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We have set out to determine the frequency ofDIIIaandDARalleles among sickle cell disease (SCD) patients. These D variants permit the unexpected development of antibodies to RhD among individuals who are otherwise classified as RhD+. DNA samples from 130 SCD patients were tested for 455A>C (specific for DIIIa), 602C>G, 667T>G (common for both DIIIa and DAR) and 1025T>C (specific for DAR) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence analysis. The PCR-RFLP showed that 12 (9·2%) of the SCD patients were carryingDIIIaandDARalleles. Genomic DNA analysis performed by sequence showed that three samples were heterozygous DIIIa (2·3%), seven heterozygous DAR (4·6%) and two (1·5%) samples carried a partial D with four mutations: 455A>C (heterozygous), 602C>G and 667T>G (homozygous) and 1025T>C (heterozygous), indicating compound heterozygosity for oneDIIIaallele and oneDARallele. The predicted phenotypes of eight (6·2%) SCD patients were DIIIa, DAR and DIIIa/DAR. Three patients were anti-D immunized (DAR,n = 1; DIIIa/DAR,n = 2). These findings suggest that SCD patients who are candidates for chronic transfusion may benefit from genotyping forDIIIaandDARto prevent alloimmunization. [ABSTRACT FROM AUTHOR]

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) byRalstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g-1 medium on soy cake in 36 h, and 0.7 mg g-1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g-1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost. [ABSTRACT FROM AUTHOR]

The GATA box single nucleotide polymorphism (SNP) at position -33 (T>C) in Blacks silences the expression of FY*B in erythrocytes, and the substitution 265 C>T, together with 298 G>A, weakens the Fyb antigen (Fyx). Individuals with these phenotypes/genotypes who receive Fy(b+) blood are unlikely to be alloimmunized to Fyb because, in the presence of 265 T, the Fyb antigen is expressed, and in the case of -33 c, other tissues express Duffy protein and probably the Fyb antigen. We studied samples from 361 blood donors (182 of African ancestry and 179 of Caucasian ancestry) by haemagglutination and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). Forty Caucasian and 130 donors of African ancestry were serologically Fy(b-); among these, the majority of the donors of African ancestry had FY*B with the GATA SNP, while the majority of Caucasians typing Fy(b-) had FY*B with 265 T/298 A SNPs. Six of the Fy(b-) donors (three Africans and three Caucasians) had both GATA and 265/298 SNPs, and six donors of Caucasian ancestry apparently had a GATA SNP. Samples from two donors — one African and one Caucasian with an unusual MspA 1I-RFLP pattern — were sequenced and found to have a novel SNP (145 G>T) co-existent with 265 C>T and 298 G>A SNPs. These findings highlight the importance of establishing the incidence and nature of molecular events that impact on Duffy expression in different populations. [ABSTRACT FROM AUTHOR]

The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCE Ce, while Trp16 is associated with RHCE ce. The presence of Cys16 in RHCE ce is associated with the R0 (Dce) haplotype in Africans, leading to a weak ‘e’ antigen expression on red blood cells (RBCs). VS is a common red cell antigen in individuals of African descent and results from a single point mutation in exon 5 of the RHCE (733C>G), leading to Leu245Val substitution; VS positivity is also associated with weak expression of ‘e’. This study investigated the association of Cys16 and/or VS with the RHCE ce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R0r or R0R0 and rr. DNA samples from 58 SCD patients were tested for the 48 G>C transversion, encoding Cys16, by allele-specific polymerase chain reaction (PCR). We also amplified exon 5 of the RHCE by PCR and subjected the amplified product to restriction fragment length polymorphism analysis, using BfaI, in order to determine the VS status. Further cDNA analysis was performed on three samples to verify whether the mutations were located on the same or on different alleles. Fifty-six of the 58 SCD patients studied (97%) were heterozygous for 48G/48C (Cys16). Of these, 18 (32%) were also heterozygous for 733C/G (245Val). All of these 18 samples showed weak ‘e’ expression on RBCs when tested with at least one monoclonal antibody to e antigen. cDNA sequencing of three of 18 patient samples showed that the genes encoding Cys16 and Val245 (VS) were on different alleles. We found a high incidence of Cys16 associated with the RHCE ce in our SCD cohort. A high percentage of these patients were also found to be heterozygous for VS. cDNA analysis showed that, in at least three samples, the two mutations were on different alleles, with consequent weakening of expression of the e antigen on RBCs. [ABSTRACT FROM AUTHOR]

Background and Objectives The blood-group antigens Dia and Dib are carried on erythrocyte band 3 and are defined by a single amino acid substitution at position 854 (Leu for Dia and Pro for Dib ). The Band 3-Memphis variant has a point mutation (166A>G) in the SLC4A1 gene, which encodes the amino acid substitution Lys56Glu. Two types of Band 3-Memphis, variants I and II, are distinguished by their susceptibility to covalent labelling with 4,4′-diisothiocyanato-1,2-diphenylethane-2,2′-disulphonic acid (H2 DIDS). Memphis II is more readily labelled than Memphis I or normal band 3. It is reported that Memphis II is associated with Dia . In a study designed to determine the frequency of the DI *A /DI *B and 166A>G polymorphisms in different populations in Brazil, we found a new DI *A allele. Materials and Methods We studied DNA samples from 70 Amazonian Indians, 71 individuals of Japanese descent, 93 random Brazilian blood donors and 84 blacks with sickle cell disease. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses were performed on all samples, using Msp I for DI *A/DI *B (exon 19) and Mnl I for 166A>G (exon 4). Exon 4 and exon 19 from four outliers were sequenced. Results Among Amazonian Indians, DI *A and 166G mutations both had a high frequency (0·57 and 0·54, respectively). In individuals of Japanese descent, these alleles were moderately frequent (0·07 and 0·19, respectively). We identified a new allele with DI *A and 166A (56Lys) in four Amazonian Indians. Conclusions Our results revealed that DI *A does not have a strict association with 166G . They also show the relevance of testing a cohort of different populations. [ABSTRACT FROM AUTHOR]

BACKGROUND: The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rhnull amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rhnull who were born to parents who were first cousins. STUDY DESIGN AND METHODS: RBCs from the Rhnull sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. RESULTS: Rhnull RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG→ GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. CONCLUSION: The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rhnull with loss of Rh antigen expression is described. [ABSTRACT FROM AUTHOR]

Background: The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCe allele, while Trp16 is associated with the RHce allele. The presence of Cys16 in RHce allele has been associated with the R0 (Dce) haplotype in Africans leading to a weak "e" antigen expression on the RBCs. VS is a common red cell antigen in individuals of African descent. It results from a single point mutation in exon 5 of the RHCE gene (733 C>G leading to the Leu245Val substitution.) VS-positivity is also associated with weak expression of e. This study investigated the association of Cys16 and/or VS with the RHce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R0r or rr. Methods: DNA samples from 58 SCD patients were tested for the 48 G>C transversion encoding for Cys16 by allele-specific PCR (Westhoff CM et al, Blood, 1999.) We also amplified exon 5 of the RHCE by PCR, and subjected the amplified product to restriction fragment length polymorphism analysis using Bfa I in order to determine VS status. This enzyme recognizes G at position 733 encoding for VS-positive (Val245), and differentiates it from the VS-negative (Leu245). Results: Fifty-six of the 58 SCD patients studied (97%) had Cys16. Of these, 50 (89%) were also heterozygous for 245Val (VS). All 50 samples showed weak "e" expression on RBCs when tested with at least one monoclonal anti-e. Conclusion: We found a high incidence of Cys16 associated with the RHce allele in our SCD cohort. Interestingly, we observed that a high percentage of these patients also were heterozygous for VS. Since the Cys16 assay does not determine zygosity, it is likely that each allele carries one of the mutations with consequent weakening of expression of the e antigen on RBCs. [ABSTRACT FROM AUTHOR]

Background: Blood group genotyping can overcome limitations of hemagglutination. However, its use has been complicated by precarious conditions of storage and transportation of samples containing DNA (e.g. blood and amniotic fluid) with consequent typing failures due to poor preservation of DNA templates. We investigated whether DNA prepared from blood, plasma and amniotic fluid spotted on a treated filter paper (gene card) were suitable for blood group genotyping after long-term storage and transportation at ambient temperature. Methods: Blood samples from 130 Brazilians: 50 patients from the Amazonian region, 30 from Northeastern and 50 from Southeastern were spotted on gene card soon after collection, stored for 10 weeks and mailed to the genotyping laboratory located over 2,500 Km away. These samples had been previously phenotyped by hemagglutination. DNA prepared with the gene card purification reagent was tested by PCRRFLP for RHC/c, RHE/e, K1/K2, JKA/JKB and, FYA/FYB-/GATA. DNA from 20 maternal plasmas and 20 amniotic fluids collected locally were prepared in the same way, stored for at 10 weeks at ambient temperature, and tested for RHD/non-D by analysis of the PCR product size associated with the RHD gene sequence in intron 4 and exon 10/3'UTR. Results: There was complete agreement between PCR-RFLP results obtained with DNA from gene card and those obtained by hemagglutination. RHD genotype results obtained with DNA from maternal plasma and amniotic fluid also agreed completely with hemagglutination results. Conclusion: DNA prepared from blood, plasma and amniotic fluid spotted onto gene card and subjected to long-term storage and travel at ambient temperatures are suited for blood group genotyping. Gene card is a practical and reliable way for genotyping studies of remotely located patient populations. [ABSTRACT FROM AUTHOR]

Background: Alloimmunization to multiple red blood cell (RBC) antigens is a common finding among patients with sickle cell disease (SCD). Antibodies to Dombrock (anti-Do[sup a] or anti-Do[sup b]) are of clinical significance in SCD, and difficult to identify, because they are usually present in serum containing other antibodies, and reliable typing reagents are not available. The Dombrock gene was sequenced and the polymorphism for Do[sup a]/Do[sup b] elucidated, and it can be determined by PCR-RFLP (Rios et al, Transfusion 2001). We employed this assay to determine the DO A/DO B type in patients with SCD and used it as tool for further serological investigations. Methods: DNA was prepared and tested for Dombrock by PCR-RFLP analysis (Rios et al, Transfusion 2001; 40). Results: Fourteen (24%) of the 58 SCD patients studied were typed as DO A/DO A, 20 (34%) as DO B/DO B and 24 (41%) as DO A/DO B. One of these patients typed as DO A/DO A and had clinical evidence of shortened red cell survival. He had anti-C, -E, -K, -S and -Fy[sup a] and he was receiving RBC products that lacked the corresponding antigens. His genotype was DO A/DO A. His serum contained anti-Do[sup b], as demonstrated by absorption-elution tests. Following genotyping, this patient received RBCs that screened negative for the Do[sup b] antigen with better in vivo survival, as documented by expected raises in hemoglobin levels after transfusion, and diminished frequency of transfusions. Conclusion: Genotyping for DO A/DO B in SCD patients who received multiple transfusions is an aid to the antibody identification process. Genotyping is also helpful for the selection antigen-negative donor RBCs for transfusion. [ABSTRACT FROM AUTHOR]

Background: The molecular basis for the RHD pseudogene or RHDY is a 37 bp insertion in the exon 4 of RHD. This insertion, found in two-thirds of D-negative Africans (Singleton et al, Blood, 2000) appears to introduce a stop codon at position 210. The hybrid RHD-CE-D[sup s], in which the 3'end of exon 3 plus exons 4 to 8 are derived from RHCE is associated with the VS+Vphenotype, and leads to a D-negative phenotype in people of African origin. We investigated whether Brazilian blood donors of heterogeneous ethnic origin had RHDY and RHD-CE-D[sup s]. Methods: DNA samples from 206 blood donors (50 of Caucasian descent, 66 of African descent and 90 Mulattos, i.e. admixture between descendants of Europeans, Africans and Native Americans) were tested for RHDY by a multiplex-PCR that detects RHD, RHDY and the C and c alleles of RHCE (Singleton et al, Blood, 2000). The RHD genotype was determined by comparison of size of amplified products associated with the RHD gene in both intron 4 and exon 10/3'UTR obtained by two different multiplex PCRs. VS was determined by amplification of exon 5 of RHCE, and restriction fragment length polymorphism analysis using Bfa I. This enzyme recognizes a G at position 733 encoding for VS-positive (Val245) and differentiates it from the VS-negative (Leu245) that has C at position 733. Results: Twenty-two (11%) of the 206 D-negative Brazilians studied had the RHDY, 5 (2%) had the RHD-CE-D[sup s] hybrid gene associated with the VS+Vphenotype, and 179 (87%, 50 of Caucasian descent, 49 of African descent and 80 Mulattos) lacked entirely RHD. Conclusion: RHD was deleted in all individuals of Caucasian descent, as expected. Among the 156 individuals of African descent and Mulattos, 17% had altered RHD. Of these, 22 (14%) had inactive RHD and 3% had the RHD-CE-D[sup s] hybrid gene, characterized by the (C)ce[sup s] haplotype that produces c, VS and abnormal C and e, but not D. These data confirm the need to perform the multiplex-PCR in fetuses of all D-negative pregnant women with D-negative phenotype. In addition, the 11% prevalence of RHDY suggests a high degree admixture of individuals of African descent in this population. [ABSTRACT FROM AUTHOR]

The article reports that cloning of blood group genes and subsequent elucidation of the molecular backgrounds to blood group polymorphisms have made it possible to predict blood group phenotypes from the DNA of a patient or donor, when a suitable red cell sample is not available. It mentions that purpose of Vox Sanguinis International Forum is to find out where fetal blood grouping is being carried out.

SUMMARY Background Red blood cell (RBC) alloimmunisation is an event that may occur due to factors such as numerous blood transfusions, age, gender and genetic factors such as human leukocyte antigen (HLA). Aims/Objectives The aim of the present study was to investigate the possibility of alloimmunisation to red blood cell group antigens associated with the HLA of individuals and to relate alloimmunisation to risk factors. Methods A total of 172 polytransfused patients with sickle cell anaemia (SCA) (44 alloimmunised, 128 non-alloimmunised) participated in this study. Blood group genotyping was performed by the DNA microarray method and HLA genotyping by polymerase chain reaction - specific sequence of oligonucleotides. Results The number of transfusions received directly influenced the incidence of alloimmunisation, and the most common alloantibodies were against Rh (48·8%) and Kell (17%) systems. The HLA-C*06 and HLA-DQB1*03 variants were significantly higher in alloimmunised patients. The HLA-DRB1*04 and HLA-DRB1*11 were more often found in individuals who developed the alloantibodies anti-Fya and anti-K, respectively. Conclusion This study suggests that polytransfused patients with SCA possessing the HLA-DQB1*03 and HLA-C*06 allele variants are more susceptible to alloimmunisation. In addition, HLA-DRB1*04 and HLA-DRB1*11 alleles were seen to be associated with the production of anti-Fya and anti-K antibodies, respectively. [ABSTRACT FROM AUTHOR]