The human platelet alloantigen 9bw (HPA-9bw, Max a ) is an epitope of GPIIb and differs by 2602G>A single nucleotide polymorphism (SNP), resulting in a Val/Met substitution at amino acid 837 (Noris et al., 1995). Alloantibodies against Max a are involved in the development of neonatal alloimmune thrombocytopenia (NAIT) (Kaplan et al., 2005;Raj et al., 2009). The frequency of HPA-9b allele is low at 0·003 in the Dutch population (Noris et al., 1995), 0·002 in United States (Peterson et al., 2005) and 0·0 in China (Xu et al., 2009). However, Peterson and coworkers found six cases involving alloantibodies anti-HPA-9bw among 217 cases not resolved on NAIT, suggesting that HPA-9bw may be the third most important trigger for NAIT, after HPA-1a and HPA-5b (Peterson et al., 2005). In such studies, HPA-3b allele was present in all individuals with HPA-9b allele.The HPA frequencies vary among distinct populations. In the literature, there are no data for the allelic frequencies of HPA-9 system in the South American population. In this study, we used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to determine the HPA-9 and HPA-3 allelic frequencies in Brazilian blood donors and Amazon Indians. The study was approved by the University Research Ethics Committee (CEP 0793/08). Blood samples from 1073 unrelated Brazilian blood donors and 120 Amazon Indians (Xikrin Kayapo Indians) were collected. DNA was isolated using a commercial kit (QIAamp DNA Blood mini kit, Qiagen, Hilden, Germany) and genotyped by PCR-RFLP using BstNI restriction enzyme for HPA-9 and Fok I for HPA-3. Control samples were used and we performed the sequencing in selected samples containing the SNPs to confirm the region studied.The results of genotype and allelic frequencies of HPA-9 and HPA-3 systems in Brazilian blood donors and Xikrin Indians are shown in Table 1. We found that the frequencies of the genotypes HPA-9a/9a, 9a/9b and 9b/9b were 99·72, 0·28 and 0% in Brazilian blood donors and 100, 0 and 0% in Amazon Indians, respectively. HPA-3a/3a, 3a/3b and 3b/3b had frequencies of 41·29, 46·50 and 12·21% in Brazilian blood donors and 41·67, 52·50 and 5·83% in Amazon Indians. The analysis by statistical χ 2 with significance level of 0·05 showed that no statistically significant differences were found regarding the HPA-9a, HPA-9b, HPA-3a and HPA-3b allelic frequencies between Brazilian blood donors (0·999, 0·001, 0·645, 0·355) and Xikrin Indians (1·0, 0·0, 0·679, 0·321). Using sequencing methods for selected samples we confirmed the corresponding SNPs; however, we did not find any other polymorphisms such as those described by the study of Hallé and coworkers in which they found mutations on GPIIb exon 26 (2614C>A and 2645C>T) and intron 26 (IVS26+89G>A) (Hallé et al., 2008). All individuals carrying the HPA-9b allele also had the HPA-3b allele.The HPA-9 allelic frequencies found in Brazilians are quite similar to those reported by studies performed with Dutch, North American and Chinese individuals as mentioned e...
Background: Neonatal alloimmune thrombocytopenia (NAIT) results from feto-maternal platelet antigen incompatibility leading to the production of maternal antibodies and destruction of fetal platelets during pregnancy. Similarly neonatal alloimmune neutropenia (NAN) is caused by maternal alloimmunization to incompatible fetal neutrophil antigens followed by transplacental transfer of the maternal anti-neutrophil antibody causing fetal or neonatal neutropenia. Although there are many reports in the literature describing these rare disorders, the simultaneous occurrence of NAIT and NAN has not been systematically evaluated. In this study we report a case of NAIT associated with NAN. Material and Methods: A healthy 46-year-old primiparous woman gave birth to triplets in the 30.9th gestational week, by C-section, after a fertility treatment using an egg donation banking. The first neonate, a 1,600g female newborn with a first/fifth minute Apgar score of 8/9, healthy at physical examination, showed severe thrombocytopenia (platelet count = 30x109/L) and a neutrophil count of 1.6X109/L at birth. The second neonate, a 1,275g male newborn had a first/fifth minute Apgar score of 7/7, was healthy at physical examination and showed thrombocytopenia (platelet count = 82x109/L) and neutropenia (neutrophil count = 0.66x109/L). Intracranial bleeding, perinatal infections, genetic and hematologic disorders were ruled out and the neonates showed a spontaneously normalization of the platelets and neutrophils counts. Unfortunately, blood sample from the third neonate, a 1,525g female newborn with first/fifth minute Apgar score of 9/9, was not available. Mother and neonates were genotyped for platelets antigens (HPA-1-11,-15) and neutrophil antigens (HNA-1,-3). HPA genotyping was performed by PCR-SSP, PCR-RFPL and xMAP technology (IDHPAXT, Progenika-Grifols, Spain). HNA genotyping was performed by PCR-SSP and PCR-RFPL. Platelets antibodies were investigated in maternal serum by platelet enzyme-linked immunosorbent assay (PaK12G, Immucor GTI Diagnostic Inc, USA); neutrophil antibodies were investigated by granulocyte agglutination test (GAT) using a panel of donors previously genotyped for HNA-1,-3 systems, while anti-HLA antibodies were investigated by ELISA (LAT Mixed, One Lambda Inc., USA and PaK12G, Immucor GTI Diagnostic Inc, USA). Results: Genotyping studies revealed mismatches for HPA-1,-5 and HNA-1,-3 systems between mother and neonates (Table1). Serologic antibody screening tests showed presence of anti-HPA-5b, anti-HNA-3b and anti-HLA in maternal serum. TABLE 1.GENOTYPING RESULTSHPA-1HPA-5HNA-1HNA-3MOTHERHPA-1a1aHPA-5a5aHNA-1a1aHNA-3a3aNEONATE 1HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3bNEONATE 2HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3a Conclusion: We described the occurrence of NAIT associated with NAN in preterm neonates with important thrombocytopenia and neutropenia due a multiple maternal sensitization to incompatible fetal antigens. As these syndromes pose a risk to the neonate’s life, the early and correct diagnosis is very important in order to apply appropriate treatment to the neonates and allow an effective prenatal therapy for future pregnancies. Disclosures No relevant conflicts of interest to declare.
3151 Poster Board III-88 Background Loss of red blood cell (RBC) antigens may occur in solid tumors and in hematological diseases; however the mechanisms involved in these changes are not fully understood. Aberrant DNA hypermethylation is thought to be involved in acute myeloid leukemia (AML) as well as in myelodysplastic syndromes (MDS), and the methylation of cytosines residues in the dinucleotide CpG may account for the expression patterns of the ABO genes. In this study we investigated loss of RBC ABH antigens and the possible role of DNA methylation in the ABO promoter gene in patients with myeloid diseases. Methods Forty-three patients were included in our study (MDS=16, chronic myeloid leukemia (CML)=12, AML=4, polycythemia vera (PV)=3, essential thrombocythemia (ET)=3, chronic myelomonocytic leukemia (CMML)=3, myelofibrosis (MF)=1 and chronic neutrophilic leukemia (CNL)=1). Forty-one patients were evaluated according to their ABO group using serological immunohematological tests (Diamed Inc., Brazil). ABH secreted antigens were investigated in saliva and a PCR-based ABO genotyping using restriction enzyme digestion (Alu and Kpn) was performed to confirm the ABO blood type. In addition, the expression of the A, B and H antigens was analyzed by flow cytometry in 26 patients (median age=63 yrs; 6M/17F) and 81 healthy controls (median age=33 yrs; 38M/43F). Methylation of CpG islands was investigated in 45 bone marrow samples using methylation specific PCR (MSP) technique with methylated and unmethylated primer sets for region from -200 to +26 sequence of the ABO gene. Results The investigation of the secreted antigens in saliva and the genotyping studies confirmed the results of the ABO serological tests in 40 patients, but we found that the RBCs of one patient were not agglutinated by anti-B while his genotype result was compatible with BO indicating loss of the B antigen in his RBCs. Overall, loss of A, B or H antigen was detected by flow cytometry in 11/26 (42%) patients. Hypermethylation of the ABO promoter gene was detected in 51% (23/45) of the analyzed bone marrow samples. Among patients with hypermethylation in the ABO promoter gene, 44% had loss of A or B antigens confirmed by serological or flow cytometric tests compared with 28% in the group of patients with unmethylated ABO promoter gene (p=0.632). Conclusions Epigenetic changes including methylation of cytosine residues are recognized as major contributors to gene silencing, disease progression and worse outcome in several cancer patients. Our data showed that the hypermethylation of the ABO gene is frequent in patients with myeloid malignancies corresponding to the pathogenesis already described for AML and MDS. However, not all patients showing loss of RBCs antigens had ABO methylation evidence, suggesting that other mechanisms may take place. Patients with myeloid malignancies often need blood transfusion support and loss of RBCs antigens can lead to changing in ABO blood group increasing the risk of serious blood transfusion reactions. The relatively high rate (42%) of loss of ABH antigens found in this study demonstrated that these patients have to be carefully managed to avoid such severe transfusion reactions. (Grants supported by CNPq, 478814/2006-2). Disclosures No relevant conflicts of interest to declare.
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