Search

Background and Objectives: In the recent years many studies on the expansion of cord blood (CB)-derived progenitor cells have been performed, whereas less information is available on their cycling status. The objective of this study was to evaluate the cycling status of CB-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle in response to a combination of cytokines., Design and Methods: CB-derived CFC and LTC-IC were first quantified by standard clonogenic assay and long-term culture, respectively. In a second set of experiments, CB-derived progenitor cells were incubated with interleukin(IL)-3, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) and their cell cycle status assessed both by the cytosine arabinoside (Ara-C) suicide approach and by flow cytometric DNA analysis., Results: We found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96% +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6% +/- 0.4% in the S-phase. Staining of CD34+ cells with an anti-statin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase, 68% +/- 7% of cells were in the G0 phase of the cell cycle. Twenty-four hour incubation with IL-3, SCF and G-CSF significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in their number. Flow cytometric DNA analysis also showed an increase of CD34+ cells in the S-phase upon continuous exposure to these cytokines., Interpretations and Conclusions: Our findings indicate that: i) a small number of CB-derived CFC and LTC-IC are in the S-phase of the cell cycle; ii) a substantial number of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction are cycling, as they are found in the G1 phase of the cell cycle; iii) 24-hour incubation with IL-3, SCF and G-CSF can drive a proportion of progenitor cells into the S-phase without reducing their number.

Background and Objective: Several in vitro and animal studies have shown that IL-3 primes hematopoietic stem cells to become more sensitive to later acting growth factors. We wanted to compare the toxicity and the synergistic stimulatory effect of interleukin-3 (IL-3) followed by granulocyte colony-stimulating factor (G-CFS) or granulocyte-macrophage colony-stimulating factor (GM-CSF) on white blood cell (WBC) and platelet counts, after standard-dose chemotherapy (CT) in patients with solid tumors., Design and Methods: Fifty consecutive cancer patients with thrombocytopenia and/or leukopenia registered during a previous course of CT were randomized to receive, after the following course, IL-3 (10 microg/kg/day, s.c., day 1-5) followed by G- or GM-CSF (5 microg/kg/day, day 6-8)., Results: The nadir of WBC in the cycles supported with the combination of IL-3 and G-CSF was significantly higher than that observed in the CT cycles not supported by growth factors (p < 0. 005). Furthermore, severe leukopenia was abrogated in all the cycles supported with IL-3+G-CSF, while in the cycles without cytokines, this event was registered in 62.5% of the cases (p < 0.0005). Finally, the recovery of WBC was achieved a mean of 4 days earlier in the cycles supported with IL-3+G-CSF. As for thrombocytoprotection, no significant differences were evidenced, but severe thrombocytopenia was abrogated in all the cycles supported by IL-3+G-CSF (p < 0.05). Furthermore, platelet recovery after CT was achieved on average 3.5 days earlier in the IL-3+G-CSF group than in the previous cycles. The nadir of WBC count in the cycles supported by the combination of IL-3 and GM-CSF was significantly higher than that observed in the CT cycles not supported by growth factors (p < 0.005). Furthermore, severe leukopenia was abrogated in 40% of the cycles supported by IL-3+GM-CSF, while in the cycles without cytokines, this event was registered in 80% of the cases (p < 0.005). Finally, the recovery of WBC was achieved a mean of 3.5 days earlier in the cycles supported by IL-3+GM-CSF. As far as thrombocytoprotection is concerned, there were no significant differences in the nadir between the cycles supported by the association IL-3+GM-CSF and the cycles not supported by cytokines. However, severe thrombocytopenia was registered in 20% of the cycles not supported by growth factors but in only 10% of the cycles supported by IL-3+GM-CSF (p < 0.05). Furthermore, platelet recovery after CT was achieved on average 3 days earlier in the IL-3+GM-CSF group. The combination of IL-3 and G-CSF would appear to be more effective than the combination of IL-3 and GM-CSF in the control of both severe thrombocytopenia and leukopenia. Indeed, severe leukopenia was abrogated in all the cycles in arm A, but only in 40% of the cycles in arm B (p < 0.0005). Furthermore, considering a platelet count below 49

In this study, blast cells from 15 patients with acute myeloid leukemia resistant to induction therapy were examined with two monoclonal antibodies that identify, respectively, the nuclear protein specifically expressed in non proliferating cells (statin) and the proliferating cell nuclear antigen (PCNA). We found that statin values varied widely, ranging from 0.6% to 14.7% (mean value 6.4%). When the patients were subdivided according to the mean value, those presenting with higher statin values survived for a shorter period of time than the ones characterized by lower levels (p = 0.003). We observed a wide variation in the range of PCNA values; however, if an agreement between survival duration and at least one of the proposed markers was considered, all but one case displayed concordance between survival duration and PCNA and/or statin values (in addition, 4/15 cases showed agreement for both markers). These preliminary data could indicate a possible discriminating prognostic factor between categories of patients characterized by different aspects of resistance, perhaps susceptible to different salvage therapy approaches.

Background: Blood cell transplantation has become a new type of support in high-dose chemotherapy (HDC) for several oncologic and hematologic diseases. Over the last few years the demand for circulating progenitor cell (CPC) collection by blood cell separators has grown dramatically, and transfusion services must manage new CPC programs., Materials and Methods: A protocol for optimizing the collection and clinical use of CPC is described. The results of 275 harvestings were studied: 128 patients were divided into 5 groups according to tumor type (A: breast cancer; B: Hodgkin's disease; C: non-Hodgkin lymphoma; D: multiple myeloma; E: various solid tumors). An additional group (F) consisted of 11 healthy donors. Factors affecting collection (mobilizing regimen or previous radiation therapy) and side effects were investigated., Results: The mean values of mononuclear cells (MNC x 10(7)/kg) and CD34+ cells (x 10(6)/kg) collected per leukapheresis in the 6 respective groups were: 31.4 and 4.6 in group A; 26.4 and 3.4 in group B; 21.8 and 5.8 in group C; 24.6 and 2.4 in group D; 26.8 and 2.9 in group E; 60 and 6 in group F. Previous chemotherapy and/or radiation therapy were the main factors influencing CPC harvesting. The different chemotherapy regimens employed demonstrated no significant differences in their mobilizing efficacy. Side effects related to leukapheresis were few (2.3% of the procedures) and manageable., Conclusions: CPC collection is feasible in a wide range of clinical situations. Careful clinical evaluation of patients, accurate monitoring of progenitor cell release and collection timing are important for obtaining a sufficient number of CPC for hemopoietic recovery. Previous chemotherapy and radiotherapy are the main factors influencing CPC harvests. The mobilizing regimens employed showed no substantial differences in their efficacy.

The techniques of flow cytometry are becoming more and more important for the clinical hematology laboratory. No longer a novelty confined to a few specialized institutions as it was 10 years ago, flow cytometry has blossomed into a mature discipline. The methodology is well-known, the mechanical apparatus is readily available, and the role it plays in clinical hematology is increasingly appreciated. The burgeoning number of scientific articles devoted to this topic attests to the interest it has aroused as a tool for both medical research and patient care. In fact, more than a thousand such papers are now published each year and it would be impossible to deal with all the methodologies and applications of FCM currently utilized or under development. Throughout this paper four relevant hematologic fields are briefly discussed, in which FCM appears to be of great help at present: the immunophenotyping of leukemias and lymphomas, the measurement of proliferative activity and DNA ploidy in hematological malignancies, the detection of drug resistant leukemic cells and the use of FCM in the study of platelets.

Seventeen consecutive patients with previously untreated poor prognosis Hodgkin's disease (clinical stage II and III with systemic symptoms, and stage IV) received 6 courses of aggressive chemotherapy, with (9 patients) and without (8 patients) the addition of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Chemotherapy (MOPP/ABV/CAD regimen) included full doses of nitrogen mustard, lomustine (CCNU), vindesine, melphalan, prednisone, epidoxorubicin, vincristine, procarbazine, vinblastine and bleomycin, and was administered between days 1 and 15 of each course. Course were planned for 28-day intervals. rhGM-CSF was given at a dose of 5 micrograms/kg/day subcutaneously from day 16 to 26 of each course. With cytopenia (i.e. white blood cell, WBC, count < 3.0 x 10(9)/L and/or platelet count < 100 x 10(9)/L) delaying courses was preferred to administering reduced drug dosages. Substantial delays (ranging from 7 to 28 days) in delivering cytostatics were necessary between 70% of courses. The cumulative mean number of days for which the courses had to be delayed before completing the 6 MOPP/ABV/CAD courses was 57. The percentage of planned doses of cytotoxic drugs (nitrogen mustard, melphalan, epidoxorubicin, procarbazine) actually administered was 92%. Causes of treatment delay were presented by leucopenia in 82% and by leuco-thrombocytopenia in 23% of the courses. The WBC nadir was constantly encountered at day 20-21 following completion of courses, and slightly worsened with subsequent courses. The decrease in platelet values was milder than that in WBC counts. There were no differences in any of the above parameters between patients treated with MOPP/ABV/CAD alone or followed by rhGM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Several nuclear and surface proteins are expressed in varying amounts in the different phases of the cell cycle. For some of them the coding gene is not known and changes in their expression could simply be secondary to changes in the proliferative activity of the population. Other proteins are oncogene products, probably having a direct regulatory function in cell proliferation, differentiation and malignant transformation. Studying these proteins may both permit a better understanding of the mechanisms regulating proliferation and differentiation and provide kinetic parameters for describing the cell cycle. Based on antibodies against these proteins, bivariate flow cytometry (FCM) is able to quantitate their expression simultaneously with DNA distribution. This allows protein expression to be related precisely with each cell cycle phase in populations having different proliferative activity. Further advantages of bivariate FCM are that few cells are required for the analysis and the percentage of cells expressing the (onco) gene product can be determined. Several cellular proteins have been investigated with bivariate FCM, and the data are reviewed. Some proteins not coded by oncogenes (such as cyclin, the Ki-67 reactive antigen and DNA polymerase alpha) are expressed in cycling, but not in G0 cells and are of special interest for the kineticist, since they could identify cells which are able to initiate DNA synthesis, i.e. those representing the "growth fraction" of the population. Statin, on the contrary, is apparently expressed only in G0 cells. The expression of some proteins coded by oncogenes, such as p53 and the c-myc product is high in proliferating G1 cells and decreases with differentiation. The expression of the c-ras product is not strictly related to cell cycle phases and increases with differentiation. Technical improvements (allowing, for example, the monitoring of the changes in protein expression following the microinjection of a protein-blocking substance into the cells and the inclusion of phenotype markers into the analysis) will expand the role of bivariate FCM for these research works.

The quantitative analysis of the buffy coat (QBC II system) of centrifuged whole blood samples allows one to obtain hematocrit (Ht), platelet (PLT) and white blood cell (WBC) values. We have tested the QBC II system in a hematological/oncological Outpatient Section, where it is necessary to know patient's hematological condition immediately before administering chemotherapy. One hundred ten blood samples from 110 patients with solid or remission hematological tumors were measured in duplicate with the QBC II system and with the counter instrumentation (Sysmex TOA 800) of the Central Laboratory, where samples were sent for emergency determination. Reliable values for Ht and WBC were obtained in all samples, and for PLT in 101 samples. The QBC II System failed to furnish very low and very high PLT counts. The time needed for the QBC II system analysis was shorter than that required to obtain the Central Laboratory data. The QBC II system analysis is a useful means for rapidly evaluating the basis hematological condition in most chemotherapy patients.

Peripheral cytopenia has been reported in a number of patients with the acquired immunodeficiency syndrome (AIDS), but the mechanism of bone marrow (BM) failure is unclear. We have examined the BM morphology and cytokinetics of 16 untreated HIV-positive patients whose clinical condition ranged from asymptomatic (stage 1 WR and II CDC classifications) to overt AIDS (stage 6 WR and IV CDC classifications). BM aspirates and iliac crest threphine biopsies were obtained for myelogram and histologic examination, as well as for propidium iodide flow cytometric (FMC) DNA analysis. FCM data were compared with those from the BM of patients with solid tumors without BM involvement. Four patients had normal peripheral blood counts, 2 were anemic, 2 had granulocytopenia, 2 thrombocytopenia, 4 bicytopenia and 2 pancytopenia. BM cellularity was normal or increased, but only 2/16 patients had normal BM morphology. Ten patients had atypical lymphoid aggregates, relative plasmacytosis and eosinophilia, and 4 had typical myelodysplastic changes. There was no correlation between morphology and WR or CDC grade. The mean proliferative fraction (i.e. the percentage of cells in the S phase of the cell cycle) of the HIV-positive patients was 11% (range 5.5-18.3%). The mean value for the control patients was 15.1% (range 7.7-26.9%) (p less than 0.05). All patients had modal diploid DNA content without aneuploid clones. These data suggest that the mechanism of BM failure in HIV-positive patients lies in a reduced proliferative activity whose exact cause is still unclear.