Secondary alloanti-D immunization post transfusion of "Asia type" DEL red blood cells.

BACKGROUND: "Asia type" DEL red blood cells (RBCs) express a very weak D antigen and cannot be detected by routine RhD typing. Thus, it is routinely typed as D-negative (D-) blood group and transfused to D- recipients. Here we described a case of secondary alloanti-D immunization that was associated with transfusion of DEL RBCs to D- recipients and was initially considered as primary alloanti-D immunization. CASE PRESENTATION: A 44-year-old D- woman (G2P2) with adenomyosis and anemia underwent transabdominal hysterectomy. She received four units of D- RBCs before operation. Before transfusion, the alloantibody screening test was negative. Four days after the first transfusion, she needed another RBC transfusion. Unexpectedly, the routine pre-transfusion alloantibody screening test became positive and anti-D (titer, 128-fold) was identified, indicating an alloanti-D immunization. The anti-D developed four days after the first transfusion was unexplained, so alloantibody identification was performed on the sample collected before the first transfusion, and weak anti-D combined with anti-E, which was not detectable during the previous routine pre-transfusion alloantibody screening test with non-enzyme-treated screening cells, was identified using bromelain-treated panel cells. The remaining blood samples of first transfusion in bag tails from two donors were collected for RHD genotyping analysis. One donor was later identified as "Asia type" DEL having RHD* 1227 A/01 N.01 genotype. CONCLUSION: Caution should be applied when we conclude that transfusion of "Asia type" DEL RBCs to true D- recipients could induce primary alloanti-D immunization, especially if the short time interval between transfusion and detection of anti-D is observed.

Routine testing of fetal Rhesus D status in Rhesus D negative women using cell-free fetal DNA: an investigation into the preferences and information needs of women.

OBJECTIVE: The goal of this study is to investigate women's preferences and information needs for routine implementation of fetal Rhesus D (RhD) typing using cell-free fetal DNA. METHODS: A questionnaire was developed following focus groups and interviews with both health professionals and RhD negative (RhD-) women offered fetal RhD genotyping within a research study and distributed to RhD- women attending routine antenatal appointments in four National Health Service hospitals. Current knowledge of blood types, anti-D administration, fetal RhD genotyping and future practices were explored. RESULTS: A total of 19 respondents participated in interviews and focus groups, and 270 respondents completed the questionnaires. Questionnaire respondents overwhelmingly felt that the test should be offered to all RhD- women (92.1%), and 75.9% said that they would accept this test. Most were happy to have the test even if it involved extra blood tests (89.3%) or appointments (79%). The knowledge of blood groups was poor. Although 90.7% knew that the baby could have a different blood group from themselves, only 34% knew that blood groups are inherited from both parents. More than 40% were not aware that anti-D would not be required if their baby was RhD-. CONCLUSIONS: Women would welcome the introduction of routine fetal RhD genotyping. Information leaflets and training of midwives will be essential for implementation to ensure good understanding regarding testing.

Anti-D in pregnant women with the RHD(IVS3+1G>A)-associated DEL phenotype.

BACKGROUND: Pregnant women with the DEL phenotype appear to be D- by routine serology. Women with DEL phenotypes that show a partial D-like epitope loss may develop anti-D. It has been proposed that this alloantibody could have a deleterious effect with respect to hemolytic disease in the fetus and newborn. CASE REPORTS: Two pregnant women, one in Australia and one in Germany, were serotyped as D- and were sensitized to the D antigen. Noninvasive fetal RHD genotyping was performed to plan pregnancy management. RESULTS: In both cases the fetal RHD status could not be assigned due to the presence of a maternal DEL allele. This was suspected through detection of high RHD amplicon levels during quantitative polymerase chain reaction. For both cases extended molecular typing of the maternal genomic DNA revealed a RHD(IVS3+1G>A) allele. For case one, the D+ infant developed a mild hemolytic disease requiring phototherapy. In the second case a D- (or DEL) newborn was unaffected. CONCLUSION: Fetal genotyping from maternal plasma reveals RHD variants in pregnant women with anti-D. Fetuses and newborns of sensitized pregnant women carrying the RHD(IVS3+1G>A) allele are at risk of hemolytic disease.

The RHCE allele ceSL: the second example for D antigen expression without D-specific amino acids.

BACKGROUND: The example of ceRT proved that the expression of some D epitopes does not require D-specific amino acids. This allele denoted as RHce(R154T) caused the "false-positive" reactions that were observed in ccddee blood donors who typed positive for the D antigen with some monoclonal anti-D. No other example exposing a similar molecular mechanism was known. STUDY DESIGN AND METHODS: Eleven donor and 1 patient ccddee samples were collected in Switzerland that typed "false-positive" with some monoclonal anti-D in bromelain technique. Their RHCE alleles were determined by nucleotide sequencing from genomic DNA and by a polymerase chain reaction with sequence-specific priming. The D epitope profile was compared to ceRT. The population frequencies were estimated in Switzerland and Germany by serology or at the molecular level, respectively. RESULTS: The "false-positive" reactions were caused by the RHCE allele RHce(S122L) occurring in the cde haplotype. Its ceSL phenotype expressed few D epitopes that belonged to the D epitope 6 group. The frequency of ceSL among D- donors was about 1:675 in the region of Bern, Switzerland. No ceSL donors were found elsewhere in Switzerland or in southwestern Germany. CONCLUSION: ceSL represented the second molecular mechanism for D antigen expression without any D-specific amino acids. ceSL and ceRT were useful to delineate the molecular mechanisms of D expression by RhCE proteins carrying amino acids not representative for the RhD proteins. The ceSL population frequencies differed significantly among three Swiss and German populations.

[Foetomaternal erythrocyte incompatibilities: from immunohaematologic surveillance of pregnant women to haemolytic disease of the newborn]. / Incompatibilités foetomaternelles érythrocytaires (IFME): de la surveillance immunohématologique des femmes enceintes à la maladie hémolytique du nouveau-né (MHNN).

Despite the generalization of prevention measures against foetomaternal alloimmunization with anti-D immunoprophylaxis since 1970s, retrospectively 30 years later, its complications (new-born child's severe haemolytic disease, foetal death by anemia or nuclear icterus by bilirubin encephalopathy) have not disappeared. At the same time, alloimmunizations against antigens other than D increase with no possible prevention. As part of the set up in France of regional files analysing and making an inventory of serious foetomaternal incompatibilities requiring in utero or neonatal transfusion, we felt the need to synthesize current data, biological profiles (early screening of erythrocytic alloimmunization and its follow up during pregnancy, father's immunohaematologic status, evaluation of in utero immune haemolysis and impact of new non invasive techniques of diagnosis-RH1 foetal genotypage from ADN foetal of RH1--mothers' maternal plasma), clinical and paraclinical data (evaluation of foetal haemolysis by echography, recording of foetal movements and foetal cardiac rhythm), therapeutic indicators (in utero foetal transfusions or exsanguinotransfusions, neo and postnatal transfusions or exsanguinotransfusions, induced premature labour, newborn's intensive continue phototherapy and Rhesus immunoprophylaxis) in order to enable medical and paramedical professionals to carry out the specific supervision of pregnancies with foetomaternal incompatibility, the in utero, neo- and postnatal treatment of child and the efficient therapeutic prevention of anti-D alloimmunization, in a cooperative way.

Severe HDN due to anti-Ce that required exchange tranfusion.

BACKGROUND: Rh system antibodies are commonly encountered in blood bank practice as well as during pregnancy. Nevertheless, no examples of anti-Ce (RH7) have been reported as a cause of HDN that requires exchange transfusion. CASE REPORT: A 38-year-old woman in her fourth pregnancy was typed as blood group O D+, C-, c+, E+, e-. Anti-C and anti-e were detected in her serum during a routine prenatal work-up. Further evaluation, including flow cytometric analysis, revealed the presence of a strong anti-Ce and a weak anti-e. Her partner was typed as group A D+, C+, c-, E-, e+. A seemingly healthy male infant was delivered at 40 weeks of gestation. The infant's RBCs were typed as group O D-, C+, c+, E+, e+ with a positive DAT (titer 128). Twenty-five hours after birth, the baby had to be transferred to the neonatal intensive care unit because of rapidly rising total serum bilirubin. Despite intensive treatment, including double phototherapy, albumin infusion, and the administration of furosemide and IVIG, the total serum bilirubin level increased during the following day and exchange transfusion with 2 units of type O D-, C-, c+, E+, e- had to be performed; this resulted in a prompt decrease in total serum bilirubin without relapse. CONCLUSION: Anti-Ce caused severe HDN requiring exchange transfusion. This highlights the need for a close follow-up throughout pregnancy if unexpected RBC antibodies are present, to permit the provision of compatible blood in case of a rare antibody.

Use of a PCR-based assay for fetal Cw antigen genotyping in a patient with a history of moderately severe hemolytic disease of the newborn due to anti-Cw.

Anti-Cw is an uncommon cause of clinically significant hemolytic disease of the newborn (HDN). We report an unusually severe case of HDN due to anti-Cw that required phototherapy and exchange transfusion. We also describe a novel PCR-RFLP method for Cw typing of fetal genomic DNA that was used for prenatal diagnosis in a subsequent pregnancy. Following PCR amplification of a 163 bp segment of the RHCE gene containing the nucleotide 122 G to A substitution that corresponds to the Cw allele, Cw types were distinguished by TaqI digestion. PCR-RFLP analysis confirmed that the father and previously affected child were Cw-positive. The fetus was Cw-negative, thus excluding HDN in the current pregnancy and obviating the need for further invasive or noninvasive diagnostic procedures for the remainder of the pregnancy. This case illustrates the utility of PCR-based fetal genotype determination in pregnancies at risk of HDN due to uncommon red cell antibodies such as anti-Cw.

Rh50 glycoprotein gene and rhnull disease: a silent splice donor is trans to a Gly279-->Glu missense mutation in the conserved transmembrane segment.

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at the RH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G-->A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G-->A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G-->A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy in trans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical from Caenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.

Identification of 5' flanking sequence of RH50 gene and the core region for erythroid-specific expression.

The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes, RCHE and RHD. Rh50 glyco-protein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression of RH50 gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5' flanking region of RH50 gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence of RH genes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter of RH50 gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Krüppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms for RH vs RH50 genes.

Anti-E as a cause of hemolytic disease of the newborn.

A case of HDN caused by anti-E antibody is reported. A group A, E-positive, hemoglobin E trait female infant was born from a group A, E-negative, beta-thalassemia/hemoglobin E mother. Hyperbilirubinemia was noted at the first day of life. The DAT was positive. Anti-E was detected in the maternal serum. Jaundice and anemia occurring to the baby were severe enough to require phototherapy intervention for 9 days and 50 ml of group A, E-negative packed red blood cells was transfused. The baby's condition improved. She was discharged at 12 days of age. Follow-up of the baby at 1 year old showed that she was alive and in good health.

Involvement of Gly96 in the formation of the Rh26 epitope.

BACKGROUND: The Rh system, a complex blood group system, comprises at least 45 antigens. Red cells expressing c usually express Rh26. Rare cells that are c+ Rh:-26 give variable reactions with anti-c and may have weak expression of f (ce). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed with red cells from persons with the c+ Rh:-26 phenotype occurring in two unrelated Dutch families. Red cells of 11 members of these two families were typed for Rh26, for c (with monoclonal and polyclonal reagents), and for f (ce). The cDNA of three donors was sequenced, while restricted DNA analysis was carried out on material from available members of the two families. RESULTS: Serologic tests showed that the rare c+ Rh:-26 phenotype was associated with a weak expression of c and a normal expression of f. The cDNA analysis of three members of one family revealed a single-point mutation (G286A) in exon 2 of the ce allele. Allele-specific primer amplification, polymerase chain reaction followed by allele-specific restriction analysis, and single-strand conformation polymorphism showed the same polymorphism in all other members of both families, whereas it was absent in 80 control donors. CONCLUSION: The c+ Rh:-26 phenotype, identified in two families, is associated with a single-point mutation at nucleotide 286 (G286A) in the ce allele, which predicts a Gly96Ser amino acid substitution. This substitution also affects c, because all anti-c reagents reacted more weakly. Other polymorphic sites apparently are involved in the formation of the Rh26 epitope as well, because Rh26 is expressed only on the c polypeptide, whereas Gly96 is expressed on all polypeptides.

Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization.

OBJECTIVE: To review the specificity and sensitivity of diagnostic techniques using the polymerase chain reaction (PCR) on amniotic fluid (AF) samples for the determination of fetal RhD status. DATA SOURCES: A MEDLINE computerized search was conducted for January 1991 through March 1996 using the key terms "polymerase chain reaction," "rhesus," and "RhD typing." METHODS OF STUDY SELECTION: All articles describing the use of PCR in AF for RhD typing were reviewed. Only cases in which the results of PCR testing were confirmed by fetal or neonatal serology were included in the final analysis. TABULATION, INTEGRATION, AND RESULTS: The results of PCR typing were compared with serology to determine the sensitivity, specificity, and positive and negative predictive values of DNA-based techniques. A total of 500 cases were reviewed, in which four different sets of oligonucleotide primers were used. The sensitivity and specificity of PCR typing were 98.7% and 100%, respectively, and the positive and negative predictive values were 100% and 96.9%, respectively. In five cases, an RhD-positive fetus was incorrectly diagnosed: Two fetuses died, one neonate needed exchange transfusions, and another neonate needed phototherapy in conjunction with a simple transfusion. The remaining infant was lost to follow-up. A theoretical model indicated that amniocentesis with PCR-based techniques for fetal RhD typing would be associated with a fourfold reduction in perinatal loss compared with funipuncture and serology for fetal typing. CONCLUSIONS: This lower rate of procedure-related loss makes RhD typing using AF the preferred method for assessing the fetal Rh status in cases of a heterozygous paternal genotype.

Maternal CW alloimmunization.

The Winnipeg Rh Laboratory has reviewed its experiences with maternal CW alloimmunization. From September 24, 1956, to March 31, 1992, 12 women with significant CW alloimmunization underwent 18 pregnancies. In 3 (4 pregnancies) the antibody, despite its strength, was 'naturally occurring' (i.e. there was no known exposure to CW-positive red cells). The remaining 9 women (14 pregnancies) had CW-positive husbands. Two had CW-negative babies and a third infant, probably CW negative, was stillborn and macerated at 43 weeks gestation. Eleven babies were CW positive and had hemolytic disease of the newborn (HDN), with antiglobulin-positive red cells. Five did not require treatment; 2 needed phototherapy only, and 4 (born between 1956 and 1963) required exchange transfusions. No anti-CW screening was carried out until 1977; thereafter it was sporadic, 11 of 51 screening red cells being CW positive in the 39-month period ending March 31st, 1992. From November 1, 1977, to March 31, 1992, 24 women (30 pregnancies, 31 conceptuses) with insignificant anti-CW alloantibodies were identified. Extrapolating these figures to the entire period from September 24, 1956, to March 31, 1992, we estimate that at least 430 women (at least 573 pregnancies) were CW alloimmunized, most of the antibodies being 'naturally occurring'. Only 2% of the conceptuses were CW positive and affected; none were severely affected. Anti-CW is relatively common, occurring in about 1 pregnant Manitoban woman in 1,100. On very rare occasions (11 times in Manitoba in 36 years and 5 months) anti-CW HDN occurs which, although not severe, may end in kernicterus with brain damage or neonatal death unless it is detected promptly and treated appropriately.

ABO and Rh(D) blood group distribution and their implication for feto-maternal incompatibility among the Palestinian population.

ABO and Rh(D) blood group distribution was evaluated among Palestinian women in the southern area of the West Bank and east Jerusalem. Eleven per cent of women were Rh(D) negative. The review of the last 12,169 deliveries at Makassed Hospital showed that 4.8% of Rh(D)-negative mothers gave birth to Rh(D)-positive infants with haemolytic disease of the newborn. Thirty per cent of A or B infants born to O Rh(D)-positive mothers had a positive direct antiglobulin test with the presence of allo-immune A or B antibody in infant serum. ABO incompatibility was a major reason for phototherapy during the 1st week of life. Results and possibilities for prevention are discussed.

Blood groups as genetic markers in glaucoma.

A series of 474 mixed cases of glaucoma was assessed to determine whether there were any genetic differences between different types of glaucoma. A careful distinction was made between chronic open angle glaucoma (COAG), acute and chronic angle closure glaucoma, ocular hypertension, low tension glaucoma, patients with large cup disc ratios, and various types of secondary glaucoma including pseudoexfoliation of the lens capsule, uveitic and traumatic glaucoma. Using ABO blood groups, Rhesus groups, ABH secretion or non-secretion, and phenylthiourea tasting we identified certain differences. The differences from normal were significant decrease in Rh-negative patients in chronic closed angle glaucoma (p less than 0.05), a decrease in ABH secretors in ocular hypertension (p less than 0.01), and fewer HB secretors in patients with COAG (p less than 0.02). There was a significant decrease in AH secretors and increase in HB secretors in both pseudoexfoliation with raised intraocular pressure compared with COAG (p less than 0.01) and in secondary glaucomas as a group compared with COAG (p less than 0.01). Tasters of phenylthiourea were more common in traumatic and uveitic glaucoma than in normal controls (p less than 0.05). These results suggest that secondary glaucoma develops in different subjects from COAG, while patients who develop a rise in intraocular pressure proceed to cupping and field loss if they have a certain genetic constitution. The groups of patients are too small for the differences to be of great prognostic value.

[Indicators of risk of reagin allergy. IV. HLA markers]. / Les indicateurs de risque de l'allergie réaginique. IV. Marqueurs de risque HLA.

The concept of a genetic score was described in a previous publication by our group. It indicated that patient groups separated by this score may help to identify a possible genetic factor of allergic risk. In the present study, 1,800 children under the age of 13 were reviewed. Groups were classified by genetic score. The determination of loci A and B of HLA system was performed in 93 allergic children and revealed a highly significant correlation between HLA B12 and genetic score. On the other hand, the frequency of HLA A2 decreased with age. This fact can be interpreted as a prognostic indicator of allergic disease in children.