Nucleolar abnormalities--a defect of the nucleolar preribosome assembly--in ringed sideroblasts in refractory anaemia with ringed sideroblasts (RARS) of myelodysplastic syndrome (MDS). An electron microscopic study.

Ringed sideroblasts were studied by means of transmission electron microscopy in patients suffering from refractory anaemia with ringed sideroblasts (RARS) of myelodysplastic syndrome (MDS) to provide more information on the structural organization of nucleoli in these abnormal erythroblasts. For control of the electron microscopic observations nucleoli in erythroblasts were also visualized by two widely used cytochemical procedures for the demonstration of RNA and AgNOR proteins. In contrast to previously described ultrastructure of nucleoli in "normal" erythroblasts, nucleoli of ringed erythroblasts in RARS of MDS were frequently characterized by a reduced incidence or lack of dense ribonucleic acid (RNA) containing granular components. Since the dense RNA containing granular components represent preribosomes, such sideroblasts in RARS of MDS exhibit a further nucleolar abnormality, which reflects a severe alteration of the nucleolar ribosome assembly in these abnormal cells. On the other hand, the alteration of the preribosome assembly was not noted in early developmental stages of ringed sideroblasts such as proerythroblasts. In addition, nucleoli in advanced or terminal stages of few ringed sideroblasts also did not exhibit such nucleolar abnormality and thus confirm a great structural and functional variability of these cells. The defect of RNA containing structures in nucleoli of advanced and terminal stages of erythroblasts are in a hormony with the light microscopic cytochemistry, which demonstrated a significantly smaller incidence of micronucleoli in specimens stained for RNA than in those stained for AgNOR (silver stained nucleolus organizer region) proteins.

A cytochemical note on nucleoli of granulocytic precursors and granulocytes in patients suffering from the refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS).

Nucleoli were studied in the proliferation as well as maturation granulopoietic compartment in patients suffering from refractory anemia with excess blasts (RAEB) of the myelodysplastic syndrome (MDS) by means of simple cytochemical procedures for the demonstration of nucleolar RNA and silver stained proteins of nucleolus organizer regions. Regardless of the procedure used for the nucleolar visualization, early stages of the granulopoietic compartment and particularly myeloblasts of RAEB patients were characterized by reduction of the nucleolar number expressed by the nucleolar coefficient the values of which resembled those described previously in acute myeloid leukemias. The reduced values of the nucleolar coefficient of these cells in silver stained specimens of RAEB patients were accompanied by a decreased number of clusters of silver stained particles representing interphasic silver stained nucleolus organizer regions (AgNORs). The reduction of these clusters was also described previously in leukemic cells. In addition, the differences in the values of the nucleolar coefficient of granulocytic precursors between specimens stained for RNA and those stained with the silver reaction might reflect changing composition and proportions of nucleolar components in the course of the granulocytic development.

Myelodysplastic and myeloproliferative type of chronic myelomonocytic leukemia--distinct subgroups or two stages of the same disease?

Several authors have tried to solve the problems in the classification of CMML. A fully suitable classification does not exist. The goal of our study was to determine common and different signs of MD and MP type of CMML and to observe frequency of shifts from MD to MP-CMML. Sixty nine CMML patients were divided according to FAB proposal into two groups: 31 patients into the MD group (WBC < or = 13 x 10(9)/l) and 38 patients into the MP group (WBC < or = 13 x 10(9)/l). Presenting features and the course of the disease in both groups were evaluated. The median age of patients was not different in both groups (71.5 and 74 years, respectively), male/female ratio was 1.1 and 2.4, respectively. The median follow-up time was 15.5 months (1-58.8) in MP group and 24 months (2-118) in MD group. In MP group splenomegaly, hepatomegaly, lymphadenopathy, abnormal karyotype and skin involvement were found more often than in MD group. Median LDH value was higher in MP group. Probability of survival was higher in the MD group than in MP group (median 30 and 11 months, respectively). Leukaemia transformation frequency was similar in both groups. In 12 out of 24 (50%) MD group patients WBC increased during the course of the disease over 13 x 10(9)/l. Oscillation of WBC values below and over 13 x 10(9)/l was observed in three patients. During the follow-up time number of patients with splenomegaly and/or immature granulocytes in the PB increased. After inclusion of 12 patients who shifted from MD to MP group a new CMML group resulted characterised by longer median survival (17 months) due to a higher number of patients in an earlier stage of the disease. Failure of evolution of myeloproliferative signs and lower frequency of AL in the remaining group might be explained by an early stage of CMML, untimely deaths due to unrelated causes and/or by patients suffering of RA with monocytosis rather than of CMML. In summary, our data suggest, that evolution from MD-CMML to MP-CMML is a frequent event and that MD-CMML could be the early stage of CMML in most of cases. The WBC at diagnosis as the single criterion for subclassification of CMML does not seem to be fully justified. We propose that CMML should not be divided in MD and MP types and that monitoring of patients and search for other signs of myeloproliferation such as PB immature granulocytes, splenomegaly, lymphadenopathy, skin involvement, pleural or peritoneal effusions, spontaneous growth of CFU-GM in vitro should be taken in consideration for a better classification of CMML, which would have an impact on the therapeutic approach.

Near-tetraploid poorly differentiated acute myeloid leukemia M0 diagnosed by short-term cultures with a phorbol ester TPA.

Leukemic blasts of two patients with acute leukemia exhibited similar characteristics. They were heterogeneous in size with a diameter of 14-30 microns in smears and unclassifiable by morphological, cytochemical, immunophenotypic and ultrastructural examinations. Cytogenetic examinations of both revealed a near-tetraploid karyotype. Blasts from both patients differentiated into macrophages in cultures with 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) which is a feature specific for myeloid blasts and the cases were thus classified as poorly differentiated acute myeloid leukemias (AML M0). Near-tetraploid poorly differentiated acute myeloid leukemias M0 seem to be a special category of AML in the morphologic, immunologic and cytogenetic (MIC) classification. The presence of very large blasts in the heterogeneous blast population in acute unclassified leukemias could be a morphological sign of near-tetraploid leukemias AML M0.

[Monocytosis and myelomonocytic dysplasia in myelodysplastic syndrome]. / Monocytóza a myelomonocytární dysplazie u myelodysplastického syndromu.

Myelodysplastic syndrome (MDS) is frequently associated with monocytosis in the blood and myelomonocytic dysplasia in the bone marrow. In two groups of patients with MDS, all subtypes excluding CMML, the authors demonstrated that monocytosis was present in 15% of the patients in group I according to the haemogram at the time when the diagnosis was established and in 19% in group II where it was required that at least in half the haemograms throughout the course of the disease there were more than 10% monocytes. No difference was found in the prognosis of patients with monocytosis, as compared with patients with monocytopenia as regards the life span and frequency of transformation into AL. The cytogenetic and cultivation findings did not differ either. In some instances, in particular in patients with RA and RAS significant monocytosis was not associated with the expected proliferation of monocytoid cells in bone marrow. The authors assume that proliferation and differentiation of germ cells in the monocytic series is easier than in the granulocytic series and that monocytosis can be considered a manifestation of substituted neutropenia. The work indicates the difficulties associated with the differential diagnosis of RA, RAS and RAEB with monocytosis, MDS with a dominating change of the type of myelomonocytic dysplasia and CMML proper.

[Morphocytochemical characteristics of hemopoietic cells in sideroblastic anemia]. / Morfotsitokhimicheskaia kharakteristika gemopoéticheskikh kletok pri sideroblastnoi anemii.

The findings of morphocytochemical and cytogenetic examinations of hemopoietic cells in 8 patients with sideroblastic anemia are analyzed. The authors consider that the final diagnosis of sideroblastic anemia can be made if the count of ring-shaped sideroblasts surpasses 30%. The leukemic nature of this condition is discussed.

"Slimy eyes phenomenon" in mice intraperitoneally injected with Pseudomonas aeruginosa cells or cell products.

Mice intraperitoneally treated with various Pseudomonas aeruginosa products or lipopolysaccharides of some selected Enterobacteriaceae representatives were found also to react by an increased slime secretion of the eye conjunctivae. The condition, tentatively designated as "Slimy Eyes Phenomenon", started to develop shortly post-treatment, culminated within 24-48 h when the eyes became fully glued up with slime, and receded 48-72 h later, leaving no sequelae for the eye or the general condition of the animal. No such phenomenon has to date been observed in other laboratory animal species.

Leukocyte-derived inhibitory activity in patients with myelodysplastic syndrome.

Leukocyte-derived inhibitory activity inhibiting the entry of normal progenitor cells of granulocytes and macrophages (CFU-GM) into the S-phase of a cell cycle was investigated in 16 patients with different forms of myelodysplastic syndrome (MDS). The presence of this inhibitory activity was analysed in medium conditioned with low-density cells obtained from peripheral blood of MDS patients. The inhibition rate was measured by 3H-thymidine suicide technique with subsequent cultivation of pretreated cells in semisolid agar medium. Low-density cells from MDS patients of various types were studied: from the twelve patients with refractory anaemia (RA or RAS) only three were positive, one patient with chronic myelomonocytic leukaemia (CMML) was negative while one patient with refractory anaemia with excess of blasts (RAEB) and two patients with RAEB in transformation (RAEB-T) were positive with respect of the described test. In two patients with RA, who underwent a long-term investigation, the production of leukocyte-derived inhibitory activity preceded the development of disease into RAEB or RAEB-T. In five positive cases, supernatants were incubated with antiserum against human placental ferritin; with one exception, the inhibitory activity was neutralized.

Dipeptidyl peptidase IV activity in cells of T-lymphoid origin is decreased in cultures with 12-0-tetradecanoylphorbol-13-acetate (TPA).

Dipeptidyl peptidase IV (DPP IV) is a specific enzyme for cells of T-lymphocytic lineage. It has been attempted to induce DPP IV activity in DPP IV negative T-lymphoid leukaemias by 12-o-tetradecanoylphrobol-13-acetate (TPA). Isolated cells from peripheral blood of 6 healthy blood donors and 22 patients with various types of leukaemia were cultivated in RPMI-1640 medium with 20% fetal calf serum alone (control) or supplemented with 20% human placenta conditioned medium (HPCM) or with 16 nmol/l TPA for 3 days. The percentage of DPP IV positive lymphocytes from blood donors remained unchanged in control and HPCM cultures, but decreased significantly in TPA cultures. Leukemic cells from three DPP IV positive cases of acute T-lymphoblastic leukaemia (T-ALL) reacted to the TPA treatment in a similar manner. Leukaemic cells of two DPP IV negative T-ALL cases remained negative in all three types of cultures, thus no induction of DPP IV activity was found. Other normal blood cells as well as leukaemic cells of 7 null-ALL, 1 preB-ALL, 3 B-CLL and 6 AML patients were DPP IV negative before and after cultivation in all types of culture. These findings showed that DPP IV is specifically expressed in cells of T-lymphocytic lineage even after short-term cultivation. HPCM was found to have no effect on DPP IV activity in T-lymphoid cells.

Distribution of T-lymphocytic subpopulations detected by monoclonal antibodies in peripheral blood of patients with chronic lymphatic leukaemia.

This paper reports on 93 patients with chronic lymphatic leukaemia (CLL) where CLL of B-type (E-, T-, B+, Ig+/-) was defined in 84 cases (90%), CLL of T-type (E+, T+, B-, Ig-) in 3 cases (3.2%) and in the remaining 6 cases (6.4%) neither T nor B were clearly detected. In 18 patients exhibiting lower total leukocyte count in peripheral blood T-lymphocytic population (OKT-3+), helper/inducer (OKT-4+) and suppressor/cytotoxic subpopulations (OKT-8+) were analysed using monoclonal antibodies in an indirect immunofluorescence test. The results show that the mean T-lymphocytic population counts in patients with CLL of B-type amounted to 1.7 X 10(9)/1 which is the value somewhat increased compared with the mean values observed in normal donors (1.5 X 10(9)/1). The counts of helper/inducer T-lymphocytic subpopulations (OKT-4+) were 1.7 X 10(9)/1 corresponding to those of healthy donors. In contrast, the counts of suppressor/cytotoxic T-lymphocytic subpopulations (OKT-8+) in patients with B-CLL were increased (0.7 X 10(9)/1). The immunoregulation index in patients with CLL decreased to 1.4 compared with that of a control 1.96).

Immunologic characterization of T-lymphoproliferative disorders by monoclonal antibodies.

A series of 11 mouse monoclonal antibodies reactive with human T lymphoid cells at different stages of differentiation was used for immunological classification of leukaemic cells of 16 patients with T cell lymphoproliferative disorders by using a fluorescence assay. The majority of T-ALL cells had an immature or early thymic phenotype and T lymphoblastic lymphoma had phenotypes corresponding to different levels of more mature stages of T cell differentiation, Two cases of T-CLL and one adult patient with mycosis fungoides had mature T cell phenotypes being T-3+, T-4-, T-8+, cytotoxic/suppressor cell types and one case of T-CLL had T-3+, T-4+, T-8-, "helper/inducer" cell type, too. These results suggested that surface marker analysis in T cell lymphoproliferative disorders may be used as a highly reproducible immunological classification system that will provide additional information about phenotypes of leukaemic cells in connection with morphological analysis and clinical diagnosis.

[Myeloproliferative syndrome]. / Myeloproliferativní syndrom.

Division of myeloproliferative syndrome is recommended based on 195 observer cases: A) Secondary myeloproliferative syndrome usually accompanies marrow carcinosis. It is characterized by non-destructive embryonal-type myeloproliferation occurring even outside the marrow excepting lymph nodes as a rule. There are neither specific changes in karyogram nor in alkaline phosphatase positivity. B) Idiopathic myeloproliferative syndrome is characterized (in comparison to A) by dysplastic changes especially in megakaryocytic line; it develops slowly tending to malignancy, namely leukemia or erythroleukemia that keep (unlike spontaneous leukemia) more severe dysplastic changes of megakaryocytes. Alkaline phosphatase is increased, atypical karyogram is not changed in Ph 1 region. C) Malignant neoplastic myeloproliferation of panmyelosis type is a primary destructive process akin to myelosis. Alkaline phosphatase is decreased, there are typical Ph 1 changes in karyogram and tumorous lymph node infiltration. Secondary myeloproliferative syndrome follows rarely and a mixed picture can be observed then, of course without severe megakaryocytic dysplasia. D) Myelofibrosis is an uncharacteristic final picture of various origin which neither develops in myeloproliferative syndrome or substitutionary extramedullar hemopoesis. Hesitation in oncological typing of idiopathic myeloproliferative syndrome cannot influence its nosological individuality. Exceptional and unexpected positive markers (alkaline phosphatase, Ph 1) occur in diagnostical practice from time to time; being unexplained they hinder from precise typing.

Differentiation of human myeloid leukemia cell lines induced by tumor-promoting phorbol ester (TPA). I. Changes of the morphology, cytochemistry and the surface differentiation antigens analyzed with monoclonal antibodies.

Human myeloid leukemia cell lines ML-1, ML-2, ML-3, promyelocytic leukemia cell line HL-60 and histiocytic lymphoma cell line U-937 were induced to differentiate by 0.5-10 ng/ml (0.8-16 nM) 12-O-tetradecanoylphorbol-13-acetate (TPA). After 48-72 h of induction, changes of the morphology, cytochemistry and of the antigenic phenotype of induced and control cells were studied using a panel of monoclonal antibodies against granulocytic, monocytic, HLA-ABC and HLA-DR antigens in indirect immunofluorescence. Cells of the TPA-treated cultures acquired morphological, cytochemical and antigenic markers of monocytes/macrophages, as surface adherence, alpha-naphthyl acetate esterase (alpha-NE) and acid phosphatase activity and the expression of monocytic antigens detected with monoclonal antibodies 63D3, FMC 17, B 44.1, B 52.1 and anti-Mol. During differentiation in vitro induced by TPA, also loss of HLA-DR antigens and diminution of antigen of cell activation were detected with antibodies L 243 and 4F2. The expression of granulocytic antigens was only slightly diminished and the expression of HLA-ABC antigens was not changed by TPA-treatment. There were differences in the percentage of cells induced to differentiate among the lines of different origin and even among the lines ML-1, ML-2 and ML-3, established from a single patient with acute myeloid leukemia. After treatment of cultures with 5 ng/ml TPA for 72 h DNA synthesis was inhibited to 60-80%.