[Follow-up of pregnancies with red-cell allo-immunisation: State-of-the art]. / Le point sur le suivi des allo-immunisations érythrocytaires.

Anti-RhD allo-immunisation has become rare since anti-D prophylaxis was introduced in the seventies; however, it remains the first cause of fetal anemia. It may cause severe fetal complications such as fetal hydrops, cerebral anoxic lesions and fetal death. In the neonatal period, severe jaundices and anemias requiring transfusion or exsanguino-transfusion are still common in case of severe allo-immunisation. Neonatal death and sequellae due to bilirubin encephalopathy have not fully disappeared. Follow-up of pregnancies with maternal allo-immunisation requires identification of the antibody (anti-RhD, anti-Kell and anti-c are the most frequently responsible for fetal complications), dosage and titration. In RhD allo-immunization, feto-maternal incompatibility may be confirmed by non-invasive RHD genotyping of the fetus in maternal blood. In cases at risk for fetal anemia, weekly Doppler assessment of middle cerebral artery peak systolic velocity (MCA-PSV) allows identification of fetal anemia before the occurrence of fetal hydrops. The reference treatment of fetal anemia is in utero fetal transfusion. The risk of fetal loss due to in utero transfusion (IUT) is 3% per procedure. The cumulated risk of fetal loss can thus exceed 10% in case of early occurrence of fetal anemia requiring up to five or six IUTs in a single pregnancy.

[Non invasive fetal RhD genotyping using maternal blood: time for use in all RhD negative pregnant women]. / Génotypage RhD foetal non invasif sur sang maternel: vers une utilisation chez toutes les femmes enceintes RhD négatif.

Non invasive fetal RhD genotyping, based on polymerase-chain-reaction (PCR), is an accurate and validated technique. It allows a reduction by one-third of anti-D immunoglobulin injections to prevent RhD allo-immunization. In case of maternal anti-D immunization, fetal RhD genotyping allows to focus on RhD positive fetuses only the biologic and sonographic follow-up. The wide use of this technique implies the validation and economic evaluation of a commercial RhD genotyping kit, ready for use in non specialized laboratories.

[Use of peak systolic velocity of the middle cerebral artery in the management of fetal anemia due to fetomaternal erythrocyte alloimmunization]. / Intérêt pratique du pic systolique de vélocité à l'artère cérébrale moyenne dans la prise en charge des anémies foetales par allo-immunisation érythrocytaire.

OBJECTIVE: To assess the peak systolic velocity in the middle cerebral artery (PSV-MCA) in the prediction of fetal anemia in case of severe red-cell alloimmunization. METHODS: A prospective study, from January 2003 to April 2006, of 47 consecutive pregnancies with severe alloimmunization. Fetal surveillance was based on titration and dosage of antibodies, ultrasound scans, and doppler for PSV-MCA measurement up to twice a week. A fetal blood sampling and in utero transfusion was performed in case of increase in PSV-MCA above 1.5 multiples of the median (MoM), and/or signs of hydrops on ultrasound. Severe fetal anemia was defined by fetal hemoglobin below 0.55MoM for gestational age. Analyses performed included the correlation between PSV-MCA and fetal hemoglobin, the value of PSV-MCA in the prediction of severe fetal anemia, and the determination of adequate threshold for intervention based on ROC curve analysis. RESULTS: Four hundred and eighty-five PSV-MCA were performed in 47 high-risk pregnancies, of which 125 were coupled with hemoglobin measurement by fetal blood sampling. There is a significant negative correlation between PSV-MCA and fetal hemoglobin (R2=0.6545 ; p<0.0001). Based on all prospective data, the negative predictive value of PSV-MCA was 97.8 %, sensitivity was 86.7 %, with a false positive rate of 12.2%. Area under the ROC curve was 0.85 (IC 95 %, 0.742-0.927 ; p<0.0001), suggesting an excellent value of this test. When switching the threshold for intervention from 1.5 to 1.6MoM, the positive predictive value increased, without decrease in sensitivity or negative predictive value. CONCLUSION: This study confirms the correlation between PSV-MCA and fetal hemoglobin. It allows a decrease of invasive procedures in the follow-up of pregnancies with severe red-cell alloimmunization.

Noninvasive fetal RHD genotyping from maternal plasma. Use of a new developed Free DNA Fetal Kit RhD.

Fetal RHD genotyping from maternal plasma was performed by real-time PCR amplification of exons 7 and 10 of the RHD gene and the amplified products were detected either with SYBR Green I dye according to our previously published method [Mol Diagn 8 (2004) 23-31] or with hydrolysis probes in a new Free DNA Fetal Kit RhD((R)). Plasma specimen from 300 RhD-negative pregnant women (between 10 to 34 weeks of gestation) were analysed and validation of the results was ascertained either by RHD genotyping on amniotic cells or by blood typing of the neonate at birth. We found 100% concordant results when comparing the two methods. Two false-positive but no false-negative results were found. Thus, the sensitivity of the assay was 100% and the specificity superior than 99%. These data confirm the accuracy of fetal RHD genotyping on maternal plasma using the Free DNA Fetal Kit RhD, thus allowing to propose non invasive PCR-based fetal RHD genotyping for all RhD-negative pregnant women and to restrict the use of anti-D immunoglobulins only to those bearing an RhD-positive fetus.

[Adverse effects and patient information]. / Effets indésirables et information des patientes.

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.

[Prevention of fetomaternal rhesus-D allo-immunization. Perspectives]. / Perspectives.

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.

[Prevention of fetomaternal rhesus-D allo-immunization. Practical aspects]. / Aspects pratiques.

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.

[Kell alloimmunization in pregnancy]. / Allo-immunisation anti-Kell et grossesse.

INTRODUCTION: Kell alloimmunization is a rare disease, although its incidence is the highest after after anti-D alloimmunization. METHODS: We report two recent cases and a review of the literature to describe practical management of Kell alloimmunization in pregnancy. DISCUSSION: When an immunization against the Kell antigen was diagnosed, amniocentesis was performed at 14 weeks gestation to determine the fetal blood group. If the fetus was Kell positive, a first fetal blood sample was drawn at 17 weeks gestation in case of fetal hydrops, and at 20 weeks without fetal hydrops. The diagnosis of anemia led to in utero transfusion. A second fetal blood sample was taken at 8 to 10 days, every two weeks during the second trimester and every three or four weeks during the third trimester. Fetal well-being was assessed with weekly sonography and rates of hemoglobin decline. These measures enable adapting the frequency of fetal blood sampling.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Seroprevalence of human herpes virus 8 antibody in populations at high or low risk of transfusion, graft, or sexual transmission of viruses.

BACKGROUND: The routes of transmission of human herpes virus 8 (HHV-8) remain unclear. In particular, HHV-8 transmission by blood components and organ transplantation is still debated and raises public health issues. The objective of this study was to determine the prevalence of anti-HHV-8 in selected populations of persons or patients with or without risk factors for the transmission of viral infections, in order to determine the routes of HHV-8 transmission. STUDY DESIGN AND METHODS: A total of 1431 persons or patients at low or high risk of sexually, blood-, or graft-transmitted viral infections were tested by means of a standardized immunofluorescence serologic assay detecting anti-HHV-8. RESULTS: The persons or patients could be classified into three distinct groups according to anti-HHV-8 prevalence: a low prevalence group (0.0% to 5.0%), including healthy blood donors, healthy pregnant women, multiply transfused patients with thalassemia major, and IV drug users; an intermediate prevalence group (5.0% to 20.0%), including organ donors, kidney transplant recipients, and multiply transfused patients with sickle cell disease; a high prevalence group (>20.0%), including HIV-negative persons at high risk of sexually-transmitted viral infections, and HIV-infected homosexual men and heterosexuals. CONCLUSION: The sexual route appears to be the main route of HHV-8 transmission; bloodborne transmission of HHV-8, if it exists, is rare. In contrast, organ transplantation recipients might be exposed to HHV-8 transmission by the transplanted organ, which raises the issue of systematic screening of organ donors.

[Value of RHD fetal genotyping in the prevention of anti-D immunization]. / Intérêt du génotypage foetal RHD dans la prévention de l'immunisation anti-D.

Anti-D prophylaxis is currently applied in France after birth of an RhD positive infant, after interruption of pregnancy and after some antenatal immunizing events (amniocentesis...). However this program does not cover all the prenatal exposures to fetal RhD antigen, and maternal Rh immunization continues to occur. DNA RhD genotyping of the fetus is now reliably performed on amniotic fluid, and pre diagnostic studies on fetal DNA extracted from maternal plasma are promising. The widespread use of fetal RhD genotyping on maternal blood would allow the antenatal administration of Rh immunoglobuline in all Rh negative patients bearing an Rh positive fetus, insofar as it would preclude exposing the other Rh negative patients to the above plasma derived and rather expensive blood product.

[Quantitative estimation by ELISA of IgG anti-D (RH1) antibodies in immunoglobulin preparations and in the sera of immunized donors]. / Dosage par une technique ELISA des anticorps anti-D (RH1) dans les préparations d'immunoglobulines et dans le sérum de donneurs immunisés.

Immunoglobulin preparations of anti-D (RH1) are injected to prevent haemolytic disease of the newborn. Such preparations are obtained by the fractionation of plasma from immunized donors. Measurement of the concentration of IgG anti-D is required to estimate the potency of anti-D preparations and sera from immunized donors. We have developed an ELISA method for the quantification of IgG anti-D. This method included the following steps, sensitization of red cells by anti-D, solubilization of red cell membranes by Triton, and eventually, measurement of IgG anti-D concentration by ELISA. The international reference preparation of anti-D (68/419) was used as a reference. With this method, we measured IgG anti-D concentrations in 5 immunoglobulin preparations of anti-D and in the sera of 10 donors immunized by D antigen. The ELISA results were compared with those obtained by automated hemagglutination. A mean anti-D concentration of 56.2 micrograms/mL was found by ELISA in immunoglobulin preparations. Similar results were obtained by automated hemagglutination (mean 52 micrograms/mL). In the sera of 10 D-immunized donors, anti-D IgG concentration varied from 2.2 to 59.8 micrograms/mL. A good correlation between ELISA and automated hemagglutination was observed in these sera (r = 0.98, p < 10(-7)). In conclusion, the ELISA technique offers an alternative to automated hemagglutination. It requires only the standard equipment necessary for immuno-enzymatic methods.

[Immune cytopenias in newborns]. / Cytopénies immunes néonatales.

Rhesus D haemolytic disease of the newborn (RH HDN) and neonatal PlA1 alloimmune thrombocytopenia (NAT) are the main immune cytopenias affecting fetal red blood cells or platelets through maternal antibodies. During RH HDN, fetal anaemia and neonatal hyperbilirubinaemia may progress, if untreated, towards fetal death and neonatal kernicterus. Likewise, during NAT, intracranial haemorrhage may occur antenally, at delivery or postnatally. Fetal and neonatal transfusion therapy, pre-term delivery, and intensive phototherapy avoid or greatly reduce the incidence of these complications. However, the best treatment of RH HDN is to prevent primary anti-D immunisation in Rh negative pregnant women through passive immunotherapy with Rh immune globulin.

Quantitative determination of anti-K (KEL1) IgG and IgG subclasses in the serum of severely alloimmunized pregnant women by ELISA.

BACKGROUND: Severe cases of HDN occur after the immunization of the mother with K (KEL1) antigen. To date, the only means of evaluating the concentration of anti-K in maternal serum is by titration with an indirect antiglobulin test (IAT). A more accurate estimation of the serum anti-K concentration is needed. STUDY DESIGN AND METHODS: An ELISA technique was developed for the determination of the absolute concentration of anti-K IgG and IgG subclasses in the sera of alloimmunized patients. In this technique, after absorption of anti-K on K-positive RBCs and subsequent elution at acid pH, the concentration of anti-K in the eluate was measured with a sensitive and reproducible ELISA. This method was validated with monoclonal and polyclonal anti-K. It was then used to assay the sera of eight pregnant women with anti-K immunization, associated with early fetal anemia (Hct, 7-17%) detected between the 20th and the 31st week of pregnancy. In addition, in most of these cases, the anemia was associated with fetal hydrops. RESULTS: The anti-K IgG concentration measured by ELISA in the sera of the eight women varied from 1.0 to 4.1 microg per mL (mean, 2.2 microg/mL). Therefore, severe and early forms of fetal anemia can be observed with a relatively low concentration of anti-K (as compared to the concentration of anti-D in similar cases of fetal anemia due to anti-D). The mean proportion of each IgG subclass of anti-K in these sera was IgG1, 95.9 percent; IgG2, 2.4 percent; IgG3, 1.3 percent; and IgG4, 0.4 percent. CONCLUSION: A simple method for quantitative estimation of anti-K in human serum has been developed. Low concentrations of anti-K can cause fetal anemia relatively early in pregnancy. This method should lead to a better identification of pregnant women whose fetuses are at risk for severe fetal anemia due to anti-K.

High rate of GB virus type C/HGV transmission from mother to infant: possible implications for the prevalence of infection in blood donors.

BACKGROUND: Because GB virus type C(GBV-C)/HGV (GBV-C/HGV) is blood-borne and sexually transmitted, persons at risk of infection with such viruses have a high prevalence of GBV-C/HGV markers. However, adults with no apparent risk factors, such as blood donors, frequently are positive for GBV-C/HGV markers. Mother-to-infant transmission could explain this high prevalence, but it has been studied only through small series of GBV-C/HGV-infected mothers co-infected with HCV or HIV. STUDY DESIGN AND METHODS: To determine the rate of mother-to-infant transmission of GBV-C/HGV RNA in women who are HCV- or HIV-negative, a prospective study was performed in a cohort of 288 mothers screened for viral RNA and in the infants born to GBV-C/HGV-infected mothers. RESULTS: Thirteen mothers (4.5%) were found positive for GBV-C/HGV RNA. Of the infants in whom at least one blood sample was collected between the third and the ninth months of life, 89 percent were positive for viral RNA. The majority of these newborns were negative for GBV-C/HGV RNA at birth and positive after the third month. The viral RNA titers of infants born to GBV-C/HGV-infected mothers appeared as elevated as those of their mothers. All the GBV-C/HGV-infected infants remained positive for viral RNA during the entire study period. No clinical events possibly linked to a primary GBV-C/HGV infection were reported in infants. Serum ALT level and blood count remained within normal values throughout the follow-up of all GBV-C/HGV-infected infants. CONCLUSION: The frequency of mother-to-infant GBV-C/HGV transmission is elevated and could explain the high prevalence of GBV-C/HGV markers (viral RNA and E2 antibody) in adults at low risk for blood-borne or sexually transmitted viruses, such as blood donors.

[Prevention of fetal hemolytic disease: it is time to take action]. / Prévention de la maladie hémolytique du foetus et du nouveau-né: il faut agir!

In spite of the progress made since 1970 in specific prevention by anti-rhesus immunoglobulins, and improved management of at-risk pregnancies, allo-immunization due to the erythrocytic Rh 1 antigen (formerly known as Rhesus D or Rh D) remains widespread. In fact, anti-Rh 1 antibodies currently constitute over one-third of the immune antibodies detected after pregnancy. The prevention of allo-immunization against the Rh 1 antigen is therefore still problematical, and concerns approximately one pregnant woman in seven. The etiology and pathology of fetal hemolytic disease have been recalled, and the treatment approach during pregnancy and delivery has been carefully examined. Tests for quantifying the risk of fetomaternal hemorrhage have also been described. This approach aims at improving the methods of preventing allo-immunization (e.g., during pregnancy and delivery) and the efficacy of treatment. It is also stated that if the necessary preventive action is not taken in cases of allo-immunization due to to the Rh 1 antigen, this should be considered a grave medical fault.