Immunoprophylaxis with IgG anti-D is a standard prevention of hemolytic disease of the fetus and newborn. Fetal Rhesus D (RhD) blood group genotyping from maternal plasma of RhD-negative pregnant women allows targeted prophylaxis with IgG anti-D in RhD-positive pregnancies only. We set up a reliable protocol for prenatal RHD genotyping.153 pregnant Caucasian RhD-negative women were tested in the 27th week (range 7-38th week) of pregnancy. 18 of them were alloimmunized to the RhD antigen. The fetal RHD genotype was determined based on an automated DNA extraction and real-time polymerase chain reaction method. Intron 4 and exons 5, 7 and 10 of the RHD gene and the SRY gene were targeted.The fetal RhD status and gender was 100% correctly predicted in all 153 pregnancies (55 RhD-positive males, 45 RhD-positive females; 23 RhD-negative males, 30 RhD-negative females).The accuracy and applicability of our protocol for non-invasive fetal RhD determination allows the correct management of RhD-incompatible pregnancies. Our protocol could prevent unnecessary immunoprophylaxis in 53 of 153 cases. We therefore recommend that non-invasive fetal RHD genotyping is introduced as an obligatory part of prenatal screening.

BACKGROUND:

Red blood cell (RBC) alloantibodies to nonself antigens may develop after transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans.

STUDY DESIGN AND METHODS:

We recently generated transgenic mice with RBC-specific expression of the human KEL glycoprotein, specifically the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system.

RESULTS:

Transfusion of RBCs from KEL2 donors into wild-type recipients (lacking the human KEL protein but expressing the murine KEL ortholog) resulted in dose-dependent anti-KEL glycoprotein immunoglobulin (Ig)M and IgG antibody responses, enhanced by recipient inflammation with poly(I:C). Boostable responses were evident upon repeat transfusion, with morbid-appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic after transfusion into wild-type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses.

CONCLUSIONS:

This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long-term translational goals of these studies include improving transfusion safety for at-risk patients.

Maternal red-cell alloimmunization occurs when a woman's immune system is sensitized to foreign red-blood cell surface antigens, leading to the production of alloantibodies. The resulting antibodies often cross the placenta during pregnancies in sensitized women and, if the fetus is positive for red-blood-cell surface antigens, this will lead to hemolysis of fetal red-blood cells and anemia. The most severe cases of hemolytic disease in the fetus and newborn baby are caused by anti-D, anti-c, anti-E and anti-K antibodies. There are limited data available on immunization rates in pregnant women from Turkey. The aim of the present study was to provide data on the frequency and nature of maternal RBC alloimmunization in pregnant women in a tertiary care hospital. In this study, we retrospectively evaluated the indirect antiglobulin test results of Rh-negative pregnant women performed in our Blood Bank between 2006 and 2012. Indirect antiglobulin test positive women also underwent confirmatory antibody screening and identification. During the study period, 4840 women admitted to our antenatal clinics. With regards to the major blood group systems (ABO and Rh), the most common phenotype was O positive (38.67%). There were 4097 D-antigen-positive women (84.65%) and 743 women with D-antigen-negative phenotype (15.35%). The prevalence of alloimmunization was found to be 8.74% in D-antigen negative group. Despite prophylactic use of Rh immunglobulins, anti-D is still a common antibody identified as the major cause of alloimmunization in our study (anti-D antibody 68.57%, non-D antibody 31.42%). While alloimmunization rate to D antigen was 6.46%, non-D alloimmunization rate was 2.69% among Rh-negative pregnant women. Moreover, detailed identification facilities for antibodies other than anti-D are not available in most of centers across Turkey. However, large-scale studies on pregnant women need to be done in order to collect sufficient evidence to formulate guidelines and to define indications for alloantibody screening and identification.

To determine the incidence of clinically significant anti-erythrocyte alloantibodies in pregnant women, which can cause severe hemolytic disease in the fetus and newborn.Retrospective-prospecitive clinical study.Transfusion Department, University Hospital Olomouc, Department of Obstetrics and Gynecology, University Hospital Olomouc.Between the years 2000-2011, a total of 45 435 pregnant women were examined at the Department of Transfusion Medicine at the University Hospital Olomouc. Screening for irregular anti-erythrocyte antibodies followed by identification of the alloantibody was performed in all women at the beginning of the pregnancy.Clinically significant anti-erythrocyte antibodies were diagnosed in 1.5% pregnant women (683/45435). The most common cause of maternal alloimmunization was antigen E with an incidence of 5.7 (258/45435), followed by antigen D 4.0 (181/45435), M 1.5 (70/45435), C 1.2 (54/45435), K 1.2 (55/45435), c 0.6 (26/45435), S 0.4 (20/45435), Jka 0.2 (9/45435), PP1pk (Tja) 0.1 (3/45435) and antigen Fya 0.0 (2/45435).Despite performing prophylaxis for RhD alloimmunization by administering anti-D immunoglobulin to RhD negative women during pregnancy and after the birth of an RhD positive child, antigen RhD still represents the 2nd most frequent cause of maternal erythrocyte alloimmunization. The remaining clinically significant alloimmunizations are caused by non-D antigens of the Rh system, antigens of the Kell system, and rarely observed antigens of the MNS and Kidd blood systems.

BACKGROUND:

Antibodies to Kell antigens can be clinically important but only limited data are published regarding anti-Ku. Missense nucleotide changes in KEL account for the numerous Kell antigens, the Kmod phenotype, and even the Knull phenotype.

STUDY DESIGN AND METHODS:

DNA and RNA were extracted from white blood cells and polymerase chain reaction-based assays, cloning, and sequencing were done using standard protocols.

RESULTS:

The anti-Ku in Proband 1, which caused hemolytic disease and anemia of the fetus and newborn, was a mixture of immunoglobulin (Ig)G1 and IgG2 and gave macrophage indexes ranging from 47.8 to 59.3 (>20 is clinically significant) in a monocyte monolayer assay. The proband, her daughter, and compatible sister had a heterozygous deletion of a G in Exon 18 (Nucleotide c.1972_1975delG) in a KEL*02 allele causing a frameshift. The mechanism for silencing of the other KE*02 allele was undetermined. Proband 2 was heterozygous for a nonsense change (KEL*382C/T; Arg128Stop), a missense change (KEL*244T/C; Cys82Arg), and KEL*578T/C (KEL*01/KEL*02). Direct sequencing of cDNA and cloning showed that the KEL*01 allele had 244C, 382C, 578T and the KEL*02 allele carried 244T, 382T, 578C.

CONCLUSIONS:

We report a novel single-nucleotide deletion, a novel nonsense allele, and a novel missense allele all resulting in the Knull phenotype. The anti-Ku from Proband 1 was clinically important.

Maternal allo-antibody production is stimulated when fetal red blood cells are positive for an antigen absent on the mother's red cells. The maternal IgG antibodies produced will pass through the placenta and attack fetal red cells carrying the corresponding antigen. Allo-immune hemolytic disease of the fetus and newborn caused by anti-E rarely occurs.We report two cases of anti-E hemolytic diseases in neonates. One of the neonates had severe hemolysis presenting with severe anemia, thrombocytopenia, and conjugated hyperbilirubinemia, while the other had moderate anemia and unconjugated hyperbilrubinemia. Although both the neonates were treated by phototherapy and intravenous immunoglobulin, one of them received double volume exchange transfusion.There appeared to be an increase in the occurrence of hemolytic disease of the fetus and newborn caused by Rh antibodies other than anti-D. In this case report, both patients presented with anemia and hyperbilirubinemia but were successfully treated, with a favorable outcome.

During pregnancy, a Rhesus-negative (Rh-negative) woman may develop antibodies if her fetus is Rh-positive, which can cause fetal morbidity or mortality in following pregnancies, if untreated.To assess the effects of administering anti-D immunoglobulin (Ig) after spontaneous miscarriage in a Rh-negative woman, with no anti-D antibodies.We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 December 2012).Randomised controlled trials (RCT) in Rh-negative women without antibodies who were given anti-D Ig following spontaneous miscarriage compared with no treatment or placebo treatment following spontaneous miscarriage as control.Two review authors independently assessed trials for inclusion and trial quality. Two review authors extracted data and checked it for accuracy.We included one RCT, involving 48 women who had a miscarriage between eight to 24 weeks of gestation. Of the 19 women in the treatment group, 14 had therapeutic dilatation & curettage (D&C) and five had spontaneous miscarriage; of the 29 women in the control group, 25 had therapeutic D&C and four had spontaneous miscarriage. The treatment group received 300 µg anti-D Ig intramuscular injection and were compared with a control group who received 1 cc homogenous gamma globulin placebo.This review's primary outcomes (development of a positive Kleihauer Betke test (a test that detects fetal cells in the maternal blood; and development of RhD alloimmunisation in a subsequent pregnancy) were not reported in the included study.Similarly, none of the review's secondary outcomes were reported in the included study: the need for increased surveillance for suspected fetal blood sampling and fetal transfusions in subsequent pregnancies, neonatal morbidity such as neonatal anaemia, jaundice, bilirubin encephalopathy, erythroblastosis, prematurity, hypoglycaemia (low blood sugar) in subsequent pregnancies, maternal adverse events of anti-D administration including anaphylactic reaction and blood-borne infections.The included study did report subsequent Rh-positive pregnancies in three women in the treatment group and six women in the control group. However, due to the small sample size, the study failed to show any difference in maternal sensitisation or development of Rh alloimmunisation in the subsequent pregnancies.There are insufficient data available to evaluate the practice of anti-D administration in an unsensitised Rh-negative mother after spontaneous miscarriage. Thus, until high-quality evidence becomes available, the practice of anti-D Immunoglobulin prophylaxis after spontaneous miscarriage for preventing Rh alloimmunisation cannot be generalised and should be based on the standard practice guidelines of each country.

Rh blood group system is one of the most polymorphic systems of human blood and consists of 50 antigens. Antigen D is the most important antigen in the Rh system and next to ABO, is the most clinically significant in transfusion medicine. The aim of this paper was to present a case of a rare Rh phenotype ccDEE in an immunized pregnant woman, whose fourth pregnancy ended with birth of a female newborn infant with hemolytic disease of the lower level.The history of a 42-year-old pregnant woman stated that she had had four pregnancies. She was transfused with 1500 ml of whole blood, three units of packed red cells and two units of fresh frozen plasma. Due to her high-risk pregnancy she was referred to the Clinic of Gynecology and Obstetrics in Novi Sad. Blood sample was tested in the Department of Prenatal Care of the Institute for Blood Transfusion Vojvodina. ABO and Rh were tested, antibody screening was done by indirect antiglobulin test and the detected antibodies were identified by gel technology. The results of testing were: O RhD positive, Rh phenotype ccDEE, positive screening for red blood cells antibodies by indirect antiglobulin test, alo anti-e antibody. According to the literature data, it is a very rare Rh phenotype whose incidence in the population ranges from 0.34% to 1.99%. The compatible blood products for the patient and her newborn were searched for on the basis of the immunoserology tests.Two major problems within transfusion medicine have emerged in our case: the problem of immunization of pregnant woman with a rare blood type and the problem of finding compatible blood. Health care of pregnant women can be improved by following pregnancies according to the national antenatal testing algorithm and better teamwork of gynecologists and transfusions.

The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.