Rh Hemolytic Disease Research Articles

An enzyme-linked immunosorbent assay for the quantitative determination of anti-D in plasma samples and immunoglobulin preparations.

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.

Three examples of Rh haemolytic disease of the newborn with a negative direct antiglobulin test

Typically the serological diagnosis of alloimmune haemolytic disease of the newborn (HDN) includes a positive direct antiglobulin test on the infant's red cells, and the presence of an IgG red cell alloantibody in both maternal and cord sera. HDN with a negative direct antiglobulin test has been reported with anti-A and anti-B, but not with other red-cell alloantibodies. In this report we describe four examples of HDN in infants whose red cells had a negative direct antiglobulin test. The first case was diagnosed retrospectively when the infant was admitted to hospital aged 3 weeks with severe anaemia and cardiac failure, and subsequently died. Maternal and infant sera were both shown to contain anti-C: however, the direct antiglobulin test on the infant's red cells was negative. Approximately 1 year later the mother of this infant gave birth to triplets: soon after birth one of the triplets required an exchange transfusion, one had hyperbilirubinaemia, and the third was unaffected. Anti-C and anti-e were detectable in the maternal serum at this time. The most probable Rh genotypes of the two affected infants were R1R2 (CDe/cDE), while the Rh genotype of the unaffected infant was R2R2 (cDE/cDE). Anti-c was implicated as causing HDN in a fourth infant (from a different family) who was a hydropic stillborn. The direct antiglobulin test on fetal blood was negative and other causes of non-immune hydrops were excluded. These four infants provide evidence that the direct antiglobulin test may be negative in some severely affected and even fatal cases of HDN.

A case of unexplained mild Rh (D) haemolytic disease in utero

This report describes the case of a patient with a history of HDN complicated by fetal losses, in which the alloantibody in this particular pregnancy did not appear to cause HDN in utero. No protective HLA-DR antibodies could be demonstrated, and transport of IgG across the placenta appeared to be normal. The infant's red cells possessed a normal D antigen and his mononuclear phagocyte system appeared unimpaired. However, the number of molecules of IgG bound in vivo per fetal red cell was below the level usually associated with significant haemolysis and HDN.

Neonatal Hepatitis and Excessive Hepatic Iron Deposition Following Intrauterine Blood Transfusion

The management of hemolytic disease has undergone a number of significant changes over the past few decades. Intrauterine transfusion therapy, particularly intravascular transfusions, have significantly reduced the morbidity and mortality associated with isoimmunization. This therapy results not only in the transfusion of blood, but also in the transfusion of iron. The long-term consequences of iron loading in the fetus are unknown. We report a case of a newborn with Rh hemolytic disease who was treated with in utero transfusions and subsequently developed liver disease consistent with iron overload.

An Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of Anti-D in Plasma Samples and Immunoglobulin Preparations

Here we present an enzyme-linked immunosorbent assay (ELISA) on microtiter plates for the quantitative determination of anti-D. This method is based on the solubilization of red blood cells sensitized with anti-D and the subsequent measurement of immunoglobulin G by ELISA. The measuring range of the assay is 40-5,000 IU/ml and the lowest quantifiable concentration in plasma is 0.5 IU/ml. The interassay relative standard deviation for concentrations above 130 IU/ml ranges from 3.2 to 8.1% and below 50 IU/ml from 10.5 to 19.7%. Comparison of ELISA with automated hemagglutination shows that the results of the two assays correlate well: r = 0.992, n = 26. The assay was validated for donor plasma samples and anti-D immunoglobulin preparations and it can also be used in assessing the severity of Rh (D) hemolytic disease during pregnancy.

Rh Haemolytic Disease of the Newborn and Rh genotyping by RFLP - and allele-specific — PCR

Resume L'identification du systeme de groupe sanguin RH est associee a la premiere description du phenomene d'incompatibilite fœtomaternelle responsable de la maladie hemolytique du nouveau-ne. Le systeme RH est extremement polymorphe, mais parmi les 48 antigenes serologiquement identifies, les mieux connus sont les antigenes D, C, c, E et e. Selon la presence ou l'absence de l'antigene majeur RhD sur la membrane des erythrocytes, les cellules sont dites Rh-positives (RH+) ou Rh-negatives (RH−). Le locus RH, localise sur le chromosome 1, est compose de deux genes fortement homologues (92 % d'identite), RHD et RHCE , l'un codant pour l'antigene D et l'autre a la fois pour les polypeptides C/c et E/e vraisemblablement par un mecanisme d'epissage alternatif. La presence ou l'absence du gene RHD constituent la base genetique du polymorphisme de groupe sanguin Rh+/Rh−. Le sequencage des transcrits specifiques du gene RHD et des differents alleles du gene RHCE a permis de definir les bases moleculaires qui determinent les specificites D, C, c, E et e. Sur la base de polymorphismes nucleotidiques ainsi mis en evidence, des oligonucleotides « allele-specifiquesont ete synthetises pour permettre de detecter par PCR sur de l'ADN fœtal la presence du gene RHD et des alleles RHc et RHE dans les situations a risque ou les meres sont respectivement immunisees contre les antigenes D, c et E. Cette methode diagnostique s'avere particulierement utile dans le cas de l'incompatibilite anti-RhD qui constitue la principale cause d'anemie hemolytique severe, laquelle, en l'absence de traitement transfusionnel in utero , conduit generalement a la mort fœtale. En effet, alors que la detection de cette incompatibilite necessitait jusqu'a present la realisation de multiples amniocenteses voire de cordocenteses, avec les risques attenants, le typage Rh+/Rh− de l'ADN fœtal ne necessite qu'une seule amniocentese avec un prelevement de tres faible quantite. Ce test PCR etant realise precocement au cours de la grossesse permet de plus d'eviter chez les meres Rh− porteuses de fœtus Rh− la repetition inutile d'examens prenataux ainsi que l'injection inadaptee d'immunoglobines anti-RhD. D'autre part, d'apres la base genetique du polymorphisme Rh+/Rh−, un genome homozygote pour le gene RHD contient 2 copies de ce gene et 2 copies du gene RHCE , tandis qu'un genome heterozygote pour le gene RHD contient 1 copie de ce gene pour 2 copies du gene RHCE . Ainsi, le genotype Dd ou DD d'un donneur peut etre facilement determine par « Southern bloten comparant les intensites relatives des fragments de restriction specifiques des genes RHD et RHCE apres hybridation avec une sonde ADNc Rh. Cette information s'avere tres utile pour le conseil genetique dans le cas ou une femme hyperimmunisee contre l'antigene D, et ayant deja subi plusieurs fausses couches, veut savoir si son partenaire est Rh positif homozygote ou heterozygote avant d'envisager une nouvelle grossesse.

Neonatal alloimmune thrombocytopenia.

EONATAL alloimmune thrombocytopenia (NAIT) is a relatively rare disease, occurring in 1 in 3,000 to l in 5,000 births. 1.2 The platelet equivalent of hemolytic disease of the newbom, NAIT is the result of matemai alloimmunization against a platelet antigen present on fetal and pa­ ternal platelets but absent on matemai platelets. Although less common than matemai autoimmune thrombocytopenia, matemai alloimmunization leads to much more profound neonatal and perina­ tal thrombocytopenia, resulting in intracerebral hemorrhage and death or severe neurological se­ quelae in up to 30% of affected neonates. 2-4 Unlike red blood cell alloimmunization, NAIT affects the frrst bom infant in 60% of cases. Considerable progress has been made in the treatment of NAIT, therefore it is critical to consider this diagnosis in a thrombocytopenic infant. By analogy with Rh hemolytic disease of the newbom, there is much interest in understanding the immune response to platelet alloantigens, with the ultimate goal of pre­ venting NAIT. For reasons that are not well un­ derstood, only a small percentage of mothers ex­ posed to incompatible platelet antigens form alloantibodies resulting in NAIT. 1.5 This review examines the platelet-specific antigens and anti­ bodies implicated in NAIT, and the physiology of the immune response to HPA-la, the antigen most frequently implicated in NAIT. The laboratory and clinical features of NAIT as well as its treatment and possible prevention is also discussed.

Repeated direct fetal intravascular high-dose immunoglobulin therapy for the treatment of Rh hemolytic disease

Recently, administration of high-dose intravenous immunoglobulin (HDivIG) either to the mother or the neonate has been proposed in an effort to prevent progressive hemolysis in Rh(D) perinatal disease, but no cases have been published with direct fetal HDivIG administration. We report a case in which HDivIG was repeatedly administered by cordocentesis to a fetus affected by Rh(D) disease from 28 to 36 weeks gestation, at doses of approximately 450 mg/kg. The fetus required no transfusions, and the neonatal evolution was satisfactory. The treatment, performed at three weeks intervals, seemed to be useful in preventing fetal hemolysis. No fetal complications were present. Recurrent administration of HDivIG therapy to the fetus in cases of severe Rh(D) disease, appears to be feasible and free of serious complications to the fetus or the mother.

Pregnancy, immunoconflict, neurosensitization

Immunoconflict pregnancy and hemolytic disease of the fetus and newborn remain, despite more than half a century of history, urgent problems in the field of obstetrics and neonatology and continue to be one of the causes of perinatal morbidity. The discovery by Landsteiner and Wiener in 1940 of a causal relationship between Rh isosensitization and hemolytic disease of the newborn (HDN) formed the basis of new branches in immunohematology and perinatology.

Recombinant Erythropoietin as Treatment for the Late Hyporegenerative Anemia of Rh Hemolytic Disease

Infants with Rh hemolytic disease can develop a "late" anemia characterized by low serum concentrations of erythropoietin but erythroid progenitors that remain highly erythropoietin-responsive. Erythropoietin administration was evaluated in two patients as an alternative to transfusion. Reticulocyte counts increased after 5 days of treatment, and hematocrits increased after 10 days. Neither patient received erythrocyte transfusions following erythropoietin therapy.

Maternal Kell blood group alloimmunization

Two recent paper have provided conflicting views regarding the severity of Kell hemolytic disease of the newborn.We reviewed our experience during 1944-1990 with pregnant Kell-alloimmunized Manitoban women and similar women referred from outside of Manitoba.Between 1944-1990, 311 Kell-immunized Manitoban women had 459 pregnancies, of which 63 ended in abortion or stillbirth unrelated to anti-Kell. Of the infants born, 376 were unaffected and 20 were affected. Twelve did not require treatment; two needed phototherapy, one required a simple transfusion, and one an exchange transfusion. One died of kernicterus and three were hydropic and died; all four deaths occurred between 1948-1954. Fourteen Kell-immunized women with 16 pregnancies were referred from outside Manitoba. Eleven had a history of Kell hydropic fetuses and ten had hydropic fetuses at referral. Five of the hydropic fetuses survived and five died. Five women had Kell-negative infants correctly predicted by amniocentesis (two) and by fetal blood sampling (three). Serial amniotic fluid delta OD 450 readings were 83-89% accurate in predicting the presence and severity of Kell hemolytic disease. Life-threatening inaccuracies occurred, primarily in the early and middle second trimester.Kell hemolytic disease, although rare, may be as severe as Rh(D) hemolytic disease when it does occur. When there is a history of hydrops or the father is Kell-positive and the maternal anti-Kell indirect antiglobulin titer is 8 or greater, amniocentesis should be performed at 16-20 weeks' gestation. Fetal blood sampling followed by fetal intravascular transfusion is indicated if delta OD 450 readings approach the 65% level in modified zone 2 of Liley or if amniocentesis is precluded because of an anterior placenta and there is a history of hydrops or ultrasound evidence of fetal hemolytic disease.

High-dose intravenous immune globulin therapy for hyperbilirubinemia caused by Rh hemolytic disease

We conducted a multicenter controlled trial to test the hypothesis that high-dose intravenous immune globulin (HDivIG) therapy can modulate bilirubin production and reduce the frequency of exchange transfusions in newborn infants with Rh hemolytic disease. Thirty-four patients with Rh incompatibility proved by positive direct antiglobulin test (Coombs test) results were randomly assigned to receive conventional treatment including phototherapy, with or without additional HDivIG therapy at 500 mg/kg given for a 2-hour period as soon as the diagnosis was established. Exchange transfusions were performed if serum bilirubin concentrations exceeded the modified curves of Polácek by more than 2 mg/dl. Two patients were excluded because of protocol violations. The results in 32 infants were analyzed. In the HDivIG group, 2 (12.5%) of 16 children required exchange transfusions, whereas it became necessary in 11 (69%) of 16 children in the control group (p less than 0.005). Bilirubin levels in the HDivIG group were lower despite reduced frequency of exchange transfusions. No side effects of HDivIG treatment were observed. We conclude that HDivIG therapy by a yet unknown mechanism reduces serum bilirubin levels and the need for blood exchange transfusions in children with Rh hemolytic disease.

Oliver memorial lecture. Towards the conquest of Rh haemolytic disease: Britain's contribution and the role of serendipity.

Oliver memorial lecture. Towards the conquest of Rh haemolytic disease: Britain's contribution and the role of serendipity.

Protection against immune haemolytic disease of newborn infants by maternal monocyte-reactive IgG alloantibodies (anti-HLA-DR)

The extent to which maternal anti-Rh(D) antibodies support lysis of erythrocytes by monocytes in the antibody-dependent cell-mediated cytotoxicity (ADCC) assay is closely correlated with the severity of Rh(D) haemolytic disease of the newborn infant (HDN). However, in some cases HDN is much milder than would be predicted from the ADCC value. We postulated that maternal ADCC-blocking alloantibodies against paternal antigens on monocytes can protect these infants against severe haemolysis. We studied 13 severely Rh(D)-alloimmunised mothers whose infants showed unexpectedly mild HDN (group I) and 14 women with similar ADCC values but whose infants had severe HDN (group II). 7 group-I women had monocyte-reactive IgG alloantibodies that inhibited lysis by paternal monocytes in the ADCC. No such antibodies were found in group II (p<0·01). In 6 of the 7 serum samples with monocyte-reactive antibodies, the antibodies had HLA-DR specificity. Our findings suggest that Rh(D)-positive children of some severely Rh(D)-alloimmunised women may be protected from severe HDN by maternal non-HLA-class-l, IgG alloantibodies against paternal monocyte blood-group antigens. These antibodies may inhibit the mononuclear phagocyte system of the fetus. Lancet 1992; 339: 1067-70.

Distribution of IgG subtypes in maternal anti-D sera and their prognostic value in Rh haemolytic disease of the new-born.

In 107 Rh(D)-immunized women having Rh(D)-positive pregnancy screening for IgG subtypes was carried out between the 34th and 36th week of gestation. Using polyclonal IgG subtype-specific reagents, all four IgG subclasses were detected in anti-D sera though IgG1 and IgG3 were the most predominant classes. IgG3 anti-D was usually low titre. At the same level of Rh(D) antibody titre, haemolytic disease of the new-born was more severe when anti-D was IgG1 type than IgG3 type. When the IgG1 and IgG3 anti-D subtypes existed together, the risk of having a stillborn child was very high compared to other subtypes (IgG1 + IgG3 34.8%, IgG1 19.2% and IgG3 15.4%).

Necrotizing enterocolitis following intrauterine blood transfusion

Intravascular intrauterine transfusion allows a more sophisticated and exact approach to the management of severe Rh hemolytic disease. This technique involves direct manipulation of the fetal umbilical vessels; its hazards include umbilical cord trauma and thrombosis or emboli. The consequences of such events in utero are largely unknown. In this case necrotizing enterocolitis occured in a full-term infant after three intrauterine intravascular transfusions.

Hemolytic Disease of the Newborn: Lowest Achievable Incidence Rates-Reply

In Reply. —Dr Hensleigh expresses legitimate concerns about the validity or the failure rates of RhIG, rates from which we derived our estimate for the minimum achievable incidence rates of Rh hemolytic disease. The failure rates that we used are based on the results of several studies from around the world that were conducted on populations demographically different from the population of the United States and by different methodologies. Nevertheless, the data summarized in Davey's 1 review of the McMaster Rh Conference of 1979, a more recent review by Keith et al, 2 and the Nova Scotia trials by Baskett et al 3 suggest that for antenatal and postnatal prophylaxis combined, an overall failure estimate of 0.20% of preventing women with at-risk pregnancies from becoming Rh sensitized is a reasonable estimate. The additional contribution to overall failure rates made by women already so sensitized when they were being considered for

Hemolytic Disease of the Newborn: Lowest Achievable Incidence Rates

To the Editor. —Chavez et al 1 found the estimated incidence rate of Rh hemolytic disease of the newborn to be 10.6 per 10 000 total births. They also calculate the minimum achievable incidence rates of Rh disease in the United States to be 3.1 per 10 000 total births. Based on these estimates we appear to be far from the achievable minimum, presumably due to failures to use antenatal and postpartum Rh(D) immune globulin (RhIg). An alternate explanation is that the failure rate of combined antenatal and postpartum use of RhIg that the authors took from the data and method of Bowman and Pollock may be incorrect. The studies from which these data were derived used controls that were neither contemporary nor matched. Other studies have found failure rates of antenatal and postpartum RhIg to be as high as 0.4% for primiparous patients and 0.85% for multiparous patients. 2

Deaths from Rh haemolytic disease in England and Wales in 1988 and 1989.

Deaths from Rh haemolytic disease in England and Wales in 1988 and 1989.

Portable medical record for the homeless mentally ill.

gramme, the time may be right to investigate circum? stances leading to cases of infants affected by Rh haemolytic disease rather than deaths. This is especially important if the death rate is falling while new cases are still occurring at the same rate. Computerised blood transfusion centres could provide information to interested clinicians about new cases occurring. This could be a topic for a regional obstetric audit group. In summary, we think that the overall outlook for almost complete prevention of Rh haemolytic disease deaths is good. The introduction of monoclonal anti-D is on the horizon and, when it is readily available, will contribute to a reduction in new cases of sensitisa tion.' We are grateful to Professor P L Mollison for advice on the classification of cases, and Professor Charles Rodeck and Dr L A P Tovey for commenting on the changing rates of death in 1988 and 1989. The Office of Population Censuses and Surveys kindly forwarded the death information and Mrs E Batty typed the manuscript. The work is supported by the Mersey Regional Research Committee.