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Background Blood samples from patients with sickle cell disease (SCD) present to transfusion service with numerous antibodies, making the searching for compatible red blood cells (RBC) a challenge. To overcome this problem we developed an effective strategy to meet needs of supplying RBC-compatible units to SCD patients using DNA arrays. Methods We selected DNA samples from 144 SCD patients with multiple (receiving > 5 units) transfusions previously phenotyped for ABO, Rh(D, C, c, E, e), K1, Fya and Jka. We also selected DNA samples from 948 Brazilian blood donors whose ABO/RhD phenotype matched that of the patients. All samples were analysed by DNA array analysis (HEA BeadchipTM, Bioarray Solutions) to determine polymorphisms associated with antigen expression for 11 blood group systems (Rh, Kell, Kidd, Duffy, MNS, Dombrock, Lutheran, Landsteiner-Wiener, Diego, Colton, Scianna); and one mutation associated with haemoglobinopathies. Results Based on genotype results we were able to predict phenotype-compatible donors needed in order to provide compatible units to this group of patients. Based on their ABO/Rh phenotype we were able to find in this pool of donors compatible units for 134 SCD patients. Conclusion Blood group genotyping by DNA array contributes to the management of transfusions in SCD patients by facilitating the transfusion support with antigen-matched blood. It has the potential to improve the life of thousands of SCD-transfused patients by reducing mortality due to transfusion reactions and immunization.

Because of the scarcity of anti-Hy and anti-Joa, hemagglutination typing for the Dombrock blood group system antigens, Hy and Joa, is not feasible. The molecular bases associated with these antigens have been determined, making it possible to distinguish HY and JO from wild-type DO. This provides a tool to predict the probable phenotype of patients and to screen for antigen-negative donors. PCR-RFLP assays and a microchip assay were used to determine the frequency of HY and JO alleles in donors from Brazil and New York. DNA from random Brazilian donors, 288 by PCR-RFLP and 599 by the bead array method (BeadChip, BioArray Solutions, Warren, NJ), was tested to determine 323G/T (HY+/HY–) and 350C>T (JO+/JO–) single-nucleotide polymorphisms. In New York, 27,226 donors who self-identified as being African American were tested by hemagglutination with anti-Gya. Nonreactive and weakly reactive samples were tested by PCR-RFLP for the same alleles as listed above. In Brazil, 30 (3.4%) of the samples were JO/DO and 13 (1.4%) were HY/DO. In New York, of the samples that had HY or JO alleles, 14 were homozygous HY/HY, 132 were heterozygous HY/DO, 13 were heterozygous HY/JO, 14 were heterozygous JO/DO, and 3 were homozygous JO/JO. These results show that in donors from Brazil, JO (30 alleles) is more than twice as prevalent as HY (13 alleles), whereas in donors from New York, HY (173 alleles) was more than five times more common than JO (33 alleles). Immunohematology 2008;24:135–137.

OBJECTIVE: To evaluate the usefulness of DNA methods to provide a means to precisely genotypically match donor blood units for the antigen-negative type of 35 sickle cell disease patients

Abstract 1050 Background: Tregs and proinflammatory Th17 cells have been described as two distinct subsets from Th1 and Th2 cells and have opposite effects in autoimmunity and inflammation. Th17/Treg balance controls inflammation and is important in the pathogenesis of autoimmune, infectious and chronic diseases. In view of growing evidence linking sickle cell disease (SS) and chronic inflammation and the potential anti-inflammatory role of Tregs cells, we hypothesized that SS is associated with decreased Treg subpopulation and consequent T cell dysfunction. Furthermore, the effects of hydroxyurea (HU) in this balance are not understood. To assess whether the Th17/Treg balance is impaired in patients with sickle cell disease, we investigated the peripheral blood Th17 and Treg frequencies and the expression of TGFβ1 and RORγT, a master regulator of Th17 differentiation, in sickle cell patients under HU therapy (SS-HU) and without HU (SSw/oHU). Methods. Thirty-eight patients with steady-state sickle cell disease (mean age 43 ± 10.5 yo; range 19–57 yo; 21 female/17 male), 19 SS-HU, 19 SSw/oHU and 20 healthy blood donors were evaluated. Tregs and Th17 quantification was performed by flow cytometry analysis and the expression of TGFβ1 and RORγT by q-PCR. Results. We found significant decrease in the Treg frequency (Foxp3+CD25+CD4+ population) comparing total SS patients and healthy controls (p=0.001) and this difference was more expressive between SS-HU and healthy controls (P=0.0006). Moreover, we found statistically increase of Th17 in SS-HU (p=0.02) and in SSw/oHU (p=0.01), comparing both with healthy controls. The TGFβ1 expression was increased in SS-HU (p=0.04) and SSw/oHU patients (p=0.01) when compared to control group. Interestingly, RORγT expression was significantly elevated only in SSw/oHU vs controls (p=0.03). Conclusion: Our results demonstrated a numerical imbalance of Th17/Treg in the peripheral blood of patients with SS disease. HU has no role mediating Th17/Treg plasticity and even SS patients under HU therapy remains Treg depleted. However, HU is able to modulated RORγT, corroborating the importance of other regulating factors during Th17 differentiation. Moreover, the higher expression of TGFβ1 may be an important positive instructive signal for Th17 differentiation. Th17/Treg imbalance might impact the disease presentation and phenotype and HU therapy may be involved in this issue. Disclosures: No relevant conflicts of interest to declare.

Introduction: The survival of patients with multiple myeloma varies from a few months to more than 10 years. This heterogeneity is related to the characteristics of the myeloma itself and of the host. The identification of the factors which influence the prognosis is very important to predict the result, assist in the choice of the treatment and adequately stratify the patients in clinical studies. Many prognostic factors have been identified in patients with multiple myeloma, such as anemia, renal failure, ß2 microglobulin, albumin and chromossomic alterations. Some authors have combined prognostic factors and proposed various systems of staging. However, none of them have yet substituted the Durie-Salmon staging system. Recently, the International Myeloma Working Group, with the objective of developing a simple and reliable staging system, which can be internationally applied to classify and stratify patients with multiple myeloma, identified 3 risk groups. This new system of staging, the “International Staging System” (ISS), consists of stage I: ß2 microglobulin < 3.5 mg/L plus albumin ≥ 3.5 g/dL (median survival: 62 months); stage II: neither I nor III (median 44 months); stage III: ß2 microglobulin > 5.5 mg/L (median 29 months). This study included sites in North America, Europe and Asia, but the sites in Latin America were not included. Objective: To validate the ISS in patients with multiple myeloma at Brazilian centers. Patients and Methods: Four hundred and eighty-seven patients with the diagnosis of multiple myeloma within the period of 1998 to 2004 at Santa Casa de São Paulo, Hospital Universitário Clementino Fraga Filho do Rio de Janeiro, Hospital das Clínicas de São Paulo, Hospital São Paulo, Hospital das Clínicas de Ribeirão Preto, HEMOPE, Hospital Prof. Edgar Santos de Salvador and Hospital de Clínicas de Porto Alegre, with available data on albumin and ß2 microglobulin, were stratified according to the ISS. A total of 339 patients received standard therapy and 148 received high-dose therapy as initial therapy. The survival was estimated using the Kaplan-Meier method with differences in survival examined using the logrank test. Results: The median age of the patients was 60 years, 52% male and 48% female. In Stage I (n=104), the global median survival was not reached, the survival at 60 months was 60%, in stage II (n=264), the global median survival was 61 months and in stage III (n=119), 19 months (p Conclusion: The new system of staging for multiple myeloma (ISS) is simple, based on variables easy to be applied and was possible to be validated in patients with multiple myeloma in Brazilian centers.