Flow Cytometry in Detection of Fetal Red Blood Cells and Maternal F Cells to Identify Fetomaternal Hemorrhage.

Accurate detection and quantitation of fetomaternal hemorrhage (FMH) is critical to the obstetric management of rhesus D alloimmunization in Rh-negative pregnant women. The flow cytometry is based on the detection of fetal red blood cells using a monoclonal anti-HbF antibody, and is the method most indicated for this estimation. The objective of this study was to quantify fetal red blood cell levels of pregnant women using flow cytometry. We analyzed 101 peripheral blood samples from Rh-negative and Rh-positive women, whose mean age was 24 years (20-32 years), after vaginal delivery or cesarean section. Our study showed that 53% of pregnant women had fetal red blood cells levels <2.0 mL, 31% between 2.0-3.9 mL, 16% between 4.0-15.0 mL, and 1% >15.0 mL. Accurate quantitation of fetal red blood cells is necessary to determine the appropriate dose of anti-D (RHD) immunoglobulin to be administered to pregnant or postpartum women.

[From the discovery of microbial Mep-Amt ammonium transporters to human Rhesus factors]. / De la découverte des transporteurs d'ammonium Mep-Amt microbiens aux facteurs Rhésus humains.

Ammonium, ubiquitous on Earth, plays major and distinct roles in most organisms. While it can be a nitrogen source for many microorganisms and plants, it is a cytotoxic metabolic product actively detoxified by the liver in animals. Furthermore, in the latter, ammonium synthesis in the kidney is involved in acid/base homeostasis. Ammonium transport is ensured by a family of proteins, called Mep-Amt-Rh. This family is conserved in all domains of life and comprises the human Rh factors, notably known in transfusional medicine. While the study of bacterial, fungal and vegetal Mep-Amt transporters reveals a fine-tuned and rapid regulation of these proteins in function of environmental changes, the regulation of animal Rh proteins has been poorly addressed. This review notably highlights the importance of the yeast model in the study of the regulation of these proteins as well as in the functional characterization of Mep-Amt-Rh members of diverse origins.

Extensive functional analyses of RHD splice site variants: Insights into the potential role of splicing in the physiology of Rh.

BACKGROUND: Among more than 300 mutated alleles identified so far within the RHD gene, almost 40 are assumed to alter cellular splicing and therefore may have a direct effect on Rh phenotype both at the quantitative and at the qualitative levels. Functional data are, however, mostly unavailable to assess the direct involvement of splicing defect in the underlying physiology. STUDY DESIGN AND METHODS: We generated plasmid constructs to carry out an exhaustive investigation of 38 RHD variants located within or in the vicinity of exon-intron junctions by a minigene splicing assay, further characterized the transcript structures by sequencing, and identified cryptic sites activated by the genetic defect. Bioinformatics predictions were carried out in parallel and compared with the functional data. RESULTS: For the first time we demonstrate that a product including the full-length Exon 9 is transcribed in the presence of the c.1227G>A substitution frequently carried by Asians with DEL phenotype and confirmed that splicing is altered in the RHD*weak D Type 2 allele, a rare variant most commonly found in Caucasians. CONCLUSION: Overall we 1) show significant correlation between functional analyses, bioinformatics predictions, and phenotypes, when available, especially for variants in close proximity of the consensus splice sites; 2) classify the variations as splicing or nonsplicing variants; and 3) provide functional data to further improve bioinformatics splicing tools. Conversely assessment of seven silent exonic variants was mainly inconclusive.

ABO and RhD blood groups and gestational hypertensive disorders: a population-based cohort study.

OBJECTIVE: To examine the association between ABO and RhD blood groups and gestational hypertensive disorders in a large population-based cohort. DESIGN: Cohort study. Risks of gestational hypertensive disorders, pre-eclampsia, and severe pre-eclampsia, estimated by odds ratios for maternal ABO blood group and RhD status. SETTING: National health registers of Sweden. POPULATION: All singleton deliveries in Sweden born to first-time mothers during the period 1987-2002 [total n = 641 926; any gestational hypertensive disorders, n = 39 011 (6.1%); pre-eclampsia cases, n = 29 337 (4.6%); severe pre-eclampsia cases, n = 8477 (1.3%)]. METHODS: Using blood group O as a reference, odds ratios of gestational hypertensive disorders, pre-eclampsia, and severe pre-eclampsia were obtained from logistic regression models adjusted for potential confounding factors. MAIN OUTCOME MEASURES: Gestational hypertensive disorders, pre-eclampsia, and severe pre-eclampsia. RESULTS: Compared with blood group O, all non-O blood groups had modest but statistically significantly higher odds of pre-eclampsia. Blood group AB had the highest risk for pre-eclampsia (OR = 1.10, 95% CI 1.04-1.16) and severe pre-eclampsia (OR = 1.18, 95% CI 1.07-1.30). RhD-positive mothers had a small increased risk for pre-eclampsia (OR = 1.07, 95% CI 1.03-1.10). CONCLUSIONS: In the largest study on this topic to date, women with AB blood group have the highest risks of gestational hypertensive disorders, pre-eclampsia, and severe pre-eclampsia, whereas women with O blood group have the lowest risks of developing these disorders. Although the magnitude of increased risk is small, this finding may help improve our understanding of the etiology of pre-eclampsia.

Diagnostic laboratory technologies for the fetus and neonate with isoimmunization.

Maternal-fetal blood group incompatibility is common but less commonly results in hemolytic disease of the fetus and newborn (HDFN). HDFN is associated with greater peak bilirubin, at an earlier age, and for longer duration than other causes of hyperbilirubinemia. It poses a substantial risk for kernicterus and accounts for the majority of exchange transfusions for hyperbilirubinemia. Advances in diagnosis and management are described, from identification of the alloimmunized pregnancy by maternal ABO and Rh typing, antibody screen (indirect Coombs test), identification and titration; laboratory evaluation of the maternal-fetal unit with a critical maternal antibody titer to prompt fetal antigen status determination; assessment of fetomaternal hemorrhage by conventional Kleihauer-Betke testing or by flow cytometric methodology; to antenatal management of isoimmunization and fetal status assessments using the systems of Liley, Queenan, and serial Doppler fetal middle cerebral artery peak velocity measurements. The utility of laboratory diagnostics in the approach to hemolysis in the neonate, including hematology, chemistry, and peripheral blood smear review, is reviewed. The goal of management, to deliver a healthy infant at or near term, is attained for the majority of cases using current modalities; future directions include noninvasive genotyping of fetal blood from maternal serum to fully eliminate RhD alloimmunization and HDFN; and development of prophylaxis and intervention strategies for non-RhD alloimmunizations for which immune globulin is currently unavailable.

Molecular genetics and clinical applications for RH.

Rhesus is the clinically most important protein-based blood group system. It represents the largest number of antigens and the most complex genetics of the 30 known blood group systems. The RHD and RHCE genes are strongly homologous. Some genetic complexity is explained by their close chromosomal proximity and unusual orientation, with their tail ends facing each other. The antigens are expressed by the RhD and the RhCE proteins. Rhesus exemplifies the correlation of genotype and phenotype, facilitating the understanding of general genetic mechanisms. For clinical purposes, genetic diagnostics of Rhesus antigens will improve the cost-effective development of transfusion medicine.

Substrate specificity of Rhbg: ammonium and methyl ammonium transport.

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

Generation and characterisation of Rhd and Rhag null mice.

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

The effect of Rhesus status on first-trimester pregnancy screening markers free beta human chorionic gonadotropin, pregnancy-associated plasma protein-A and nuchal translucency.

OBJECTIVE: To examine whether maternal Rhesus status has any effect on the levels of first-trimester markers free beta-human chorionic gonadotropin (beta-hCG), pregnancy-associated plasma protein-A (PAPP-A) and nuchal translucency (NT). METHODS: First-trimester free-beta-hCG and PAPP-A levels from pregnant women attending three hospitals in Kent were converted into MoMs and corrected for maternal weight, ethnicity, and smoking status. Maternal Rhesus status data were merged with screening data and free-beta-hCG and PAPP-A medians multiples of medians (MoMs) and NT from the Rhesus positive and Rhesus negative group were compared. RESULTS: Totally, 15 045 normal, singleton pregnancies were retrieved with full records. Altogether, 16.0% of the population were Rhesus negative. There was no difference between the medians MoMs of both free-beta-hCG nor PAPP-A nor NT in the two Rhesus status groups (p > 0.05). Demographic analysis on the distribution of Rhesus status in different ethnic origins showed that Caucasians have lower percentages of RhD-positive antigen compared to Asians and Afro-Caribbeans. CONCLUSION: Maternal Rhesus status does not influence the levels of free-beta-hCG and PAPP-A in the first trimester of pregnancy in this almost exclusively Caucasian population studied; therefore correction for maternal Rhesus status is not suggested.

Neurodevelopmental disorders, maternal Rh-negativity, and Rho(D) immune globulins: a multi-center assessment.

BACKGROUND: Many formulations of Thimerosal (49.55% mercury by weight)-containing Rho(D) immune globulins (TCRs) were routinely administered to Rh-negative mothers in the US prior to 2002. OBJECTIVES: It was hypothesized: (1) if prenatal Rho(D)-immune globulin preparation exposure was a risk factor for neurodevelopmental disorders (NDs) then more children with NDs would have Rh-negative mothers compared to controls; and (2) if Thimerosal in the Rho(D)-immune globulin preparations was the ingredient associated with NDs, following the removal of Thimerosal from all manufactured Rho(D)-immune globulin preparations from 2002 in the US the frequency of maternal Rh-negativity among children with NDs should be similar to control populations. METHODS: Maternal Rh-negativity was assessed at two sites (Clinic A-Lynchburg, VA; Clinic B-Rockville and Baltimore, MD) among 298 Caucasian children with NDs and known Rh-status. As controls, maternal Rh-negativity frequency was determined from 124 Caucasian children (born 1987-2001) without NDs at Clinic A, and the Rh-negativity frequency was determined from 1,021 Caucasian pregnant mothers that presented for prenatal genetic care at Clinic B (1980-1989). Additionally, 22 Caucasian patients with NDs born from 2002 onwards (Clinics A and B) were assessed for maternal Rh-negativity. RESULTS: There were significant and comparable increases in maternal Rh-negativity among children with NDs (Clinic: A=24.2%), autism spectrum disorders (Clinic: A=28.3%, B=25.3%), and attention-deficit-disorder/attention-deficit-hyperactivity-disorder (Clinic: A=26.3%) observed at both clinics in comparison to both control groups (Clinic: A=12.1%, B=13.9%) employed. Children with NDs born post-2001 had a maternal Rh-negativity frequency (13.6%) similar to controls. CONCLUSION: This study associates TCR exposure with some NDs in children.

Molecular biology of Rh proteins and relevance to molecular medicine.

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

Rh proteins vs Amt proteins: an organismal and phylogenetic perspective on CO2 and NH3 gas channels.

Rh (Rhesus) proteins are homologues of ammonium transport (Amt) proteins. Physiological and structural evidence shows that Amt proteins are gas channels for NH(3), but the substrate of Rh proteins, be it CO2 as shown in green alga, or NH3/NH4+ as shown in mammalian cells, remains disputed. We assembled a large dataset generated of Rh and Amt to explore how Rh originated from and evolved independently of Amt relatives. Analysis of this rich data implies that Rh was split from Amt first to emerge in archaeal species. The Rh ancestor underwent divergence and duplication along speciation, leading to neofunctionalization and subfunctionalization of the Rh family. The characteristic organismal distribution of Rh vs. Amt reflects their early separation and subsequent independent evolution: they coexist in microbes and invertebrates but do not in fungi, vascular plants or vertebrates. Rh gene-duplication was prominent in vertebrates: while epithelial RhBG/RhCG displayed strong purifying selection, erythroid Rh30 and RhAG experienced different episodes of positive selection in each of which adaptive evolution occurred at certain time points and in a few codon sites. Mammalian Rh30 and RhAG were subject to particularly strong positive selection in some codon sites in the lineage from rodents to human. The grounds of this adaptive evolution may be driven by the necessity to increase the surface/volume ratio of biconcave erythrocytes for facilitative gas diffusion. Altogether, these results are consistent with Rh proteins not being the orthologue of Amt proteins but having gained the function for CO2/HCO3- transport, with important roles in systemic pH regulation.

Structural and functional insights into the AmtB/Mep/Rh protein family.

X-ray crystallography revealed a similar architecture of the ammonium transport protein AmtB from Escherichia coli and the homologous protein Amt-1 from Archaeoglobus fulgidus. Furthermore, the atomic structures suggest that the proteins conduct ammonia (NH3) rather than ammonium ions (NH4+). These findings indicate that the more than 350 members of the ammonium transporter/methylamine permease/Rhesus (Amt/Mep/Rh) protein family found in archaea, bacteria, fungi, plants and animals are ammonia-conducting channels rather than ammonium ion transporters. The essential part of these proteins is the narrow hydrophobic ammonia-conducting pore with two highly conserved histidine residues located in the middle of the pore. A specific ammonium ion binding site is found at the extracellular entry site of E. coli AmtB. E. coli AmtB and its regulator GlnK form an effective ammonium sensory system that couples intracellular gene regulation by the nitrogen control system to external changes in ammonium availability. Based on structural and functional analysis of various mutants, two conserved histidine residues were found to be essential for substrate conductance also in the functional eukaryotic ammonium transporters. The next big challenge in the field surely is to determine the atomic structure of Rh proteins.

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins.

The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.

Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.

LW protein: a promiscuous integrin receptor activated by adrenergic signaling.

The LW blood group antigen glycoprotein, although part of the Rh macromolecular complex, is nonetheless a member of the intercellular adhesion molecule (ICAM) family. Thus, while it is only rarely clinically important in the setting of transfusion and pregnancy, LW is likely to contribute to red cell adhesion in a variety of settings, including during hematopoiesis, as well as in vascular disorders. The best documentation of a pathophysiological role for LW in human disease is in sickle cell disease, where it contributes to red cell adhesion to endothelial cells and the development of vaso-occlusion, the hallmark of that disease. LW may also contribute to other intravascular processes, such as both venous and arterial thrombosis, due to its ability to interact with both activated platelets as well as leukocytes. The evidence that LW itself can undergo activation on red cells holds promise that pharmacotherapeutic maneuvers may be found to prevent such pathophysiologic interactions.