Background and Objective Previous studies have shown that volunteer, community-recruited donors have a higher prevalence of human immunodeficiency virus (HIV) infection in Sao Paulo, Brazil, than replacement donors. One hypothesis which may explain this unexpected finding is that some individuals donate blood because they are seeking HIV testing. The objective of this study was to characterize test-seeking blood donors and to determine whether they are at higher risk for HIV infection compared with other donors.Materials and Methods Subjects presenting for blood donation were asked to participate in a study assessing their motivations (including test seeking) to donate, as measured by perceiving donation as a means to obtain infectious disease test results. Participants completed the standard blood bank predonation screening questions plus our additional survey, and were tested for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell lymphocytotrophic virus (HTLV) I/II, syphilis and Chagas' disease. As a result of anticipated low statistical power to directly measure the association between test-seeking motivation and HIV infection, we tested for herpes simplex virus type 2 (HSV-2) as a marker of sexual risk for HIV. Our survey includes accepted donors as well as persons whose risk-behaviour histories would result in their exclusion from donation according to routine screening procedures.Results Of 1720 potential blood donors randomly selected and approached, 1600 (93.0%) participated. Overall, 141 (8.8%) were classified as test seekers; 15.6% of these were HSV-2 positive. The proportion of test seekers was the same among community-recruited and replacement donors. Test seekers had a higher prevalence of HSV-2 [adjusted odds ratio (AOR) 1.66; 95% confidence interval (CI): 1.06-2.59] adjusting for age, gender and prior donation. The association was significant among community-recruited blood donors whose previous donation was more than 1 year ago (i.e. 'lapsed donors') (AOR 2.55; 95% CI: 1.20-5.44). Test seekers were not more likely to be rejected from blood donation as a result of health reasons, self-reported HIV risk-related behaviour, or by their own confidential unit exclusion. We found no difference in HSV-2 prevalence between persons accepted for donation (15.7%) and those rejected because of self-reported sexual risk (16.7%).Conclusions We did not detect a difference in the proportion of test seekers across different types of blood donors; however, we did detect an association between HSV-2 infection and test seeking, especially among community-recruited lapsed blood donors. Of note, questions on test-seeking behaviour detected donors with increased prevalence of HSV-2, but the self-reported sexual risk behaviours currently used for deferral criteria did not. Incentives to get tested at sites other than blood banks may decrease the residual risk of HIV in the blood supply.

Drugs can result in broad variety of hematologic abnormalities including positive direct antiglobulin test. In this study, we evaluated gel microcolumn assay for the detection of drug-induced antibodies. Direct antiglobulin test was performed by conventional tube and by gel microcolumn assay in 139 hospitalized patients. Drug in vitro studies were done in 34 patients with positive direct antiglobulin test by tube test and gel microcolumn assay using serum and eluate. None of them had signs of hemolytic anemia. A total of 1,000 blood samples from donors were used as control group. Gel microcolumn assay was more sensitive than in tube test for direct antiglobulin test (P < 0.01). Positive direct antiglobulin test was more. frequent in patients than in donors (P < 0.01). Drug in vitro studies were positive with at least one drug in 76.5% of patients with positive direct antiglobulin test by immune complex and/or adsorption mechanisms. We found a high incidence of positive drug in vitro tests in positive direct antiglobulin test patients. Gel microcolumn assay showed appropriate results for drug in vitro studies. The combination of tube and gel microcolumn assay can improve detection of drug-induced positive direct antiglobulin tests.

Background: A3 blood subgroup is heterogeneous at molecular level. To date only two cases of ABO*A301 allele containing a single substitution 871G>A (Asp291Asn) were found in A3B individuals exclusively. The purpose of this study was to use a sequence specific PCR (PCR-SSP) to detect 871G>A mutation in A3 and A3B samples. Material and methods: Twenty blood samples from unrelated donors were classified serologically as A3 and A3B using tube test technique and gel-test simultaneously. Agglutination of red cells by lectins was performed according to manufacturer’s instructions using anti-A1 lectin from Dolichos biflorus and anti-H from Ulex europaeus . Genomic DNA was prepared from peripheral blood cells by a salting-out method. ABO genotyping was performed using a polymerase chain reaction method with sequence specific primers: 95s (5′-CAC CAG GCC ATG ATG GTC A-3′) for consense G detection, 96s (5′-CAC CAG GCC ATG ATG GTC A-3′) for mutation A detection and 244as (5′-CGC AGT GAA CCT CAG CTT C-3′) for both detections on gene ABO exon VII (Seltsam, Transfusion 2003, 43(4): 428-39). PCR amplification was carried out under the following conditions: 5 initial cycles at 94°C for 10 minutes and 65°C for 1 minute; 20 cycles of 94°C for 10 seconds and 65°C for 1 minute; 20 final cycles of 94°C for 20 seconds, 61.5°C for 50 seconds and 72°C for 30 seconds. PCR amplification of 0.1 μg of genomic DNA was performed in a total volume of 25 μL containing 0.2 mM of each dNTP, 5 units Taq DNA polymerase in the buffer supplied, 2.5 mM MgCl 2 and 12.5 pmoles of each primer. Fragment of 177 bp derived from each mutation or consense assay was separated for 90 minutes at 102V using 0.5 μg.mL −1 ethidium bromide-stained 2.5% agarose gels and visualized in a UV light apparatus (Eagle Eye II). Results: We found 871G>A mutation in heterozygosis in three blood donors (15%), two of them typed as A3B and one as A3. Conclusion: The 871G>A mutation is relatively commom in A3B and A3 individuals and can be easily detect by PCR-SSP.

Gel microcolumn assay (GMA) is a modified serological technique that has been used for ABO and Rh typing, direct antiglobulin test (DAT), detecting alloantibodies, red cell phenotyping, and other applications. However, for DAT, the role of GMA is controversial. The purpose of this large study was to compare the performance of the conventional tube test (CTT) to GMA for detecting potentially significant antibodies coating red blood cells in vivo. From January 1996 to May 2002, we performed DATs by GMA and CTT on 9,862 blood samples submitted to our reference laboratory, using LISS/Coombs cards (DiaMed‐Latino America, Lagoa Santa‐MG, Brazil) for GMA and polyspecific and monospecific anti‐IgG reagents for CTT. Acid eluates were prepared from all positive DAT samples. The specificity of eluates was determined by GMA. We detected nonconcordant results in 2,079 out of 3,163 positive DATs (65.7%). All of these tests were only positive in GMA. Sensitivity and specificity for DATs was 100% and 83.0% for gel, and 50.7% and 97.8% for tube, respectively. Based on this study GMA showed to be more sensitive than CTT for detecting potentially significant antibodies coating red blood cells in vivo. J. Clin. Lab. Anal. 18:255–258, 2004. © 2004 Wiley‐Liss, Inc.

Background: Human platelet antigens (HPA), mainly HPA-1 and HPA-2, play a significant role in alloimmune thrombocytopenic syndromes. Accurate HPA typing is important for the diagnosis and therapy of these patients. HPA gene frequencies vary in different populations. Thus, it is relevant to determine HPA genotype in distinct populations. The purpose of this study was to perform HPA-1,-2 genotyping in apheresis Brazilian blood donors. Material and Methods : Genomic DNA was prepared from whole blood of 57 unrelated repeated apheresis Brazilian blood donors using a commercial DNA isolation kit (Qiagen,. GmbH, Germany). Sequency specific PCR (SSP-PCR) was performed amplifying HPA - 1A/HPA-1B and HPA-2A/2B genes using a pair of primers for each allele as described elsewhere (HPA-1A ACT TAC AGG CCC TGC CTC T; HPA-1B ACT TAC AGG CCC TGC CTC C; HPA-1AS AGC CGG AGT GCA ATC CTC TG; HPA-2A CCC CCA GGG CTC CTG AC; HPA-2B CCC CCA GGG CTC CTG AT and HPA-2AS GCC AGC GAC GAA AAT AGA GG). A fragment of the human growth hormone gene (HGH) served as internal positive control. PCR was carried out in a final volume of 20 μL, containing 0.2 mg of genomic DNA, 0.2 mM of each dNTP, 5% of glycerol, 1.5 units of platinum Taq polymerase (Invitrogen, Brazil) in the buffer supllied, 5.0 mM MgCl2 and 10.0 pmoles of each primer. Fragments of 196 bp derived from HPA-1A/1B mutation and of 241 bp for HPA-2A/2B were separated for 90 minutes at 102V using 0.5 μg.mL−1 ethidium bromide-stained 2.0% agarose gels and visualized in a UV light apparatus (Eagle Eye II, EUA). Results : The genotype frequencies presented 77.2% of HPA-1A/1A , 19.3% of HPA-1A/1B and 3.5% of HPA1B/1B ; for HPA-2 the frequencies were 56.1% of HPA-2A/2A , 35.1% of HPA-2A/2B and 8.8% HPA-2B/2B. The gene frequencies in apheresis blood donors are 0.87 for HPA-1A , 0.13 for HPA-1B , 0.74 for HPA-2A and 0.26 for HPA-2B . Conclusion: In this studied population we found high gene frequency homozygous HPA-2B when compared with other data already published. (Castro et al, European Journal of Immunogenetics,1999;26:355–60).

Background: Alloantibody and autoantibody formation to red blood cell (RBC) antigens is one of the observed complications in sickle cell disease patients (SCD). The incidence of alloimmunization and autoantibodies in this selected group of patients is particularly high, although the clinical implication of autoantibodies in sickle cell disease patients is not clear. The purpose of this study is to evaluate the rate of alloantibody and autoantibody formation in SCD patients. Study design and methods: A retrospective analysis of transfused sickle cell disease patients followed at Fundacao Pro-Sangue Hemocentro de Sao Paulo between 1988 and 2004 were retrieved. Data on transfusion history, were correlated with development of alloantibodies and autoantibodies. Results: The study group was composed by 43 sickle cell disease patients followed for a mean of 89 months (22–116). The number of RBC units transfused (mean) was 64 (4–208). The development of the first alloantibody was detected after a mean of 40 months (1–107) after the first transfusion in our institution. Out of these patients, 31 (72.1%) were identified with RBC alloantibodies; 9 of these patients (21%) had both allo and autoantibodies to RBC antigens, whereas 5 (55.6%) developed autoantibodies after alloimmunization. The one remainder had only autoantibodies. Conclusion: The alloimmunization rate was extremely high (72.1%) and can be partially explained because of the extended time of follow-up (mean of 89 months). Different from the literature the development of autoantibodies preceeded alloantibodies in 44.4%. The impact of this observation in clinical practice warrants further investigation.

The diagnostic approach to venous thromboembolism has many difficulties and limitations. Its clinical picture is not specific, and it has a very low positive predictive value, so the use of complementary examinations is necessary to confirm the presence of venous thromboembolism. To implement a better diagnostic investigation, reducing its inconvenience, this protocol was developed to evaluate D-dimer test efficacy in excluding acute venous thromboembolism.One hundred and twenty three patients were studied (70 women), who presented to the emergency department with signs and symptoms suggestive of pulmonary thromboembolism (PTE) or deep venous thrombosis (DVT), or both of these. Acute arterial occurrences were excluded.The prevalence of acute thrombosis among the study patients was 29%. The D-dimer examination (VIDAS) is effective and safe in excluding acute events of venous thromboembolism.The clinical picture of acute venous thromboembolism has a very low positive predictive value; therefore, it is essential to conduct complementary examinations to confirm its presence.

Background: Mutations critical for ABO group phenotypes which encode the catalytic domain of ABO glycosyltransferases have been studied in healthy blood donors. Weakening of A and B transferases have been reported in patients with leukemia, particularly those in which the myeloid lineage is involved. The purpose of this study was to use sequence specific PCR (PCR-SSP) to perform ABO genotyping of leukemic patients.

Background: Primary immune response against red blood cell (RBC) antigens often takes weeks or months to be detected. In previous reports, for children receiving multiple units of blood components, ranging from five to 81 units, the elapsed time between the first RBC transfusion and antibody detection ranged from 18 to 78 days. Cytomegalovirus (CMV) is sometimes associated with immunohaematologic findings and may modulate immune response.Case report: A 24-week-old male infant with interstitial pneumonia and hepatitis because of CMV developed an RBC auto antibody and two RBC alloantibodies: anti-Jk(a), detected in tube 11 days after a single RBC transfusion, and anti-K, detected only in papain gel test 18 days later.Conclusion: As anti-Jk(a) is not a naturally occurring antibody, this is the most rapid primary immune response against an RBC antigen after a single RBC transfusion ever described, in the youngest child ever described.

Dear Sir, Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon, acquired clonal hematopoietic stem cell disorder characterized by hemolysis, propensity to bone marrow failure and tendency to thrombosis [1]. On occasion of thrombosis heparin is the drug of choice followed by oral anticoagulation. In special conditions, thrombolytic therapy can be used [1]. Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated adverse effect of heparin therapy. It is supposed that in this event antibodies are directed against platelet factor 4 (PF4) [2]. Arixtra® (Fondaparinux) is a synthetic pentasaccharide active in the presence of antithrombin, producing selective factor (F)Xa inhibition [3]. As Arixtra® does not bind to PF4 [4], it is supposed that this drug can be an option to be used in the event of HIT. We report the case of a patient with the diagnosis of PNH, who developed Budd–Chiari syndrome and HIT and was successfully treated with Arixtra®. Due to a hemolytic anaemia that had started in 1966, a 32-year-old male African-Brazilian patient was evaluated in the Department of Haematology in July 1997. Laboratory evaluation showed Ham test and sucrose lysis test positive, neutrophils immunophenotype 93% CD55– and 75% CD59–. The diagnosis of PNH was made and since then the patient has maintained a platelet count ranging from 157 to 92 × 103 mm−3. In March 2002 the patient presented with a 2-month complaint of increased abdominal volume and local tenderness. The abdominal ultrasound Doppler study showed ascites, hepatomegaly and thrombosis of suprahepatic veins and inferior cava vein (Budd–Chiari syndrome). The therapeutic approach proposed was thrombolysis with recombinant tissue type plasminogen activator (rt-PA) associated with heparin. The patient was put on dalteparin (5000 IU s.c. bid) which was changed to enoxaparin (60 mg s.c bid) 2 days later. At admission the patient's platelet count was 93 × 103 mm−3, but on the day 11 of low-molecular weight heparin (LMWH) his platelet count showed 50% decrease without associated signs of a new vaso-occlusive event. Platelet aggregation induced by ADP in the presence of different concentrations of heparin showed platelet hyperaggregation, what supports the diagnosis of HIT. With these data, LMWH was stopped and thrombolytic therapy was postponed. The patient showed spontaneous progressive increase of the platelet count, which soon reached 101 × 103 mm−3. A new therapeutic regimen was proposed using rt-PA associated with Arixtra® (donated by Sanofi-Synthelabo, Brazil) (2.5 mg s.c. daily). The patient received two systemic infusions of rt-PA without showing ultrasonographic improvement of the venous flow in the inferior cava vein and in the suprahepatic veins. While on Arixtra® prophylaxis, warfarin was introduced and when the International Normalized Ratio reached 2.2 Arixtra® was discontinued (5 days of drugs overlapping). During all the treatment period with Arixtra® the platelet count remained within the patient's normal values. Recently it was suggested that PNH patients would be at risk for HIT [5]. In our case, thrombocytopenia appeared in the expected period of time, but with a type of heparin (LMWH) normally associated with lower frequency of HIT. Nowadays there are several alternative agents for the treatment of HIT, but all of these drugs must be carefully dosed (activated partial thromboplastin time or anti-FXa assay) [2]. In this case, fondaparinux (Arixtra®) was successfully used for the treatment of HIT based in some in vitro studies that have shown absence of cross-reactivity between fondaparinux and PF4 [4]. Moreover, Arixtra® showed some advantages, since it was administered once a day and without requirement of laboratory control. To the best of our knowledge, this is the first time that Arixtra® was used for HIT, showing good results. Nevertheless, the administration of Arixtra® in an increased number of patients with HIT is necessary to assure its use as a therapeutic option for patients with HIT.

Anti-D titration is the first step in the evaluation of the RhD-sensitized patient. Traditionally, anti-D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti-D titer needs to be established. Seventy-nine known blood samples with anti-D (titers 1-32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R0r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2-2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti-D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4-fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti-D titration by the gel microcolumn assay.

BACKGROUND:Few immunohematological studies have been done in myelodysplastic syndrome (MDS).METHODS:Twenty-nine MDS patients were retrospectively evaluated with a direct antiglobulin test (DAT), antibody screening, serum electrophoresis and immunoelectrophoresis. Clinical and laboratory studies (hemoglobin level, reticulocyte count, DHL, total and indirect bilirubin) were done simultaneously, as well as the French-American-British subtype and bone marrow biopsy findings.RESULTS:Alloantibodies were demonstrated in 17 patients (58.6%), autoantibodies in 10 (34.4%) patients and cold agglutinin in 18 (62%) patients. DAT was mediated by only IgG in 8 patients (80%), by IgG and C3 in 1 patient (10%) and by IgG, IgA and C3 in 1 (10%) patient. No hemolytic disease occurred in patients with autoantibodies. Increased serum gammaglobulin was observed in 16 (54.4%) patients. There was no correlation between the incidence of allo-/autoantibodies and the gammaglobulin level (p = 0.937) and the presence of lymphocyte infiltrates in bone marrow biopsies (p = 0.156). No significant difference was observed when the incidence of autoantibodies and number of red blood cell transfusions were compared (p = 0.334). Patients with refractory anemia and refractory anemia with ringed sideroblasts subtypes had a higher incidence of allo-/autoantibodies than other MDS subtypes (p = 0.03).CONCLUSION:Patients with MDS, in particular refractory anemia and refractory anemia with ringed sideroblasts have a high incidence of allo- and autoantibodies, probably related to intrinsic immune disorder, without clinical or laboratory hemolysis.

In families afflicted with haemophilia A, genetic counselling is often requested. Inversion mutations and polymorphic sites of the FVIII gene have been examined in a Brazilian population, with the aim of developing a strategy that would be accurate and informative for carrier analysis and prenatal diagnosis in Brazil. Patients with haemophilia A and families were studied. Inversion mutations in the FVIII gene were detected in 39.4% of severely affected patients, 85% of them being of distal type. No inversions were observed in patients with mild or moderate forms of the disease. Two bi‐allelic polymorphisms were studied. Intron 18 SSCP and intron 19 RFLP analyses indicated the presence of a restriction site in 39.5% and 42.9% of haemophilics, respectively. Two multiallelic microsatellite polymorphisms in introns 13 and 22 were also studied; eight different alleles were detected in each system with a heterozygosity rate of 83.08% and 78.77%, respectively. When all four intragenic loci were examined in linkage analysis, the cumulative reliability was 100%. In conclusion, inversion mutation analysis should be the first‐line test for Brazilian patients with severe haemophilia A. In cases of severe haemophilia A where no inversion could be detected or in mild or moderate haemophilia A, the use of all four polymorphisms is very informative for linkage analysis and should be used for carrier detection and genetic counselling in the Brazilian population.

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.