Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.

Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis.

Phosphatidylinositol phosphate kinases (PIPKs) are enzymes that participate in diverse intracellular signaling pathways. They are classified into 3 functionally distinct subfamilies - PIPKI (α, ß, γ), PIPKII (α, ß, γ), and PIPKIII - located in various subcellular compartments. Recently, the PIPKIIα and ß-globin genes were found to be overexpressed in reticulocytes from 2 siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIa and the production of globins. The main aim of this study was to determine the expression profiles of PIPK genes in healthy individuals during in vitro erythropoiesis using quantitative real-time polymerase chain reaction and to compare these profiles with profiles of globin genes. Our results showed that expression of all PIPKs increases as the cells differentiate, coinciding with the expression profiles of globins. Analysis of the effects of globins on PIPK genes revealed that they varied significantly between the globins, the most noticeable being the effect of α-globin on PIPKIIα (P < 0.0001) and γ-globin on PIPKIIγ (P < 0.0001). The relationship between the expression of PIPKs and globin genes was statistically significant, particularly between PIPKIIα and α-globin (P = 0.0002) and PIPKIIγ and ß-globin (P < 0.0001). Linear correlation analysis revealed a strong relationship between PIPKIIα and α-globin genes. This study is the first to establish the expression profiles of PIPK genes during in vitro erythropoiesis in healthy individuals and suggests a parallel between the expression of PIPK and globin genes, reinforcing the hypothesis that they may be related.

Ex vivo expansion of CD56+ NK and NKT-like lymphocytes from peripheral blood mononuclear cells of patients with ovarian neoplasia.

Methods for ex vivo expansion of natural killer (NK) cells have allowed obtaining enough numbers of human NK cells for clinical trials. However, the evaluation of these methods has been mostly limited to haematological malignancies. This study aimed at evaluating a method for selective expansion of NK cells when applied in peripheral blood mononuclear cells (PBMC) of patients with ovarian neoplasia. PBMC from 13 volunteer patients with ovarian neoplasia, seven benign and six malignant tumours, were cultured in CellGro medium supplemented with anti-CD3 (9-10 initial days), IL-2 and foetal bovine serum for 21 days. The resulting effector cells were evaluated for their phenotype, cytotoxicity and cytokine secretion. PBMC cultures resulted in multiple populations (NK, NKT and T) of effector cells, enriched with CD56(+) lymphocytes. NK cells from patients with benign and malignant ovarian neoplasia were expanded 139.6 ± 63.4 and 82.7 ± 25.3-fold, respectively, being the largest lymphocyte subtype among CD56(+) population. Effector cells expanded from patients with malignant ovarian neoplasia had higher proportion of T lymphocytes and altered cytokine production patterns, characterized by lower INF-γ, TNF-α and higher IL-4, compared with patients with benign ovarian neoplasia. Effector cells were cytotoxic against K562 and OVCAR3 cell lines. Cytotoxicity was significantly higher (P < 0.05) using magnetically separated CD56(+) effector cell fractions compared with CD56-deprived ones. The present study demonstrates the feasibility of the culture system employed to generate effector cells, enriched with CD56(+) lymphocytes, from PBMC of patients with ovarian neoplasia. NK cells were the largest lymphocyte subtype among the CD56(+) population and the main variable among the final effector cell preparation affecting target cell killing.

The impact of several phenotypic features at diagnosis on survival of patients with myelodysplastic syndromes.

Multiparametric flow cytometry is a useful co-criterion for diagnostic confirmation of MDS in patients with peripheral cytopenias and a normal karyotype. We examined the impact on patients' survival of several phenotypic aberrancies detected by a small 4-color panel of monoclonal antibodies (MoAbs). Diagnosis of the patients (54) was made by WHO criteria using peripheral blood counts, bone marrow (BM) morphology and karyotype. Flow cytometry was performed at diagnosis, and features obtained were compared to normal BM (24). We could detect 16 alterations: 4 in granulocytic precursors, 4 in monocytes, 6 in CD34+ cells, beside changes in plasmacytoid dendritic cells and basophil precursors. The total number of changes in RAEB was higher (median 8) than in cases with of abnormalities) were independent risk factors for a shorter survival. Our panel was sufficient to confirm the diagnosis of MDS and permitted to detect independent prognostic features.

A simple score derived from bone marrow immunophenotyping is important for prognostic evaluation in myelodysplastic syndromes.

Immunophenotyping of bone marrow (BM) precursors has been used as an ancillary diagnostic tool in myelodysplastic syndromes (MDS), but there is no general agreement about which variables are the most relevant for prognosis. We developed a parsimonious prognostic model based on BM cell populations well-defined by phenotype. We analyzed 95 consecutive patients with primary MDS diagnosed at our Institution between 2005 and 2012 where BM immunophenotyping had been performed at diagnosis. Median follow-up: 42 months (4-199). Median age: 67 years (33-79). According to IPSS-R, 71 cases were low or intermediate risk. Flow variables significant in the univariate Cox analysis: "%monocytes/TNCs", "% CD16+ monocytes/TNCs", "total alterations in monocytes", "% myeloid CD34+ cells", "number of abnormal expressions in myeloblasts" and "% of B-cell progenitors". In the multivariate model remained independent: "% myeloid CD34+ cells", B-cell progenitors" and "% CD16+ monocytes/TNCs". These variables were categorized by the extreme quartile risk ratio strategy in order to build the score: % myeloid CD34+ cells" (≥ 2.0% = 1 point), B-cell progenitors" (< 0.05% 1 point) and "CD16+ monocytes/TNCs" (≥ 1.0% 1 point). This score could separate patients with a different survival. There was a weak correlation between the score and IPSS-R. Both had independent prognostic values and so, the flow score adds value for the prognostic evaluation in MDS.

Microparticles in deep venous thrombosis, antiphospholipid syndrome and Factor V Leiden.

Microparticles (MPs) are blebs released from cellular surfaces during activation/apoptosis. They are procoagulant, pro-inflammatory and could contribute to pathogenesis of deep venous thrombosis (DVT). This study compared the number, cellular origin and procoagulant activity of MPs on DVT patients in different clinical situations: at diagnosis (n = 9, 5F/4M; mean age = 41.11), 1-3 years after warfarin withdrawal (n = 10, 7F/3M; mean age = 32.90), associated to antiphospholipid syndrome (APS; n = 11, 9F/2M; mean age = 33.82), or asymptomatic carriers of Factor V Leiden (FVL; n = 7, 7F/0M; mean age = 34.00) vs healthy controls (CTR). The quantification and characterization were performed by flow cytometry using CD235, CD61, CD45, CD31, CD14, CD45, anti-TF and Annexin V. The plasmatic procoagulant activity was investigated by prothrombin fragment 1 + 2 (F1 + 2) determination. The MPs procoagulant activity was analyzed by D-dimer (DD2) and Thrombin Generation Test (TGT) on a healthy pool of plasmas adjusted or not by their number (10,000 MPs). The MPs percentages were not different between the groups, but absolute number was increased in patients 1-3 years after warfarin withdrawal vs CTR (P = 0.02). There was no difference of the MPs cellular origin comparing patients to controls. TGT using 10,000 MPs was lower on these patients (P = 0.01). APS patients showed a reduction of plasmatic procoagulant activity (P = 0.004), but they were under warfarin therapy. DD2 in the presence of MPs, independently of its number, was higher in patients with DVT at diagnosis (P < 0.0001). MPs of patients with spontaneous DVT at diagnosis can promote coagulation activation demonstrated by increased DD2. Even the increased MPs from patients 1-3 years after thrombotic episode generated lower amount of thrombin, they can have a protective effect by activation of Protein C anticoagulant pathway.

Variation of bone marrow CD34+ cell subsets in myelodysplastic syndromes according to who types.

Bone marrow (BM) hematopoietic progenitor cells (CD34+) are a heterogeneous population with varying degrees of commitment and maturation to several cell lineages. In myelodysplastic syndromes (MDS), this population is increased. We examined the major cell types found in the blast gate by flow cytometry in newly diagnosed patients with MDS, compared them to normal BM and studied their variation according to WHO type. Two subsets defined by SSC were found both in normal BM and MDS, corresponding to myeloblasts and B-cell precursors. The number of B-cell precursors among all nucleated cells was equally low, independent of WHO type. However, the subset with an intermediate SSC, but CD117, CD13 and CD19 negative increased with the rise of myeloblasts. Concomitantly, the ratio between CD34+/CD117+/CD34-/CD117+ cells was increased. These two features are consistent with the maturation block occurring in the progression of the neoplastic clone. We conclude that the quantitative analysis of the cell types present in the BM blast gate by flow cytometry is not only important for the diagnosis of MDS in patients with peripheral cytopenias and a normal karyotype, but gives also important prognostic information of the patients.

Flow cytometry diagnosis in myelodysplastic syndrome: Current practice in Latin America and comparison with other regions of the world.

BACKGROUND: Flow cytometry (FC) is a valuable tool for the diagnosis of myelodysplastic syndromes (MDS). We present results of a survey carried out to evaluate FC current practice for MDS diagnosis in Latin America (LA), focusing on markers used and characteristics of the clinical diagnostic report. Compliance to IMDSflow recommendations was also evaluated. These practices were then compared with those used in other countries. METHODS: An online survey was sent through the Grupo Latino-Americano de Mielodisplasia to LA cytometrists and other international scientific societies. RESULTS: 91 responses from 15 LA countries were received. The median of the number of markers used was 20 ± 4.5, but only 8.1% of participants adopted the complete panel proposed by the International/European LeukemiaNet Working Group (IMDSflow). We received 140 eligible answers from regions other than LA (66 Europe, 59 USA-Canada, 8 Oceania, 6 Asia and 1 Africa). LA utilized more markers for MDS diagnosis than USA/Canada (p = 0.006), but similar to Europe. The use of MDS scoring systems differed among regions: 10.3% in LA, 0% USA/Canada and 25.7% Europe reported the "Ogata score". Finally, 52.0% of all participants included a general interpretation statement in the final report about the consistency of the FC results with MDS diagnosis, with no statistical differences between regions. CONCLUSIONS: This survey shows a low compliance with the IMDSflow recommendations and a scarce use of the scoring systems proposed in the literature. However, the number of surface markers used is high. We will work to develop a FC consensus for MDS diagnosis adapted to the clinical practice requirements in LA.

The prognostic value of maturation-associated phenotypic abnormalities in myelodysplastic syndromes.

Several phenotypic abnormalities of bone marrow (BM) hemopoietic precursors have been associated with disease progression in myelodysplastic syndromes (MDS). We analyzed the influence on overall survival of the expression of lineage and maturation-associated antigens of BM hemopoietic cells quantified in a previous study. In the univariate Cox regression the peripheral platelet count was a significant favourable factor for overall survival. Unfavorable prognostic factors were: WPSS, increase in BM CD34+ cells, increased mean fluorescence intensity (MFI) of CD13 on myelocytes, metamyelocytes and mature neutrophils as well as increased CD45 of myelocytes and mature neutrophils. In a model containing platelet count, WPSS and MFI of CD45 and CD13 on mature neutrophils, only hyperexpression of CD13 and degree of thrombocytopenia were independent risk factors. Therefore, phenotypic features that can also be obtained from PB might be useful for predicting survival in MDS.

Normal variation of bone marrow B-cell precursors according to age - reference ranges for studies in myelodysplastic syndromes in Brazil.

BACKGROUND: Normal B lymphoid maturation occurs in bone marrow (BM) throughout life, but immature B-cell progenitors (BCPs) are more numerous in children than in adults. To assess the normal values according to age became important as BCPs are decreased in myelodysplastic syndromes and have been considered an important diagnostic and prognostic feature in these clonal disorders. METHODS: in a multicenter retrospective study from the Brazilian Group of Flow Cytometry we analyzed the variation of BCPs in normal BM according to age and technical peculiarities of each laboratory. We analysed of 45 BM donors and 89 cases examined for elucidation of transitory reactive cytopenias presenting a normal BM immunophenotyping. BCPs were enumerated as CD19+ /CD34+ /CD45dim /CD10+ cells (panel 1) or CD19+ /CD34+ /CD45dim cells (panel 2) among the total nucleated non-erythroid cells and as percentage of CD34+ cells. RESULTS: we included 134 cases. Panel 1 was applied in 88 cases and panel 2 was used in 46. Age range: 10 months to 89 years. In a multiple regression, % BCPs/total nucleated cells was an exponential function of age. Age explained alone 49.4% of the variance, while 'panel used' explained 1.8% and 'laboratory' explained 0.7%. Age explained only 24.9% of the variance of BCPs/CD34+ cells. CONCLUSIONS: in normal individuals, BM B-cell precursors varied mainly according to age, but were also dependent on technical peculiarities of operators and equipments. Analysis by phenotype and as percentage of total nucleated cells was more accurate and less susceptible to variation than evaluating % BCPs/total CD34+ cells. © 2017 International Clinical Cytometry Society.

Evaluation of reticulated platelets in patients with sickle cell diseases.

BACKGROUND AND PURPOSE: Reticulated platelet (RP) count provides an estimate of thrombopoiesis. The objective was to evaluate RP in patients in different stages of sickle cell disease (SCD) and to determine the relationship between interleukin-6 (IL-6), interleukin-3 (IL-3) and thrombopoietin (TPO) and RP count and degree of activation. METHODS: Eighty-nine adult patients with SCD were studied: 38 were in the steady state, 27 in hemolytic crisis (HC) and 24 in vaso-occlusive crisis (VOC). RPs and activated platelets were analyzed by flow cytometry. Soluble P-selectin, IL-6, IL-3 and thrombopoietin (TPO) levels were measured by ELISA tests. RESULTS: The patients in VOC had a higher absolute number of RPs and CD62P+ platelets than did the control group or patients in the steady state. A significant correlation was observed between the absolute number of CD62P+ platelets and RPs in patients in the steady state, HC and VOC. In the steady-state group of patients, the level of soluble P-selectin was found to be dependent on the RP values. IL-3 and TPO serum levels were higher in patients in the steady state, HC and VOC than in the control group. IL-6 serum levels were higher in HC and VOC patients than in the control group and higher in patients in the steady state than in the VOC group. CONCLUSION: Our results suggest that PRs contribute to the vaso-occlusive process in sickle cell disease. Increased interleukin serum levels probably indicate that inflammatory process is involved in the vascular-occlusive phenomenon. However, it appears that these inflammatory mediators do not have an effect on thrombopoiesis in sickle-cell-disease patients.

Diagnosis of chronic lymphoproliferative disorders by flow cytometry using four-color combinations for immunophenotyping: A proposal of the brazilian group of flow cytometry (GBCFLUX).

BACKGROUND: Multiparametric flow cytometry (MFC) is a powerful tool for the diagnosis of hematological malignancies and has been useful for the classification of chronic lymphoproliferative disorders (CLPD) according to the WHO criteria. Following the purposes of the Brazilian Group of Flow Cytometry (GBCFLUX), the aim of this report was to standardize the minimum requirements to achieve an accurate diagnosis in CLPDs, considering the different economic possibilities of the laboratories in our country. Most laboratories in Brazil work with 4-fluorescence flow cytometers, which is why the GBCFLUX CLPD Committee has proposed 4-color monoclonal antibody (MoAb) panels. METHODS/RESULTS: Panels for screening and diagnosis in B, T and NK lymphoproliferative disorders were developed based on the normal differentiation pathways of these cells and the most frequent phenotypic aberrations. Important markers for prognosis and for minimal residual disease (MRD) evaluation were also included. The MoAb panels presented here were designed based on the diagnostic expertise of the participating laboratories and an extensive literature review. CONCLUSION: The 4-color panels presented to aid in the diagnosis of lymphoproliferative neoplasms by GBCFLUX aim to provide clinical laboratories with a systematic, step-wise, cost-effective, and reproducible approach to obtain an accurate immunophenotypic diagnosis of the most frequent of these disorders. © 2016 International Clinical Cytometry Society.

Optimization of CD34+ collection for autologous transplantation using the evolution of peripheral blood cell counts after mobilization with chemotherapy and G-CSF.

BACKGROUND: Peripheral blood progenitor cells (PBPC) collection after high dose chemotherapy can be influenced by several factors. We searched for parameters that may predict the best day to start harvesting of PBPC in order to collect most CD34+ cells with the least number of aphereses. METHODS: We studied patients who underwent mobilization chemotherapy for autologous transplantation. The influence of age, sex, diagnosis, number of previous chemotherapy cycles, peripheral blood (PB) counts at day of mobilization (D0), day of neutrophils <1.0 x 10(9) l(-1) and day of nadir and interval between both (delta) on harvesting was investigated. Multivariate linear correlation models were built to predict the best harvesting with principles of parsimony. In patients where sequential CD34+ cell count was performed, the theoretical day of peak was calculated by interpolation in polynomial regression. RESULTS: One hundred and thirty four patients entered the analysis: 36 Hodgkin's lymphoma (HL), 65 B-large cell lymphoma (NHL) and 33 multiple myeloma (MM). Day of harvesting correlated with nr CHT, hemoglobin on D0, day of granulocytes <1.0 x 10(9) l(-1), delta and dosis of mobilization therapy. The day of CD34+ peak could be calculated by the formula = (-0.41) x Hemoglobin D0 + (day peripheral CD34+ cells = 10 x 10(6) microl(-1)) x 0.99 + 7.8. This model could explain 81% of the variance of the peak day and was stable by bootstrap resampling. Day of peripheral CD34+ cells = 10 x 10(6) microl(-1) preceded the calculated peak by 3-9 days. CONCLUSIONS: Although the day of best collection can be predicted using only sequential PB counts after mobilization chemotherapy, a model of prediction using peripheral CD34+ cell count is important especially for optimizing collection in poor mobilizing patients.

Phenotypic quantitative features of patients with acute myeloid leukemia.

The recent WHO classification for acute myeloid leukemias (AML) separates entities by recurrent cytogenetic abnormalities and immunophenotypic features presenting prognostic impact. We have examined the expression of several lineage and maturation linked antigens used in routine immunophenotyping of patients with de novo AML, using a 3-color two-step panel. Cases were diagnosed by peripheral blood counts, bone marrow cytology, cytochemistry, cytogenetics and immunophenotyping (CD2, CD3, CD7, CD19, CD13, CD33, myeloperoxydase -- MPO, CD14, CD15, HLA-DR, CD34, CD56 and CD45). Antigen expression was measured by mean fluorescence intensity (MFI) by flow cytometry (Paint-a-gate software). Thirty five patients were analyzed. Median age: 51 years (15-79). Predominant FAB types were M2 and M4. In 6 cases more than one phenotypically distinct blast subpopulation could be detected. Although our set was small, we tried to analyze the impact of MFI of the examined antigens on the overall survival of the patients. In Cox univariate analysis, age, peripheral leukocytes (WBC) at diagnosis, MFI of CD45, and MPO were significant for worse a survival. In the multivariate analysis only MFI of CD45 and WBC remained in the model (p=0.018 and p=0.014 respectively). After bootstrap resampling, MFI of CD45 entered the model in 69%, WBCin 60%, age in 42% and MFI of MPO in 35% of the sets. Analysis of antigen expression by MFI permitted to detect cases presenting phenotypically distinct blast subpopulations. This may represent a pitfall in studies of minimal residual disease by flow cytometry, as chemotherapy may select one of these subsets.

Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface.

1. The nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated. 2. Human peripheral blood eosinophils were treated with L-NAME (0.01 - 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0+/-4.6 and 43.8+/-4.7%, respectively, when eosinophils were treated with 1.0 mM L-NAME. Increased adhesion by L-NAME could be abolished when cells were co-incubated with VLA-4- and Mac-1-specific monoclonal antibodies (mAbs). 3. The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3+/-4.5 and 45.2+/-5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP. 4. Flow cytometrical analysis demonstrated that L-NAME induced an increased expression of CD11b (Mac-1) on the eosinophil cell surface of 36.3+/-7.4%. L-NAME had no effect upon CD49d (VLA-4) expression. 5. Treatment of human eosinophils, in vitro, with H-[1,2,4] oxadiazolo quinoxalin-1-one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5+/-17.9 and 91.7+/-12.9%, respectively. This increase in adhesion could also be inhibited by co-incubation with the Mac-1 and VLA-4-specific mAbs. 6. In conclusion, results indicate that NO, via a cyclic GMP-dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophil's adhesion molecules, in particular, the expression of the Mac-1 integrin and the function of the VLA-4 integrin.

Molecular analysis of the retinoblastoma (RB1) gene in acute myeloid leukemia patients.

The pathogenesis of acute leukemia is still poorly understood. In the past few years several groups have reported deletion of the RB1 gene or altered pRB expression in certain hematologic malignancies, suggesting a possible role of RB1 gene inactivation in the process of leukemogenesis. Most studies regarding structural abnormalities of the RB1 gene indicate that gross deletions or rearrangements are present in a small percentage of patients with acute myeloid leukemia (AML), as is the case with retinoblastoma, where the majority of RB1 gene abnormalities are attributed to point mutations. To investigate if such point mutations in the RB1 gene may have a role in leukemogenesis in AML, we screened the RB1 gene of 36 AML patients using conformation-sensitive gel electrophoresis (CSGE). No point mutations were found in the 27 exons, their flanking intron regions or in the promoter region in any of the 36 patients. Thus, according to our findings, the susceptibility in these patients for developing AML does not appear to be related to point mutations in the RB1 gene. While screening for point mutations, we identified a number of new and previously noted neutral sequence variations indicating the efficiency and sensitivity of CSGE in identifying small changes in the RB1 gene.

A randomised, prospective comparison of allogeneic bone marrow and peripheral blood progenitor cell transplantation in the treatment of haematological malignancies.

We present the results of a prospective, randomised study comparing PBPC and BM focusing on engraftment, acute and chronic GVHD and survival. Forty patients with haematological malignancies received HLA-identical sibling BM (group A) or PBPC (group B). Evaluable patients were 19 (A) and 18 (B). Median age was 35 (17-56) in A and 29.5 (9-51) in B. Conditioning was mainly Bu-Cy2; GVHD prophylaxis was CSA-MTX. PBPC were harvested after 5 days of G-CSF 10 microg/kg/day. Median days for an ANC >0.5 x 10(9)/l was 18 (13-30) in A and 16 (11-25) in B (P = 0.10). Platelets >20 x 10(9)/l occurred at +17 (10-40) in A and +12 (9-36) in B (P = 0.01). The probability of > or =2 grade a-GVHD was 19% (A) and 27% (B) (P = 0.53). The probability of all grade c-GVHD was 70% with BM. In spite of the small number of patients in group B (PBPC), our data suggest the great majority of them will have c-GVHD (P = 0.08); extensive disease was present in 50 and 100%, respectively (P = 0.05). The estimates of overall survival for A and B at 1000 days are 51 and 47%, respectively (P = 0.67); DFS at 1000 days are 52 and 58%, respectively (P = 0.50). PBPC resulted in faster platelet engraftment. The incidence of acute and chronic GVHD was similar in both groups, but the severity of c-GVHD was higher with PBPC. No differences in survival and DFS have been observed to date.

Adult T-cell leukemia (ATL) with an unusual immunophenotype and a high cellular proliferation rate.

A patient with adult T-cell leukemia (ATL) characterized by a suppressor phenotype is reported. A 52-year-old mulatto male presented with symptoms and signs of hypercalcemia. His laboratory finding disclosed a peripheral blood specimen with abnormal cells characterized by a rather pleomorphic morphology and polylobated nucleous typical of ATL cells. Serum calcium and LDH were 18.2 mg/dl and 1373 IU, respectively. The phenotype of these cells was CD2+, CD4-, CD8+, CD28+ associated with the expression of activated antigens such as CD25, CD38, CD71 and CD30. Ki-67 positive were found in 20% of cells. The argyrophilic stain for nuclear organizer regions (AgNORs) was shown one cluster in 35% of abnormal cells. The serum antibodies were positive against human T-cell lymphotropic virus type I (HTLV-I) and clinical features were compatible with the diagnosis of ATL acute type. The combination therapy with cyclophosphamide, vincristine, prednisone decreased the number of leukemic cells but the clinical course was aggressive. He only responded transiently to treatment and died of multiorgan failure due to uncontrollable septicemia two weeks after admission.

N-ras gene point mutations in Brazilian acute myelogenous leukemia patients correlate with a poor prognosis.

The frequency of ras gene mutations varies from 11 to 27% in AML populations from the United States and Europe but it seems that there is no study regarding the frequency of mutated N-ras gene in patients with AML in South America. In order to study the frequency of N-ras gene mutations (exons 1 and 2) in Brazilian patients with AML and to evaluate the possible correlation between the presence of the mutation and clinical features, 40 patients were analyzed. N-ras mutations were identified in DNA samples from eight of 40 AML patients (20%). No significant correlation was found between N-ras mutation and age, sex, race, response to therapy, FAB subtype or occupational exposure. However, the overall survival and AML-free survival were significantly shorter in patients with N-ras mutations than in those without these abnormalities.

Acute Megakaryoblastic Leukemia: Importance of Bone Marrow Biopsy in Diagnosis.

Five cases of megakaryoblastic leukemia, presenting as "de novo" acute leukemias are reported. They represented 7.2% of all cases of adult acute non-lymphoblastic leukemias (ANLL) seen in our Hematology Service during the past 30 months. These cases showed features of predominantly blast proliferation with a marked increase of reticulin fibres in the bone marrow. One case had features of acute leukemia with trilineage myelodysplasia and one had a more chronic evolution with splenomegaly initially resembling a myeloproliferative syndrome. In all cases, the definitive diagnosis was made on bone marrow histology as cytology was poor and the blast cells were positive for factor VIII with the immunoperoxidase technique. The importance of bone marrow histology is emphasized.