongly increased. The patients in the CMML and CMoL groups showed a mean survival of more than 13 months (2 out of 7 are still alive), whereas the SMML patients survived an average of 8 months. Deaths were frequently due to advanced age rather than to leukemia. In other cases a terminal accumulation of blasts marked a transition to acute leukemia. During the smouldering phase of the disease no beneficial effect of combined chemotherapy could be noted. Supportive and symptomatic therapy might improve length and quality of survival.

[Prolymphocytic and hairy cell leukemias as special forms of chronic lymphatic leukemia]. / Prolymphozytäre und Haarzellen-Leukämie als Sonderformen der chronischen lymphatischen Leukämie.

Cytochemical and immunological studies were performed on "hairy" cells from 5 patients. The results, primarily the detection of surface immunoglobulins after incubation in immunoglobulin-free medium support the hypothesis that hairy cells are B-cells. Further parameters for B-cell lineage are the failing suppression of unspecific esterases by NaF, the presence of tartrate-resistant acid phosphatase and the absence of phagocytic capacity and glass adherence (skin window). A lysozyme could not be demonstrated. Of two patients with prolymphocytic leukemia one was characterized as T- and one as B-cell leukemia. Therefore, the prolymphocytic leukemia has to be discussed as intermediate form of ALL and CLL. In both patients the therapeutic results were discouraging with cytostatics (e. g. chlorambucil), splenectomy is considered as the treatment of choice.