Search

Despite the generalization of immunoprophylaxis by anti-RH immunoglobulins over 40 years, fetomaternal incompatibility due to RH1 antigen (RhD) is not completely eradicated, although perinatal consequences might be extremely serious. Additionally, allo-immunizations against other antigens, especially anti-RH4 (anti-c) and anti-KEL1 (anti-Kell), may cause severe haemolytic disease. Follow-up of allo-immunization during pregnancy and its prevention are therefore still a concern for all pregnant women. Immunohaematological tests used in antenatal patients are under practice for a long time. However, despite significant progress, it is clear that these tests provide only an indirect indication and will only help the obstetrician, in conjunction with over fetal parameters, to assess the severity of the haemolytic disease. Since almost two decades, fetal RHD genotyping became a reality, first using amniocytes, but more recently by analyzing fetal DNA present in the maternal plasma. RH prophylaxis concerns RH:-1 women, who are non-sensitized against RH1 antigen during and at the end of their pregnancy with a RH1 child. RH prophylaxis includes targeted prophylaxis after foetomaternal haemorrhage and now routine antenatal RH prophylaxis at 28 gestation weeks. Indications for RH prophylaxis and immunohaematological testing to assure an efficient therapeutic prevention have been summarized in France through specific recommendations of the National College of Gynecologists and Obstetricians.

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Context: Rh(D)-negative women with a large fetomaternal hemorrhage (FMH) from an Rh(D)-positive fetus are at risk for anti-D alloimmunization if they do not receive adequate Rh immune globulin (RhIG). Determination of the adequate RhIG dose for these women is a critical laboratory procedure for protecting their future Rh(D)-positive children., Objective: To determine how often laboratories recommended an inaccurate dose of RhIG for excess FMH., Design: Nearly 1600 laboratories using the College of American Pathologists' proficiency testing for fetal red blood cell detection were surveyed to determine (1) their calculation method and (2) the number of RhIG doses recommended for a survey specimen, based on their measured percentage of fetal red blood cells. We surveyed nearly 1450 laboratories for their accuracy in determining RhIG dose, using 2 common calculation methods we provided., Results: The AABB Technical Manual method was used by 67% of responding laboratories. However, 20.7% of laboratories using this method would have recommended an inaccurate dose of RhIG--11.5% too much and 9.2% too little--for the level of FMH reported in the survey specimen. If all laboratories had used the common recommendation of 300 microg/30 mL of fetal blood present, 2% would have recommended RhIG doses too low for the volume of FMH they measured. In 3 of the 4 calculation exercises we provided, 20% to 30% of laboratories underestimated the necessary dose of RhIG., Conclusions: Based on our surveys, some mothers with excess FMH may be receiving inaccurate doses of RhIG. Laboratories performing quantification of FMH should review their procedures and training for calculating RhIG dosage.

Background: The successful prevention of RhD disease has brought attention to other red blood cells' antigens causing alloimmunisation including RhC/c and RhE/e. Prenatal diagnosis of fetal Rh genotype from maternal blood is in clinical use in Europe but not in the USA., Objective: To estimate the collective reported diagnostic accuracy of fetal RhCE genotyping from peripheral maternal blood and compare the results of genotyping when fetal cells and free fetal DNA (FfDNA) are used., Search Strategy: English-written literature describing fetal RhCE determination from maternal blood using fetal cells or FfDNA was performed using medical subject headings and text words. The sources included Pubmed (1966-2007), Ovid (1966-2007), CINAHL, The Cochrane Library, ACP Journal Club and OCLC. Key words were prenatal diagnosis, fetal RhCE, fetal DNA in maternal blood and alloimmunisation., Selection Criteria: A study was considered eligible if it described fetal RhCE type determination using maternal peripheral blood reported in the English literature. Abstracts were excluded., Data Collection and Analysis: From each study, we determined the number of samples tested, fetal RhCE genotype, the source of the fetal DNA, gestational age, presence of alloimmunisation and confirmation of fetal RhCE type. Exclusions and inclusions were noted. We calculated composite estimates of accuracy using a weighted random effects model. We assessed the papers against an international quality, STARD checklist which is standards for reporting studies of diagnostic accuracy., Main Results: We identified 20 protocols in six English-written publications reporting fetal RhC/c (seven protocols) and/or E/e (13 protocols) genotyping using DNA obtained from maternal blood for a total of 369 samples. For RhC/c, 176 samples were tested and for RhE/e, 193 samples were tested. Accuracy was determined for each study and for all studies. The combined accuracy of fetal genotype was 96.3% for RhC/c and 98.2% for RhE/e. Only a few samples of the sorted cells were found to be a source for accurate diagnosis, but plasma was consistently the best source of fetal RhCE genotyping in 147/147 (100%) for RhC/c and 168/168 (100%) for RhE/e., Conclusions: The combined accuracy of noninvasive fetal RhC/c or RhE/e determination using maternal peripheral blood is 96.3% and 98.2%, respectively. FfDNA in maternal plasma is a better source for genotyping reported to be 100% correct for both RHCE genotypes. Further studies and reports of accuracy from laboratories performing the tests are required before prenatal determination of fetal RhC/c or RhE/e genotypes from maternal blood can safely replace the current methods used in the management of the RhC/c or RhE alloimmunised pregnancies.

The last decade has witnessed an abundance of information detailing the genetic diversity of the RH locus which has exceeded all estimates predicted by serology. Well over 120 RHD and over 60 different RHCE alleles have been documented, and new alleles are still being discovered. For clinical transfusion medicine, RH genetic testing can now be used to determine RHD zygosity, resolve D antigen status, and detect altered RHD and RHCE genes in individuals at risk for producing antibodies to high-incidence Rh antigens, particularly patients with sickle cell disease (SCD).

Background: Aim of this study was to establish the method yielding the highest sensitivity routinely used to determine fetal RhD type and gender from maternal cell-free plasma DNA in different periods of gestation., Methods: Plasma DNA concentrations were measured from 46 pregnant women in different gestational periods and tested for RhD using three different PCR methods on exon 7: Thermal Cycler, Taqman method on LightCycler, and melting curve analysis on LightCycler. In addition, fetal gender was determined by PCR. Cell-free plasma DNA was measured in 100 healthy volunteers as a reference group., Results: The mean value of cell-free plasma DNA in the reference group was 10.9 pg/microL mean, (standard deviation (SD): 3.66) in 50 healthy women and 12.7 pg/microL (SD: 8.2) in 50 healthy men. In the first trimester of pregnancy cell-free plasma DNA was 14.9 pg/microL mean, (SD: 4.2), in the second trimester 15.4 pg/microL mean, (SD: 4.96), and the maximum was achieved in the third trimester of pregnancy 15.6 pg/microl mean, (SD: 6.49). TaqMan probes had the same accuracy, when compared with Thermal Cycler technology (46 samples, 6 failures). Using real-time PCR with melting curve analysis 12 of 17 samples were correctly tested. Gender determination was correctly in 41 of 46 samples., Conclusion: RhD determinations with TaqMan and Thermal Cycler technology are useful methods for fetal RhD prediction. To increase the accuracy of RhD determination it is necessary to test on other exons in addition., (Copyright 2009 Wiley-Liss, Inc.)

The aim of this study was to determine how many UK maternity units have implemented National Institute for Clinical Excellence (NICE) guidance for routine antenatal anti-D prophylaxis (RAADP). In May 2002, the NICE recommended a policy of RAADP for RhD-negative pregnant women. The policy has the potential to reduce maternal sensitization and prevent deaths from haemolytic disease of the foetus and newborn, but implementation entails considerable clinical, financial and organizational challenges. A postal survey of all 324 UK maternity units was completed in 2005.Responses were received from 91% of units (294 of 324). RAADP was offered by 220 of 294 (75%) and in England and Wales 19% of those offered a single-dose regime. At 12% of maternity units, routine paternal blood group testing was offered. For 84% of maternity units, staff education was offered at the time of implementation. Written patient information was provided at 97% of maternity units and 147 of 217 (69%) returned a copy. We received 60 different leaflets. Three years after NICE guidance was issued, one in four maternity units did not offer RAADP. Among those that do offer RAADP, practice with regard to anti-D administration, paternal testing, provision of written information and staff education varied. Unit and clinician level research is required to understand why.

Transfusion of D+ red blood cells (RBCs) into D- recipients, whether through whole blood, RBC, or platelet (PLT) transfusion, can lead to alloimmunization with associated risks of hemolytic reactions from subsequent mismatched transfusion. The incidence of D alloimmunization in various transfused patient populations may be different from that reported in normal subjects or in pregnancy, but prevention of D alloimmunization after mismatched transfusion can be achieved using RhIG. An optimal approach to the use of RhIG, however, has not been identified for the United States. Case histories and studies of volunteers reported over the past 40 years have established that alloimmunization to mismatched RBC transfusion can be successfully prevented with a dose of 20 microg of RhIG per 1 mL of D+ RBCs (per 2 mL of whole blood) when given within a window of opportunity that extends to at least 72 hours. Evidence from prospective studies of RhIG as a therapy for immune thrombocytopenic purpura suggests that such doses can be tolerably given by intravenous injections over short periods, with adverse event rates minimized when pretransfusion medication is given. For mismatched PLT transfusions, the lowest dose of standard preparations of RhIG (e.g., 125 or 300 microg) should be sufficient to prevent alloimmunization given the small D+ RBC volumes involved. This article reviews how our understanding of prevention of alloimmunization in mismatched transfusion has progressed over the years and outlines some practical considerations based on the currently available evidence.

Background: The objective was the evaluation of the effect of the Dutch national routine antenatal RhIG (anti-D) immunization prevention (RAADP) program comprising one single dose of 200 microg (1000 IU) of RhIG in the 30th week of pregnancy, restricted to women without a living child., Study Design and Methods: A nationwide historic control study was performed. All newly detected anti-D-immunized para-1 in 1999, 2002, and 2004 were included and classified on the basis of received prophylaxis during the first pregnancy: antenatal and postnatal versus only postnatal RhIG. The numbers of D- parae-1 who delivered a D+ first child before the introduction (control group) or after the introduction (intervention group) of the RAADP were calculated from Vital Birth Statistics (8,700 and 12,000, respectively)., Results: Fifty-eight newly detected anti-D immunizations in the first trimester were observed in the control group and 39 in the intervention group, which resulted in a significant reduction of the prevalence of new anti-D immunizations from 0.67 percent (95% confidence interval [CI], 0.50%-0.84%) to 0.31 percent (95% CI, 0.21%-0.41%). No reduction was observed in anti-D immunizations newly detected at the 30th-week screening (0.25%). A nonsignificant risk reduction of the risk of severe hemolytic disease of the fetus and newborn (HDFN) was found (0.23% vs. 0.10%). The numbers needed to treat to prevent one anti-D-immunized pregnancy and one case of subsequent severe HDFN were 357 and 1255, respectively., Conclusions: RAADP of one single dose of 200 microg of RhIG in addition to postnatal RhIG (200 microg) halves the risk of anti-D immunization and subsequent severe HDFN.

Rhesus immune globulin has decreased the prevalence of rhesus D alloimmunization in pregnancy so that only approximately six cases occur in every 1,000 live births. The rarity of this condition warrants consideration of consultation with or referral to a maternal-fetal medicine specialist with experience in the monitoring and treatment of patients with red cell alloimmunization in pregnancy. Evaluation for the presence of maternal anti-D antibody should be undertaken at the first prenatal visit. First-time sensitized pregnancies are followed with serial maternal titers and, when necessary, serial Doppler assessment of the peak systolic velocity in the middle cerebral artery. In cases of a heterozygous paternal genotype, new DNA techniques now make it possible to diagnose the fetal blood type through free fetal DNA in maternal plasma. When there is a history of an affected fetus or infant, maternal titers are no longer predictive of risk in subsequent pregnancies. Serial peak middle cerebral artery velocities using Doppler ultrasonography can be used in these pregnancies to detect fetal anemia. In some situations, intrauterine transfusion is necessary through ultrasound-directed puncture of the umbilical cord with the direct intravascular injection of red cells. Perinatal survival rates of more than 90% have been reported; hydrops fetalis reduces the chance for a viable outcome by up to 11%. Neonatal and infant outcomes are complicated by the need for repeated transfusions secondary to suppressed erythropoiesis. Long-term studies have revealed normal neurologic outcomes in more than 90% of cases. Future therapy will involve selective modulation of the maternal immune system, making the need for intrauterine transfusions a rarity.

Background: The understanding of the molecular bases of blood groups makes possible the identification of red cell antigens and antibodies using molecular approaches, especially when haemagglutination is of limited value. The practical application of DNA typing requires the analysis of the polymorphism and allele distribution of the blood group genes under study since genetic variability was observed among different ethnic groups. Urban populations of Argentina are assumed to have a white Caucasian European genetic component. However, historical and biological data account for the influence of other ethnic groups. In this work we analyse FY and RH blood group alleles attributed to Africans and that could have clinical implications in the immune destruction of erythrocytes., Methods: We studied 103 white trios (father, mother and child, 309 samples) from the city of Rosario by allele specific PCRs and serological methods. The data obtained were analysed with the appropriate statistical test considering only fathers and mothers (n = 206)., Results: We found the presence of the FY*BES and RHCE*ce(733C>G) alleles and an elevated frequency (0.0583) for the Dce haplotype. The number of individuals with a concomitant occurrence of both alleles was significantly higher than that expected by chance. We found that 4.68% of the present gene pool is composed by alleles primarily associated with African ancestry and about 10% of the individuals carried at least one RH or FY allele that is predominantly observed among African populations. Thirteen percent of Fy(b-) subjects were FY*A/FY*BES., Conclusion: Taken together, the results suggest that admixture events between African slaves and European immigrants at the beginning of the 20th century made the physical characteristics of black Africans to be invisible nowadays. Considering that it was a recent historical event, the FY*BES and RHCE*ce(733C>G) alleles did not have time to become widespread but remain concentrated within families. These findings have considerable impact for typing and transfusion strategy in our population, increasing the pool of compatible units for Fy(b-) individuals requiring chronic transfusion. Possible difficulties in transfusion therapy and in genotyping could be anticipated and appropriately improved strategies devised, allowing a better management of the alloimmunization in the blood bank.

Objective: To investigate immediate perinatal outcome of RhD-negative patients carrying RhD-positive fetuses who received antenatal Rh immunoglobulin for the prevention of RhD-mediated hemolytic disease of the fetus and newborn., Methods: A retrospective population-based analysis was conducted comparing pregnancies of all RhD-negative women who received antenatal Rh immunoglobulin prophylaxis (anti-D), to RhD-positive parturients, during the years 1988-2003. All women were RhD-negative without evidence of RhD sensitization. Patients received anti-D during the 28-30th week of pregnancy, and an additional dosage within 72 hours following delivery after confirmation of the newborn's RhD status., Results: Of 145,437 deliveries during the study period, 6.8% were of RhD-negative women (n = 9961). Perinatal mortality rate was significantly higher among the RhD-negative women who received antenatal prophylaxis rhesus immunoglobulin as compared with the controls (17/1000 vs. 12/1000, OR = 1.3, 95%CI 1.2-1.6; p < 0.001). This higher mortality rate was related to a higher rate of intrauterine fetal demise (IUFD) (10/1000 vs. 6/1000, OR = 1.5, 95%CI 1.2-1.9; p < 0.001). The association remained significant after controlling for RhD isoimmunization leading to hydrops fetalis, using the Mantel-Haenszel technique (weighted OR = 1.3; 95% CI 1.1-1.5; p = 0.001). The rate of RhD isoimmunization was 0.6% (n = 58). Using a multivariable analysis with IUFD as the outcome variable, controlling for known confounders for fetal demise, RhD-negative status was an independent risk factor for IUFD., Conclusion: RhD-negative women carrying RhD-positive newborns are at an increased risk for IUFD despite Rh immunoprophylaxis.

Objectives: To study the incidence of red cell immunization and to evaluate the use of low-risk invasive procedures in the management of alloimmunized during pregnancy., Design: A 14-year retrospective study of all immunized mothers and their newborns. Population. All reported alloimmunizations between the years 1992 and 2005 in our catchment area were examined., Methods: Background factors, maternal antibody classification, antibody titers, anti-D quantitation, procedures and maternal treatments instituted during pregnancy, fetal outcome and treatment of the newborn were evaluated., Results: There were 78,145 deliveries in the region. Alloimmunization during pregnancy was detected in 0.4% of all pregnancies, excluding ABO immunizations. A significant alloimmunization (titer level > or =8) was detected in 0.16%. Anti-D immunizations were responsible for 60% of significant immunizations followed by anti-Fy(a) in 10%, anti-c in 7% and anti-K in 4%. Maternal plasma exchange and high-dose intravenous immunoglobulin were used as low-risk invasive treatments in 12 cases. Delivery was in > or =38 weeks in 93% of cases. Twenty-nine newborns were treated with exchange transfusions (ETs) after delivery, whereof 21/29 were due to anti-D, seven due to anti-c and anti-E and in one case anti-Fy(a). No deaths occurred due to severe alloimmunization., Conclusion: Anti-D still accounts for the most severe immunizations and for most of the cases where ET was necessary. Low-risk invasive techniques to evaluate and treat pregnancies complicated by alloimmunization seem possible and accurate, avoiding invasive procedures that may exacerbate the immunization during pregnancy.

Rhesus D (RhD) blood group incompatibility between mother and fetus can occasionally result in maternal alloimmunization where the resultant anti-D can cross the placenta and attack the fetal red cells, which in worse case scenarios can cause fetal anemia and ultimately death. Fetal RHD genotyping was introduced in the mid-1990s after the molecular characterization of the RH genes as an aid to the clinical management of these cases. Initially, these tests used fetal DNA extracted invasively from chorionic villus and amniocyte samples. RHD genotyping of fetuses carried by RhD-negative women has become the first large-scale application of noninvasive prenatal diagnosis (NIPD). Initially the real-time polymerase chain reaction (PCR)-based tests were devised to characterize free fetal DNA in maternal plasma and serum, and RHD genotyping was a convenient assay to develop this exciting new technology, because the accuracy of tests could easily be confirmed after the simple RhD phenotyping of fetal cord blood cells after birth. "First generation" RHD genotyping tests were based on the incorrect concept that all D-negative phenotypes were caused by a complete RHD gene deletion. Thus, it was a relatively simple task to develop diagnostic PCR strategies based on the detection of RHD where D-negative genomes will completely lack RHD. Subsequent research into the molecular basis of D-negative phenotypes revealed that a significant number of D-negative genomes possess fragments of, or mutated RHD genes, the most notable of which is the RHD pseudogene found in Africans. Thus, more comprehensive RHD genotyping tests have evolved to differentiate these alleles, and are more appropriate in the diagnosis of multi-ethnic population groups such as those found in Europe and North America. Many European Union countries have suggested the mass application of RHD NIPD for all fetuses carried by D-negative women. This is of clear benefit, because most RhD prophylaxis programs have switched to antenatal administration. This will help conserve anti-D stocks and it will prevent unnecessary administration of a human-derived blood product to a vulnerable patient group. Although anti-D stocks are inherently safe, there is a moral obligation to eliminate unnecessary administration of it because there have been instances of hepatitis C infection due to contamination. Furthermore, as yet undescribed viruses may be contaminants of blood products. Mass-scale RHD NIPD will shortly be implemented in several countries in the European Community as a consequence.

The aim of this study was to determine the compliance rates for women being offered routine antenatal anti-D prophylaxis in two obstetric units in the UK. Haemolytic disease of the newborn (HDN) is a potentially serious condition that can result in substantial morbidity and sometimes death. Current guidelines recommend that 500 IU anti-D immunoglobulin G (IgG) should be offered to all non-sensitized RhD-negative women at 28 and 34 weeks' gestation in order to prevent the risk of RhD sensitization in pregnancy. Implementing guidance, however, remains a challenge. We conducted a retrospective audit of 207 RhD-negative, non-sensitized pregnant women attending obstetric units during 2004 to assess compliance with national guidance on the provision of antenatal anti-D prophylaxis. Informed consent for routine antenatal anti-D prophylaxis was documented for 185 of these women. In total, 86.5% of women received the two doses of anti-D IgG. The majority of women received their first and second doses within 1 week of 28 and 34 weeks' gestation (87.0 and 86.0%, respectively). Accurate records of prophylactic anti-D IgG were maintained and updated. This audit demonstrates that the level of patient compliance with the two-dose regimen was high.

Since the beginning of RHD genotyping in maternal plasma, no Rh D positive baby was diagnosed RHD negative in our institution. Genotyping from circulating DNA in maternal plasma is as efficient as genotyping on amniocyts but without the associated risks. We propose a prophylactic injection based on fetal genotyping RHD in maternal blood with 300 microg anti-D immunoglobulin at 28 weeks of amenhorrea in all of Rh D negative pregnant women whitch fetuses positive RHD.

Documented routine antenatal anti-D prophylaxis was given to 90% and 81-87% of eligible women at 28 and 34 weeks of gestation, respectively, during the early 1990s and early 2000s. With increasing experience and education, a significant improvement in the timing of the first (OR 0.26, 95% CI 0.16-0.41: P < 0.0001) and second injections (OR 0.40, 95% CI 0.26-0.61: P < 0.0001) occurred during the latter period. Despite these improvements, there was no reduction in the sensitisation rate at 0.4%. However, this low rate occurred despite significant proportions of women delivering more than 42 days after the second injection. Fifteen of the 16 sensitised women had received routine antenatal prophylaxis.

Objective: To examine whether literature supports the use of Rh immune globulin in Rh-negative women with first trimester spontaneous abortions to prevent maternal sensitization to the fetal Rh antigen and subsequent fetal morbidity and mortality., Methods: We searched MEDLINE (1966-2005), the Cochrane Central Register of Controlled Trials, EMBASE (1990 to 2005), and the reference sections of the articles found. The search is considered updated to December of 2005. Search terms included vaginal bleeding, Rh negative, Rh immune globulin, RhoGAM, isoimmunization, sensitization, first trimester pregnancy, threatened, and spontaneous abortion., Results: The evidence to support the use of Rh immune globulin for a diagnosis of first trimester spontaneous abortion is minimal. There is a paucity of well-designed research that examines maternal sensitization or hemolytic disease of the newborn as an outcome in patients receiving, versus not receiving, Rh immune globulin in first trimester bleeding. There is significant evidence to demonstrate fetomaternal hemorrhage in first trimester spontaneous abortions; yet, no studies demonstrate subsequent maternal sensitization or development hemolytic disease in the fetus as a result of this hemorrhage., Conclusion: In summary, there is minimal evidence that administering Rh immune globulin for first trimester vaginal bleeding prevents maternal sensitization or development of hemolytic disease of the newborn. The practice of administering Rh immune globulin to Rh-negative women with a first trimester spontaneous abortion is based on expert opinion and extrapolation from experience with fetomaternal hemorrhage in late pregnancy. Its use for first trimester bleeding is not evidence-based.

Objective: The objective of this review was to assess the effects of antenatal anti-D immunoglobulin on the incidence of Rhesus D alloimmunization when given to Rh-negative women without anti-D antibodies and assess the effects of giving anti-D to Rhesus negative women, with no anti-D antibodies, who had given birth to a Rhesus positive infant., Design: A review article., Setting: Department of Obstetrics and Gynecology, Department of Medical Genetics and Fetal Medicine, University Hospital, Olomouc, Ministry of Health, Czech Republic., Subject and Method: We searched the Cochrane Pregnancy and Childbirth Group trials register, refence lists of relevant articles and bibliographies., Conclusion: The risk of Rhesus D alloimmunization during or immediately after a first pregnancy is about 1%. Administration of 100 microg (500 IU) anti-D to women in their first pregnancy can reduce this risk to about 0.2% without, to date, any adverse effects. Anti-D, given within 72 hours after childbirth, reduces the risk of RhD alloimmunization in Rhesus negative women who have given birth to a Rhesus positive infant. However the evidence on the optimal dose is limited.

In the past 20 years, numerous monoclonal anti-D antibodies have been developed in order to replace the human plasma derived anti-D immunoglobulins, using different in vitro functional assays as screening methods. Some of these monoclonal antibodies have been evaluated in exploratory in vivo clinical trials, notably for their ability to mediate the clearance of D-positive red cells. A review of these reported trials is presented and the results are analyzed in the light of the newly published hypothesis conferring an important role to some Fc-FcgammaR interactions and to the glycosylation-dependent potency of the monoclonal anti-D antibodies.

At present, rhesus prophylaxis concerns RhD negative pregnant women, even though 30 to 40% of them are bearing a RhD negative child. Knowing the RhD fetal genotype could change this quite irrational practice of prophylaxis (exposing many more women than needed to blood derived products) without reducing its efficacy. RhD fetal genotype determined on amniotic fluid has an excellent sensitivity. Presence of silent D genes slightly impairs its specificity which remains acceptable. However women have to be informed of possible false positives. Fetal RhD genotyping on maternal blood is more complex. Sensitivity is good from 10 GW and excellent after 15 GW. In case of a first negative result, it is recommended to control fetal RhD on a second sample drawn a few weeks later. Another new perspective for rhesus prophylaxis is the attempt to substitute polyclonal IgG anti-D into human monoclonal IgG anti-D. The main difficulty is to elaborate monoclonal antibodies with a capacity to neutralize RhD positive red blood cells equivalent to those of polyclonal anti-D. A new generation of antibodies is in process and preliminary clinical results are suggesting a possible use of these monoclonal antibodies for future rhesus prophylaxis but long-term follow-up is required to draw further conclusions.

Any prevention policy has a cost. For anti-D immunization, the main questions concern the cost of a change in the prevention policy by the health insurance fund and more globally by the society in general. Furthermore, the analysis must also examine the cost effectiveness of systematic prevention extended to all Rhesus negative women in comparison with targeted prevention or no prevention. Studies published in Great Britain, Canada, and The United States have generally concluded that the raw cost of systematic prevention is high compared with targeted prevention. On the other hand, the cost-effectiveness would favor the systematic approach. These data are difficult to apply to the French situation because health care costs are different and because of the lack of perfectly reliable epidemiological data on the frequency and consequences of severe forms of allo-immunization. Technological advances, particularly concerning genotyping of fetal Rhesus from maternal blood samples, could have an important impact on the cost factor if a systematic approach is adopted.

RhD prophylaxis concerns RhD negative women, who are non-sensitized against D antigen during and at the end of their pregnancy with a RhD positive child. RhD prophylaxis includes targeted prophylaxis (prevention of anti-D immunization after feto-maternal hemorrhage (FMH) induced by prenatal events and delivery) and routine antenatal D prophylaxis (prevention of anti-D immunization resulting from spontaneous FMH in the last trimester of pregnancy). Targeted prophylaxis should be applied regardless of the gestational age and a dose of 100microg anti-D is usually enough (200microg is the lowest dosage currently available in France). However it is recommended to quantify the volume of feto-maternal hemorrhage to avoid administration of a dose of IgG anti-D less than 20microg per ml of fetal red blood cells. Efficacy of prophylaxis relies also on the delay between the sensitizing event and the injection of anti-D, delay should be less than 72 hours. Intravenous administration of anti-D allows immediate neutralization of D positive fetal red blood cells and should be, if possible, preferred to intramuscular administration (IM). After a first injection of anti-D, if repetition of potential sensitizing events occurs, abstention of prophylaxis is possible depending on the previous administrated dose (protection lasts 6 weeks for 200microg and 9 weeks for 300microg) and the amount of feto-maternal hemorrhage. For routine prophylaxis of the third trimester, 300microg of anti-D should be proposed IM at 281+/-GW. Abstention of Rh prophylaxis is possible if the alleged father is certified RhD negative or if the fetal RhD genotype is confirmed negative. At delivery, RhD phenotype of the newborn should be determined even if RhD fetal genotype is known. Maternal blood should be drawn for quantification of feto-maternal transfusion at least 30 min after delivery is completed.

Background: In France, anti-Rh prophylaxis is currently based on systematic postnatal prevention which has validated efficacy (relative risk=0.04 versus placebo), associated with targeted antenatal prevention in the event of situations raising a risk of fetomaternal hemorrhage. In most industrialized countries, a systematic prevention policy is applied with immunoglobulin injections for the above cited situations and during the third trimester to cover the risk of spontaneous occult fetomaternal hemorrhage occurring at the end of pregnancy., Objective: Compare the efficacy of two strategies for antenatal prevention., Material and Methods: Review of the literature of published comparative studies. Eleven studies were retained (two randomized trials, seven comparative studies, one before-after study, one population-based study) including more than 30,000 treated patients., Results: Globally, immunization rate was to the order of 1.5% (1.2-1.9%) for targeted prevention limited to situations at risk and to the order of 0.2% (0-0.9%), all parities included, for systematic antenatal prevention. Comparative analyses have reported significant odds ratios of 0.20 and 0.37 in all subgroups., Conclusion: Despite the heterogeneous nature of the published studies, available data are in favor of systematic prevention: either with a 300g dose at 28GW or 100g at 28GW and 34GW complementary to the postnatal prevention. Few data are available on the real perinatal benefit of systematic prevention.

Anti-D prophylaxis should be proposed to all RhD negative non-sensitized pregnant women, after delivering an information concerning both Rhesus disease and anti-D immunoglobulins. This information must be delivered as a written document and the patient's oral consent is required before administration of the anti-D immunoglobulins. Anti-D immunoglobulins currently used in France for prophylaxis are extracted from plasma of hyperimmunized paid donors. Even if all the conditions of viral safety are fulfilled in the preparation of anti-D immunoglobulins, they remain blood derived products. As such, prescription of anti-D immunoglobulins should follow legal rules concerning tracability and information. Refusal of rhesus prophylaxis can occur but should be transcribed and motivated in the patient's chart. Administration of anti-D immunoglobulins is usually well tolerated. Reactions to hemolysis of fetal Rhesus positive red cells can occur but remain rare and linked to important foeto-maternal hemorrhage. They can be easily prevented or treated by anti-inflammatory drugs. Patients can be vaccinated against rubella in the post-partum period even though they will receive a concomitant prophylaxis with Rh immunoglobulin. Persistence of passive anti-D in maternal circulation after injection lasts several weeks or months and could have various consequences. In the mother: it can interfere with diagnosis of active anti-D immunization. In most cases, it may be possible to differentiate passive and immune anti-D. When reliable information concerning date and dosage of antenatal anti-D prophylaxis are available. In the newborn: anti-D immunoglobulins can pass through the placenta and enter the fetal circulation, coat the D positive fetal red cells and give positive DAT. Positive DAT is reported in 5 to 15% of the newborns following rhesus prophylaxis in the third trimester but with no report of anemia or jaundice. In absence of ABO incompatibility, no additional investigation is needed in these newborns.

The number of women exposed to a risk of anti-D allo-immunization who require care depends on the number of pregnancies, the distribution of blood groups in the Rhesus D system, and the number of allo-immunization risk situations as well as the chosen prevention protocol. In 2004, there were 790,000 pregnancies in metropolitan France. The total number of conceptions (adding abortions, voluntary interruptions, ectopic pregnancies and in utero fetal deaths) would be to the order of 1,100,000 to 1,200,000. Since 15% of the French population is RhD-negative, 15% of pregnant women or women who have delivered are RhD-negative, which would correspond to 160,000 to 180,000 women exposed to a risk of anti-D allo-immunization. Two strategies could be evaluated: prevention targeting risk factors and systematic prevention from 28 weeks gestation. Both strategies involve treatment with anti-D anti-globulins to RhD-negative women with an RhD-positive newborn. Targeted prevention would involve 160,000 to 190,000 doses for 130,000 to 150,000 women. The systematic approach would consist in 300,000 to 340,000 doses for 250,000 to 280,000 women. This number could be lowered by reserving treatment for women with a RhD-positive partner (210,000 to 230,000 doses for 260,000 to 290,000 women), but this would require a precise contract between the physician and the woman. Another way to reduce the number of doses would be to determine the Rh group of the fetus either by ovular samples or by genotyping on maternal blood. This would give about 200,000 to 220,000 doses for 240,000 to 270,000 women. In light of the literature, the systematic protocol would probably reduce the number of immunized women, the current estimation being 700 women still immunized in France and the goal being a reduction to a residual 200. The number of infants at risk of in utero maternal allo-immunization (fetal death, anasark, anemia) or after birth (jaundice, severe anemia) is not known in France and could be the cause of a few deaths with involvement of about a hundred children. In light of experience in other countries, the systematic protocol would enable avoiding a certain number of these deaths.

With the development of the Rh D immunoglobulin and utilisation in clinical practice, severe rhesus iso-immunisation is rarely seen today. Antibodies against other blood groups are now more common than anti-D and while the majority do not cause significant haemolysis, there are reports of serious morbidity with antibodies other than anti-D. We reviewed retrospectively all cases (excluding anti-D and anti kell antibodies) where antibodies were detected at routine booking visit between 1997-2001 and correlated the type of antibody to clinical outcome as assessed by coombs test, postnatal phototherapy and transfusion. Our approach to these cases does not involve invasive test as amniocentesis and relies on the ultrasound monitoring, level of antibodies and timely delivery with appropriate paediatric follow up. Over five years period 150 women were identified with antibodies other than anti-D or anti-kell. There were no directly related deaths. Many authorities suggest amniocentesis for specific antibodies to assess fetal haemolysis and plan management. This study support a non-invasive approach with mortality and minimal morbidity.

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

Objective: To observe the pharmacokinetics of intramuscular anti-D immunoglobulin (IgG) given for routine antenatal prophylaxis., Design: Prospective observational study., Setting: Maternity unit and antenatal serology laboratory in a district teaching hospital., Population: Forty-five rhesus-D-negative pregnant women not sensitised to RhD., Methods: Serial serum quantitations of anti-D IgG following the intramuscular injections of anti-D IgG 100 microg (500 IU) at 28 and 34 weeks of gestation. Anti-D IgG concentrations were assayed with the RFA-300 continuous flow analyser., Main Outcome Measures: The kinetic profile and duration of detectable anti-D IgG in maternal serum following the first and second injections of anti-D IgG., Results: For the 43 women in whom serial data were collected, there were no detectable differences between pregnancies with an RhD-positive (26) or -negative (17) fetus. Maximum IgG concentrations were detected two to five days following the first anti-D IgG injection and ranged between 0 and 28 ng/mL. Only 30% of women still undelivered at 40 weeks of gestation had detectable IgG at 2 ng/mL or greater. There was a significant relationship between higher maximum values and low maternal surface body area (R2 = 0.204, P = 0.002), but this did not influence duration of persistent IgG., Conclusion: Using previously published data, 70% women are not adequately protected with anti-D IgG 12 weeks after the first prophylactic injection. Despite this, previous clinical results suggest that the antenatal prophylaxis schedule used provides adequate protection and that the recommendation for the lowest concentration of protective anti-D IgG antibody levels currently in use is probably overestimated.

Purpose of Review: Traditional methods of fetal evaluation require invasive procedures (e.g. amniocentesis) with an inherent, albeit low, risk of complications and pregnancy loss. As medical knowledge and ultrasound technology have advanced, noninvasive modes of fetal evaluation have become increasingly used. Two disorders in which this has been most applied are fetal aneuploidy and alloimmunization to red blood cell antigens., Recent Findings: First-trimester screening protocols for fetal aneuploidy combine ultrasound measurement of the fetal nuchal translucency with the measurement of two maternal serum hormones, free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A. This combination detects approximately 85% of women pregnant with fetuses with trisomy 21. An increase in the velocity of fetal middle cerebral artery blood flow occurs in the setting of moderate to severe fetal anemia and can be measured with Doppler ultrasound. Middle cerebral artery evaluation is equally sensitive in the prediction of anemia as amniocentesis for bilirubin breakdown products and has less associated risk. It has an additional benefit because it can also be applied to causes of anemia other than hemolysis., Summary: Noninvasive methods of fetal assessment are becoming increasingly commonplace. There has been a dramatic decrease in the use of prenatal diagnostic services as screening strategies have become available. Doppler assessment of the velocity of blood flow through the fetal middle cerebral artery will allow for more judicious application of invasive procedures to pregnancies at significant risk for anemia.

Background: The D antigen includes category D, partial D, and weak D types, which are important because anti-D alloimmunization can occur in some but not all persons that express a variant RHD allele. At present, there is little prospective information on the prevalence of D variants among obstetric patients and potential transfusion recipients., Study Design and Methods: The RHD alleles were prospectively examined in a large patient population identified on the basis of a difference in anti-D reactivity between two reagents., Results: Fifty-five discrepancies (0.96% of D-) were noted among 33,864 ethnically diverse patients over 18 months, of which 54 represented mutated RHD alleles. Seven obstetric patients were assigned D- status based on serology; only 1 patient had a partial RHD allele. Ten of 25 (36%) obstetric patients and 4 of 6 (67%) female potential transfusion recipients of childbearing age or younger were assigned D+ status, and they expressed a D variant known to permit anti-D alloimmunization. In total 20 RHD alleles were identified including category, DVa or DVa-like alleles (n = 7), DAR (n = 8), and four novel RHD alleles including two new DAU alleles., Conclusion: Given the complexity of D antigen expression, it is concluded that some clinically important D variants identified by standard serologic analysis phenotype as D+ and are potentially at risk for the development of anti-D.

Introduction: Guidelines for distribution and use of blood products have been established for both blood transfusion institution and hospitals, in particular for the use of Rh (D)-incompatible platelet concentrates. The aim of this study was to evaluate: 1) the rate of attribution for the Rh (D)-incompatible platelets concentrates, 2) the immunisation prophylaxis practices, 3) the immunological consequences using short and medium term follow-up of transfused patients., Methods: Patients with Rh (D)-incompatible platelets concentrate administered during the year 2003 at Rouen University Hospital were retrospectively selected. Patients on transfusion were described. The relationship of various factors with the injection as well as the appearance of allo-immunization was statistically tested., Results: During a year, 280 Rh (D)-incompatible platelets concentrates were administered to 67 patients. Immunisation prophylaxis by injection of Ig anti-D was not systematically performed. Four immunizations in the Rhesus group system were identified: 2 against D antigen (Ag), 1 against E Ag and 1 against C Ag. Immunisations against D Ag occurred for two younger women considered as immunodeficient. Immunization prophylaxis was more frequent in poly-transfused patients. However no difference was observed for the other factors., Conclusion: Compatibility concerning Rhesus (D) is not always possible. The immunization against red cells persists, in particular against the antigens of the Rhesus group system and moreover for the immunodeficient patients. Recommendations for immunization prophylaxis by injection of specific anti-D immune-globulin (Ig) could be reconsidered.