Search

An early-term, 2,860-g female infant is delivered via caesarean section by a 28-year-old gravida 3, para 1-1-1 woman. Her second pregnancy had terminated in an abortion 4 years ago with the cause unknown. Results of serologic studies at the time of her previous pregnancies are not available, but the antibody screen (3-cell panel, Immucor Inc, Peachtree Corners, GA) during the present pregnancy is positive. The mother’s red blood cells (RBCs) are typed as B Rh (D) positive. The sample reacts with all cells in the 16-cell panel (Capture-R Ready-ID, Immucor Inc) and 11 cell panels (ID DiaPanel, Biorad Labs, Hercules, CA). The auto control and direct antiglobulin test results are negative. The reaction pattern seen on screening and identification panels indicates the presence of an RBC alloantibody to a high-frequency antigen. Crossmatch with multiple B-positive and O-positive RBC units are incompatible on Coombs testing. Extended phenotyping of the mother for minor blood group antigens of high frequency reveals her minor blood group phenotype to be K−, k+, Kp (a−b+), Js (a+b−), Lu(a–b+) and In(a–b+). Allogeneic adsorption studies done using 0.01%-ficin treated RBCs of known phenotype to deliberately adsorb the anti-Jsb from the mother’s serum confirms the absence of other common alloantibodies. The antibody is confirmed to be anti-Jsb alloantibody. The anti-Jsb titer using the antiglobulin technique Jsb(+) red cells is 128. …

of the study is to research the epidemiological aspects of maternal alloimmunization against erythrocyte antigens of fetuses (AB0, Rhesus, Lewis, Kell, Duffy and others) and to identify the most common types of hemolytic disease of the newborn (HDN) in the West Herzegovina region.The 20-year retrospective epidemiological study includes all pregnant women who had been immunologically tested and newborn treated for HDN.The indirect antiglobulin (IAT) detected antibodies against antigens in 545 (1.8%) pregnant women of the 29 663 who were tested at the Department of Transfusion Medicine. During the 20-year-long study 310 (1.0%) newborn with HDN were treated. Our results indicate that 42% (230/545) of the pregnant women had AB0 immunization. The most common form of HDN is AB0 HDN 64% (199/310), whereas RhD HDN was treated in 19% (59/310) of the newborn infants. ETR was performed on 29 (19%) infants, 21 (72.4%) with AB0 HDN, and 7 (26%) with RhD HDN.This 20-year-long study concludes that, even though there has been significant progress in the prevention of immunization and proactive treatment of HDN, precautionary measures are still required as is the need for gynecologists and obstetricians to be active. The reasons for this are the non-existence of preventive measures for non-RhD immunization, the irregular immunological screening of RhD positive women in pregnancy in the region encompassed by the study in the past few years. The above raises new questions and recommends further research and monitoring of immunization and HDN treatment worldwide.ZIEL DIESER STUDIE: war die Untersuchung von epidemiologischen Aspekten mütterlicher Immunisierung gegen fetale Erythrozytenantigene(AB0, Rhesus, Lewis, Kell, Duffy und andere) und die Feststellung von häufigsten Ursachen der hämolytischen Erkrankung des Neugeborenen (MHN) in der Region der West-Herzegowina.20-jährige retrospektive epidemiologische Studie umfasste alle Schwangeren, die immunologische getestet sowie Neugeborene, die wegen MHN behandelt wurden.Durch den indirekten antiglobulin Test (IAT) wurden bei 545 (1,8%) Schwangeren Antikörper gegen Antigene nachgewiesen, von 29 663 Probanden, die an der Transfusionsanstalt UHC Mostar getestet wurden. Während der zwanzigjährigen Studie hatten 310 (1,0%) Neugeborene MHN. Unsere Ergebnisse zeigten, dass 42% (230/545) der Schwangeren die AB0-Immunisierung hatten. Die häufigste MHN bei dieser Forschung war die AB0 MHN 64% (199/310), während RhD MHN bei 19% (59/310) der Neugeborenen behandelt wurde.Die Blutaustauschtransfusion (ETR) erfolgte bei 29 (19%) Neugeborenen, 21 (72,4%) bei Neugeborenen mit AB0 MHNund 7 (26%) mit Rh D MHN.Obwohl es zu während dieser 20-jährigen Studie zu einem bedeutenden Fortschritt bei der Vorbeugung der Immunisierung und der proaktiven MHN-Behandlung kam, erfordert es weiterhin Vorsichtsmaßnahmen und die Aktivitäten der Gynäkologen und Neonatologen. Grund dafür ist das Nichtbestehen von Vorbeugungsmaßnahmen zur nicht-RhD Immunisierung (67% der Schwangeren hatten Nicht-RhD Antikörper), unregelmäßiges immunologisches Testen von RhD-positiven Schwangeren in den letzten Jahren im Untersuchungsgebiet. All dies eröffnet neue Fragen und Empfehlungen zur Erforschung und Beobachtung der Immunisierung sowie MHN-Behandlung auf der globalen immunologischen Szene.

Since its discovery in 1953, the presence of the antigen Diego (Di) has been related to the Mongoloid populations and American natives. Given its immunogenic potential, the presence of the mentioned antigen is related to the development of hemolytic disease of the newborn (HDN). We present the case of a puerperal patient with no known history who was detected the presence of antibodies to Di b during study of a mild HDN presented by her daughter. When studying the patient's family, the presence of Dib antigen was reported in a brother as well as in both parents. We consider that being a rare event, this type of case should be reported promptly to promote the exchange of knowledge with other health professionals and the community.

Background. This study is devoted to a rare variation of the -D- phenotype . The -D- phenotype was first discovered by R. Race, R. Sanger and J.G. Selwyn in 1951. In Russia, the phenotype -D- was first discovered by V. Morokov in 1985. Typically, the -D- phenotype is detected when physicians examine post-transfusion complications or hemolytic disease of the newborn, since such patients demonstrate high antibody titres to absent antigens. In the present study, the -D- phenotype was detected in a primary blood donor at the clinical laboratory of the Ulyanovsk Regional Blood Transfusion Station (Ulyanovsk, Russia). Aim. To study specific features of immunohematological and hematological blood parameters in a donor with a rare variation of the -D- phenotype.Materials and methods. The detection of the -D- phenotype by immunohematological methods was carried out using automatic analysers. Molecular DNA typing was used to confirm the -D- phenotype. The shape of erythrocytes of the donor with the -D- phenotype was evaluated using an atomic force microscope. The characteristics of the erythroid lineage were studied using an automatic hematological analyser.Results. The -D- phenotype was detected in a primary blood donor. Due to the extreme rarity of the -D- phenotype and the lack of programmed algorithms, the validation of the results by automatic analysers was incorrect. Of critical importance was the visual assessment of gel ID cards by the medical staff. Genotyping confirmed the lack of C, c, E, e, Cw specificities in the RHCE gene. The hematological parameters of the donor were within the age norm. An assessment of the image of a cytological blood preparation did not reveal changes in the shape of erythrocytes and their size.Conclusions. The primary determination of the -D- phenotype using automatic immunohematological analysers can be complicated by the impossibility of validating the results, the incorrect operation of the installed software and the need for expert evaluation of blood samples by the staff. The presented case of the -D- phenotype was not associated with changes in the shape of erythrocytes and blood hematological parameters.

Background The clinical and laboratory features of hemolytic disease of the newborn can be challenging to diagnose during pregnancy in the apparent absence of a blood group antibody. Low-frequency antibodies go undetected due to the lack of appropriate antigen-positive reagent red blood cells (RBCs). Case report A pregnant woman of Southeast Asian descent was referred to a maternal-fetal medicine outpatient clinic due to a complicated obstetric history and a negative antibody screen. This initial visit at 29 weeks and 0 days' gestational age (GA) was unremarkable. A hydropic infant, born at 29 weeks and 5 days' GA, succumbed on the seventh day of life. Comprehensive laboratory testing was performed after birth. The hospital blood bank performed a maternal antibody identification. Direct antiglobulin test was performed on the cord blood. A reference laboratory confirmed an anti-Mia , performed paternal Mia phenotyping, and identified a hybrid glycophorin B-A-B GP*Mur allele. Discussion Maternal alloimmunization to low-frequency antigens remains a challenge. Southeast Asians make up a significant percentage in some US locations. Worldwide reports on the frequency of maternal alloimmunization of the MNS system can be used to guide the use of specific reagent RBCs for testing. Such strategies rely on the identification of blood donor units for reagent manufacture and use in perinatal antibody screens. Conclusion The incidence of Mia and related antibodies is significant among Southeast Asians. In North America, prenatal antibody screening cells are not routinely chosen to match this population. The clinical and societal implications are discussed.

Background Anti-D antibody is not the common cause of Rh-isoimmunization in Chinese neonatal jaundice. Recent change in national population policy has followed by an increase in Rh-isoimmunization related hemolytic disease of the newborn (HDN). Unfortunately, regional status of Rh-HDN is unavailable. We hypothesize that Rh-HDN in our region is most commonly due to anti-E antibody. Aim To investigate the prevalence of hemolytic disease of the newborn due to Rh-isoimmunization in Hefei City. Methods Retrospective review of data obtained from Children's Hospital of Anhui and Hefei Blood Center between January 2017 and June 2019. Status of minor blood group antibody was studied in the corresponding mothers. Results Totally 4138 newborns with HDN admitted during the study period and 116 (2.8%) received blood exchange transfusion (BET). Eighteen newborns (0.43%) with proven Rh-incompatible HDN were identified. All were not the first-born baby. Thirteen mothers were RhD (+) (72%) and five were RhD (-). The distribution of Rh-related antibodies in mothers was ten anti-E (55%), five anti-D (27%), and for one anti-C, anti-c, and anti-E/c (6%) each. Thirteen (72.2%) were qualified for BET, relative risk for BET was 28.9 as compared to other types of HDN, but only 10 received due to parenteral refusal. All (100%) RhD related HDN received BET which is not significantly different from RhE related HDN (81.8%). Conclusion As expected, all Rh-incompatible HDN newborns were not the first-born. Contrary to the Caucasian population, anti-D induced HDN is not the most common etiology. In our region, anti-E (11/18, 61%) is the most common cause of Rh-HDN.

We report on a term infant with clinically significant hemolysis and hyperbilirubinemia. Testing revealed ABO incompatibility between maternal type A and infant type AB. The maternal alloantibody screen was negative. The infant’s direct antiglobulin test was positive, and anti-B IgG was eluted off the infant’s red blood cells (RBCs). Testing of the mother’s plasma revealed a high anti-B titer. The infant was successfully treated with phototherapy and intravenous immunoglobulin. The bilirubin and hematocrit stabilized, and the infant was discharged home. This case was unusual because of its severity and unusual ABO constellation. Furthermore, this report is an exemplary educational case study on how effective collaboration between the clinical team and the blood bank laboratory is critical in reaching the correct diagnosis. In summary, the differential diagnosis of more unusual and atypical ABO-incompatible constellations must be considered when an infant presents with unexplained hemolysis.

The Rh blood group system is a complex blood group which includes different antigen specificities such as c antigen. Anti-c antibody is associated with both acute and delayed hemolytic transfusion reactions as well as hemolytic disease of the newborn (HDN). Rh mediated hemolytic transfusion reactions (HTR) are mostly immunoglobulin G (IgG) mediated and results in extravascular hemolysis and delayed HTR (DHTR). However, we are presenting a case of acute intravascular hemolytic transfusion reaction due to anti-c in a patient with acute subdural hematoma. A 77-year-old woman was referred to our hospital with a loss of conscious and left-sided hemiparesis. After an emergency MRI, she was diagnosed with Acute Subdural hematoma, and an emergency craniotomy was performed. Since Acute Subdural hematoma is a neurosurgery emergency, laboratory technician performed an Immediate-spincrossmatchedd for blood bag to preserve time. During the transfusion of the first packed cell, the patient developed severe hypotension and tachycardia. Thus, the transfusion was stopped. Laboratory results raised the suspicion of an Acute Intravascular Hemolysis. Antibody identification revealed that the patient had an irregular blood phenotype (C2+/c-/E-/e3+/K-), and the presence of alloantigen-c Rh antibody confirmed the suspicion of HTR. In patients with multi transfusion history and pregnant women, pre-transfusion screening of irregular antibodies must be performed. The immediate spincrossmatchh must be prevented in patients with a history of multi transfusions, even in emergency situations.

Background: Isoimmune hemolytic disease is a major cause of neonatal severe indirect hyperbilirubinemia that requires phototherapy or exchange transfusion which is an invasive procedure to avoid brain injury. Administration of intravenous immunoglobulin (IVIG) is used as an adjunct treatment to phototherapy in order to decrease the rate of exchange transfusion. Methods: This retrospective case-control study aimed to describe the safety and efficacy of IVIG therapy in newborns with isoimmune hemolytic disease and to compare their clinical outcomes to those of a control group who were treated only with phototherapy. Criteria for IVIG treatment were variable; when phototherapy threshold was reached or when exchange transfusion level was approached, using either indication is based on the attending discretion. Results: Ninety-four infants were included in the IVIG group, compared to 108 infants in the control group. Most of the included infants were term infants and most common cause was ABO incompatibility. There were no side effects documented in all the included infants. The IVIG group had more severe hemolysis with average highest bilirubin of 14.6 ± 3.7 mg/dL in the IVIG group versus 12.6 ± 3 in the control group (P = 0.0001). Complication of hemolysis was seen more in the IVIG group with higher rate of rebound hyperbilirubinemia, blood transfusion and exchange transfusion. Conclusions: IVIG use as an adjunct treatment to phototherapy in isoimmune hemolytic disease of the newborns is safe. The favorable results of the phototherapy only group were supportive of using selective criteria for administration of IVIG in neonates with isoimmune hemolytic disease. J Clin Med Res. 2019;11(11):760-763 doi: https://doi.org/10.14740/jocmr4003

Background: hemolytic disease of the newborn is an autoimmune haemolytic disease and caused by antibodies produced due to Rh and ABO incompatibilities. Neonatal jaundice is a common clinical problem encountered during the neonatal period. To avoid the associated neurological complications; exchange transfusion and phototherapy have been traditionally used. Aim of the work: was to assess the efficacy of intravenous immunoglobulins in neonates with Rh and/or ABO incompatibility in reducing the duration of phototherapy and the need for exchange transfusion and hospital stay. Subjects and Methods: in this clinical intervention study 40 patients diagnosed with hemolytic disease of newborn were grouped into group A and B. All neonates were treated with standard protocol for phototherapy and exchange transfusion. Group A patients received only phototherapy. Group B patients received intravenous immunoglobulins in a dose of 0.5 gm/kg of body weight single dose and phototherapy. Exchange transfusion was given to the patients of both groups if the bilirubin rose by 0.5 mg/dl per hour. Results: there was a significant decrease of bilirubin level in group B [that received phototherapy plus Intravenous Immunoglobulin (IVIG)] than group A [that received phototherapy only]. Hospital stay and duration of phototherapy decreased in group B than group A. Number of cases exchanged blood in group A was higher than cases of group B. Conclusion: addition of intravenous immunoglobulins for treatment of hemolytic disease of newborn significantly reduces bilirubin levels, duration of phototherapy, need for exchange transfusion and hospital stay.

Hemolytic disease of newborn (HDN) is a condition that develops in a fetus, when the IgG molecules produced by the mother pass through the placenta and attack the fetal red blood cells. HDN can occur due to Rh and ABO incompatibilities between the mother and the fetus as well as due to other allo-immune antibodies belonging to Kell (K and k), Duffy (Fya), Kidd (Jka and Jkb), and MNS (M, N, S, and s) systems. Role of intravenous immunoglobulin in management of HDN is not clear.SARA red blood cell antigen, first discovered in 1990 is a low frequency antigen. We report, a multiparous female whose pregnancy was complicated by HDN due to anti-SARA antibodies requiring both exchange transfusion and intravenous immunoglobulin. The response was sustained after intravenous immunoglobulin (IVIG) rather than after exchange transfusion.

Background The distribution of the Mur blood group antigen is 5–7% in the south of China, and a much higher prevalence is observed in some areas of the region. Anti-Mur can cause hemolytic disease of the newborn and severe transfusion reactions. Objectives Genetic testing is more ideal than conventional serological tests because antibodies for detection are usually not available. Methods In this study, a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Mur blood group antigen was established. Results Fifteen of 275 (5·5%) samples were confirmed by LAMP as Mur antigen positive. All the Mur antigen-positive samples were GP.Mur subtype which was confirmed with sequencing. Conclusion The LAMP method has identical results with conventional serology method but more suitable for large-scale screening.

BACKGROUND Hyperbilirubinemia, which is a sign of hemolytic disease of the newborn (HDN), can irreversibly damage the central nervous system. OBJECTIVES To determine the etiology of HDN in affected patients and characterize the changing pattern of bilirubin using direct antiglobulin testing (DAT). METHODS We collected clinical data from newborns who underwent perinatal DAT and from their mothers, between August 2008 and July 2017. RESULTS Among 303 neonates, 37 (12.2%) showed positive DAT results. The positive predictive values (PPVs) and negative predictive values (NPVs) based on DAT results were 75.7% and 28.9%, respectively, for starting phototherapy. Bilirubin levels increased more rapidly in the DAT-positive group, compared with the DAT-negative group. The initial bilirubin level differed significantly according to the etiology of hyperbilirubinemia. Further, neonates with anti-D showed higher delta bilirubin per day than neonates with other antibodies. CONCLUSION Our results may help to determine the measurement period for bilirubin according to DAT results and etiology.

ABO incompatibility of red blood cells leads to brisk complement-mediated lysis, particularly in the setting of red cell transfusion. The ABO blood group is the most clinically significant blood group because of preformed immunoglobulin M (IgM) and IgG antibodies to ABO blood group antigens (isohemagglutinins) in everyone except group AB individuals. In addition to transfusion, ABO incompatibility can cause hemolysis in hematopoietic and solid organ transplantation, hemolytic disease of the newborn, and intravenous immunoglobulin infusion. It is important to prevent ABO incompatibility when possible and to anticipate complications when ABO incompatibility is unavoidable.

Background and objectives To determine the effects of intravenous immunoglobulin (IVIg) on the course of late hyporegenerative anemia and the requirement for exchange transfusions, phototherapy and top-up red blood cell transfusions due to rhesus hemolytic disease of the newborn (Rh-HDN). Patients and methods This is retrospective cohort study of newborns with Rh-HDN who were treated with or without IVIg. Relevant maternal and neonatal data were obtained and analyzed. All infants were followed up as outpatients for a period of 2–3 months, and during the follow ups, complete blood counts (RBCs) were performed every two weeks to diagnose late hyporegenerative anemia. Results Sixty-six infants with Rh-HDN were enrolled in this study (42 in the IVIg group and 24 in the no-IVIg group). We observed no significant differences between the two groups in terms of maternal age, number of previous children affected with Rh-HDN, intrauterine transfusions, gestational age (GA), birth weight (BW), Apgar scores at the 1st or 5th minutes, exchange transfusions, or days on phototherapy. The serum bilirubin immediately after birth and the maximum serum bilirubin during the hospital course were significantly higher in the IVIG group (P = 0.0035 and P = 0.0225, respectively). Although there were no statistically significant differences, there was a trend toward the late anemia in the IVIg group being diagnosed and resolved earlier than in the no-IVIg group. Additionally, a greater proportion of the IVIg group required three or more top-up transfusions compared to the no-IVIg group, but this difference was not statistically significant. Conclusion IVIg might have had an effect on the early development of late anemia and might have shortened the duration of late anemia relative to the infants who did not receive IVIg. The exchange transfusions and the days on phototherapy were not affected by IVIg.

BACKGROUND: RhIG is used worldwide to reduce the incidence of alloimmunization to D during pregnancy. We report a case of clinically significant neonatal hemolysis mediated by maternally administered RhIG. CASE REPORT: A 25-year-old, O‐, primigravid mother with a negative antenatal antibody screen delivered a 6-lb 4-oz, blood group A, D+ baby girl at 36.5 weeks’ gestation. Prenatal care included a dose of intramuscular RhIG at 28 weeks’ gestation. At delivery, the newborn was markedly jaundiced with a total bilirubin of 6.3 mg/dL, which reached more than 20 mg/dL after 6 days. The newborn’s lactate dehydrogenase (LDH) was 485 U/L (normal

OBJECTIVE: To investigate the effects of blood transfusion and number of pregnancies on ABO antibody titers and irregular antibodies in pregnant women with type O blood. MATERIALS AND METHODS: The study included 4,200 pregnant women with type O blood (their husbands were with non-O type blood) that were divided into transfusion group and non-transfusion group, according to whether they had a history of blood transfusion. The both groups were respectively divided into three subgroups (the number of pregnancies was one, two, and > or = three). The ABO antibody titers and irregular antibodies were detected at the same time. The effects ofABO antibody titers and irregular antibodies on hemolytic disease of the newborn (HDN) were discussed. RESULTS: There was no consistency of ABO antibody titers and existence of irregular antibody. The positive rates of irregular antibody of transfusion group and of the subgroup (number of pregnancies > or = three) were far higher than that of non-transfusion group and of the subgroups (number of pregnancies or = three or with history of blood transfusion, the prenatal joint detection of ABO antibody titers and irregular antibodies is helpful for accurately reflecting the in vivo antibody type and level.

Aim: This study was aimed to review and establish the practice of exchange transfusion (ET) with whole blood reconstituted (WBR) in hemolytic disease of newborn (HDN). Objectives: To observe fall in indirect serum bilirubin, correction of anemia and comparison with related studies. Background: Hemolytic disease of the Newborn is characterized by presence of IgG antibodies in maternal circulation, which causes hemolysis in the fetus by crossing the placenta and sensitizing red cells for destruction by macrophages in the fetal spleen with consequent hyperbilirubinemia. Exchange transfusion with or without phototherapy is the method of choice for treating the newborn with on going hemolysis Methods/Materials: Sample size consisted of 110 neonates in whom 119 exchange transfusions were carried out with WBR. WBR was prepared by suspending O Rhesus-D (RhD) positive/negative cells (compatible with neonate’s/ mother’s serum) in AB plasma. Double volume exchange transfusion(s) were carried out through umbilical vein by push-pull technique. Results: Out of 110 cases, 61 (55.5%) were of RhD HDN whereas ABO and other group HDN cases were 30 (27.3%) and 19 (17.3%) respectively. An average post-ET fall in indirect serum bilirubin by 54.6% and correction of anemia by3.7 gm/dl were reported in the study. Conclusion: An average post-ET fall in indirect serum bilirubin and correction of anemia was found to be more significant when compared to other studies. Hence we recommend exchange transfusion in HDN with WBR to obtain reasonable fall in indirect serum bilirubin and high average rate of correction of anemia.

Hemolytic disease of the newborn (HDN) is a clinic phenomenon which occurs during pregnancy due to the Rhesus (Rh) D alloimmunization between a Rh (-) pregnant woman, who has become sensitive to RhD antigens, and her Rh (+) fetus. As a result of the attack of maternal RhD antibodies on fetal RhD antigens, fetal anemia, HDN and fetal death may occur. % 40 of Rh (-) pregnant women carry Rh (-) fetus. However, all Rh (-) pregnant women are offered anti-D Immunoglobulin (Anti-D Ig) at 28 weeks' gestation in case of fetomaternal haemorrhage, so the pregnant women carrying Rh (-) fetus are exposed to blood products unnecessarily. Although the RhD of fetus can be detected, methods used for prenatal diagnosis recently are invasive tests and they can result in abortion in a certain percentage. The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for non invasive prenatal diagnosis. The aim of this study was to detect prenatal RhD by analysing the presence of the RhD gene of fetal DNA in maternal blood.Total free DNA was isolated from the blood of 19 Rh (-) pregnant women, who had RhD alloimmunization with their husbands, in the 11-14 th week of their pregnancy. The existence of a gene in isolated DNA was investigated with TaqMan prob and "Real-time PCR" method by using primers belonging to exon 7 of RhD gene.Using a quantitative real-time PCR assay, the presence of RhD gene sequences was evaluated in the serum of patients at the onset of pregnancy. We have analyzed 19 Rh (-) pregnant women. Twelve of them were Rh (-) and the rest of them were 7 Rh (+). After birth the baby's blood groups were concordant with our results.The results obtained by RhD primer were analysed. The possibility of detection of fetal RhD gene in maternal blood contributed to noninvasive prenatal diagnosis.Yeni doğanın hemolitik hastalığı, daha önce “Rhesus” (Rh) D antijenleri ile duyarlı hale gelmiş Rh (−) anne ile Rh (+) çocuğu arasındaki RhD uyumsuzluğuna bağlı olarak hamilelik sürecinde ortaya çıkan klinik bir tablodur. Maternal RhD antikorlarının fetal RhD antijenlerini yok etmesiyle, fetusta kansızlık, yenidoğanda sarılık veya fetal ölüm gerçekleşebilir. Rh (−) gebelerin yaklaşık % 40′ı Rh (−) fetus taşımaktadır. Buna rağmen tüm gebelere olası bir fetal kanama riskine karşı hamileliklerinin 28. haftasında anti D immünglobulin (Anti-D Ig) uygulanmakta ve Rh (−) fetus taşıyan gebeler gereksiz aşı ürününe maruz kalmaktadır. Doğum öncesi tanı ile fetusun RhD tayini yapılabilse de, günümüzde kullanılan doğum öncesi tanı metodları girişimsel tetkikler olup belli oranlarda gebelik kayıplarına neden olabilmektedir. Anne kanında fetusa ait nükleik asitlerin hücre dışında serbest dolaşabileceklerinin keşfi noninvazif prenatal tanı için yeni olanaklar açmıştır. Bu çalışmada da maternal kandan fetal DNA’da RhD geninin varlığına bakılarak doğum öncesi RhD tayini yapılabilmesi amaçlanmıştır.11–14. gebelik haftalarında bulunan ve eşleriyle aralarında RhD uyumsuzluğu olan 19 Rh (−) gebeden kan alınarak total serbest DNA izolasyonu yapılmıştır. İzole edilen DNA’larda RhD geninin ekson 7 bölgesine ait primerler kullanılarak, TaqMan prob yöntemi ile ve “Real-time PCR” aracılığıyla genin varlığı araştırılmıştır.“Real-time PCR” kullanılarak gebeliklerinin başında olan hastaların serumlarında RhD gen dizilerinin varlığı değerlendirildi. Analiz edilen 19 Rh (−) gebenin 12′sinin (Rh−), 7′sinin Rh (+) olduğu belirlendi. Doğum sonrasında bebeklerin kan grupları ile bizim sonuçlarımız birebir uyumlu çıkmıştır.RhD primeri ile alınan sonuçlar analiz edilmiştir. Fetüse ait RhD geninin maternal kanda belirlenebilmesi girişimsel olmayan doğum öncesi tanı için önemli bir katkı sağlamıştır.

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.

Doctor Parviz Lalezari, currently a clinical professor of Medicine and Pathology at Albert Einstein College of Medicine in New York, describes highlights of his research career since 1958. He became the director of the blood bank at Montefiore Hospital in New York City in 1961, director of the Division of Immunohematology until 1996, and then until 2001, was President and chief executive officer of the Bergen Community Regional Blood Center in New Jersey. Doctor Lalezari was born in Iran in 1931, and after graduation from Medical School, he came to the United States in 1956. His initial research was on leukocyte antibodies. After modifying the available antibody detection techniques, he discovered that like hemolytic disease of the newborn and neonatal immune thrombocytopenia, fetal-maternal neutrophil incompatibility can cause neonatal neutropenia. He identified the targets of these antibodies and showed that they were expressed only on peripheral blood neutrophils. Doctor Lalezari also discovered that a common form of neutropenia in early childhood was caused by development of autoantibodies, which surprisingly were directed against the same neutrophil-specific antigens involved in fetal-maternal incompatibility. In 1959, a heparin-neutralizing drug (Polybrene) was introduced to be used after open-heart surgery. Lalezari discovered that Polybrene, a quaternary ammonium polymer, reacted with sialic acid molecules on the red blood cell (RBC) surface, causing the RBCs to aggregate. Later, realizing that the repelling forces generated by the RBC surface membrane charges were responsible for failure of the small IgG antibody molecules to agglutinate the RBCs, he used Polybrene to neutralize the RBC surface negative charge to allow the IgG antibody molecules to induce hemagglutination. This became The Polybrene test, which is to be used in RBC antibody detection.

BACKGROUND: Few cases of antibodies to the Cromer (a) antigen have been described during pregnancy. Interestingly, the anti-Cr a titers decreased during pregnancy, and although the newborns were Cr a +, the direct antiglobulin tests (DATs) were negative and hemolytic disease of the newborn (HDN) was not observed. Cromer antigens reside on the decay-accelerating factor (DAF), which is expressed on the fetal derived syncytiotrophoblast layer of the placenta. It has been postulated that Cromer antibodies are not transported to the fetus, but are bound to placental DAF, thereby protecting the fetus from HDN and causing the disappearance of Cromer antibody in maternal plasma. This report is the first to demonstrate Cromer antibody sequestration by the placenta. CASE REPORT: A G4P1 woman with an anti-Cr a presented for prenatal care during her fourth pregnancy. The anti-Cr a titer decreased from 64 at 7 weeks gestation to undetectable after 25 weeks. At delivery, the infant had no evidence of HDN. The infant's DAT was negative, and the maternal plasma, cord plasma, and the cord blood eluate were negative with screening cells, the infant's cord cells, and the mother's cells. Placental eluates revealed anti-Cr a . CONCLUSIONS: This is the fourth case report of a Cromer (a-) woman producing anti-Cr a during pregnancy, and the first demonstrating anti-Cr a sequestration in the placenta. The presence of anti-Cr' in the placental eluate, but not in the cord plasma, maternal plasma, or cord blood eluate, strongly supports the hypothesis that DAF at the fetomaternal interface absorbs anti-Cr a from the maternal circulation blocking its passage to the fetus.

The quantification of IgG anti-D subclasses is one of the most important parameters considered in the assessment of the severity of hemolytic disease of the newborn. Traditionally IgG subclassing is performed using qualitative haemagglutination methods, difficult to interpret. A quantitative enzyme-linked immunosorbent assay (Elisa) was implemented for measuring IgG anti-D subclasses in 20 sera collected from 14 RhD-immunized pregnant women. All 4 IgG subclasses were detected in the 20 sera tested. The mean proportion of IgG1 was 52.8%. The mean proportion of IgG3 was 30.7%. The mean proportions of IgG2 and IgG4 were 14.5 and 1.9% respectively. A good correlation between the sum of IgG subclasses and the severity of HDN was found. Severe HDN occurred when both IgG1 and IgG3 were present. IgG1 anti-D was the predominant subclass in 4 of the 8 severe cases.

We conducted a population-based, cross-sectional study among pregnant women in Kampala, Uganda, to determine ABO and D blood types and to determine the percentage who have unexpected red blood cell (RBC) antibodies and their specificities. De-identified blood samples from routine testing of 1001 pregnant women at the Mulago Hospital antenatal clinics in Kampala were typed for ABO and D and screened for the presence of unexpected RBC antibodies with confirmation and subsequent antibody identification. Of the 1001 blood samples tested, 48.9 percent, 26.4 percent, 21.0 percent, and 3.8 percent tested positive for blood groups O, A, B, and AB, respectively. Of these samples, 23 (2.3%) were negative for D, and 55 (5.5%) showed initial reactivity with at least one screening RBC. The RBC antibody screen was repeated on these 55 samples, and antibody identification was performed at the Johns Hopkins Hospital Blood Bank in Baltimore, Maryland. Twenty-one of the 55 samples were confirmed to have evidence of agglutination. Nine of the 21 samples demonstrated the presence of clinically significant RBC antibodies with anti-S being the most common, 8 samples demonstrated the presence of benign or naturally occurring antibodies, and 4 had only inconclusive reactivity. This study revealed a relatively high frequency of D and a low frequency of demonstrable clinically significant alloantibodies that may cause hemolytic disease of the newborn or hemolytic transfusion reactions among pregnant women in Kampala, with anti-S being the most frequent antibody specificity. Immunohematology 2012;28:115–7.

Hemolytic disease of the newborn (HDN), with high potential for increased fetal loss is less common now, due to the universal screening for iso-sensitization and also because of appropriate use of antenatal anti-RhD antibody prophylaxis. There are other non-RhD antibodies that can cause HDN. In US, we occasionally encounter a highly sensitized fetus with significant morbidity and mortality. In utero RBC transfusions and Intravenous Immunoglobulin (IVIG) therapy for such an infant are effective to some extent, in the management of HDN. Partial exchange transfusions (immediately after the delivery) and double volume exchange transfusions are rarely but still, needed as rescue modes.

Abstract 3794 Introduction: Hemolytic disease of the newborn (HDN) results from maternal IgG antibodies that cross the placenta into the fetal circulation during gestation and cause red blood cell (RBC) destruction. Anemia, hyperbilirubinemia and, in severe cases, hydrops fetalis are well-recognized complications. However, much less is known about the prevalence and outcome of neutropenia as a feature of HDN. This study examined the prevalence and outcome of neutropenia in infants with HDN who were followed in the Pediatric Hematology Clinic at the Children's Hospital of Eastern Ontario (CHEO) during the last 5 years, and also investigates possible risk factors for the development of neutropenia in this setting. Methods: A retrospective chart review was perfomed on all infants with HDN followed in the Pediatric Hematology Clinic at CHEO from April 2005 to May 2010. Neutropenia was defined as a neutrophil count low for patient's age according to laboratory normal values ( Results: Seventy-four infants with a diagnosis of HDN were identified during the study time period, of which 69 were evaluable. The male to female ratio was 1:1 and the median gestational age at birth was 38 weeks. Rh antibodies were the most prevalent (48%), followed by ABO incompatibility (42%). No cases of hydrops fetalis were recorded. Fifty-three percent of the infants studied developed some degree of neutropenia, with a median neutrophil count at nadir of 0.56 ×109/L. The mean age at the nadir of the neutropenia was 56 days (range 5–181 days) and the neutropenia lasted a mean of 37 days following the nadir (range 3–153 days). No infectious complications were recorded during the period of neutropenia except for one patient born at 32 weeks of gestational age who had necrotizing enterocolitis and bowel perforation at the onset of the neutropenia. In most instances the neutropenia resolved spontaneously within days, but interestingly, 6 infants subsequently developed chronic neutropenia. Statistical analysis revealed that males with HDN were at a significantly higher risk of developing neutropenia (p=0.022) compared to females, but no statistically significant associations between neutropenia and severity of HDN, antibody specificity, treatment with IVIG or transfusions were found. Not surprisingly, 77% of infants with HDN developed thrombocytosis with a maximum platelet count of 1200 ×109/L, and this was not significantly associated with the development of neutropenia. Conclusion: This is the largest study to date to determine the prevalence of neutropenia in the context of hemolytic disease of the newborn. Our results demonstrate that neutropenia is a common feature of HDN, regardless of the severity of disease, the treatment received or the antibody specificity. Although its clinical course seems to be benign, careful follow-up of these infants should be undertaken. Given the retrospective nature of the study, possible contributing factors such as maternal conditions of pre-eclampsia, drug exposure or ethnicity were not available. Larger prospective studies should be conducted to learn more about the pathophysiology of this phenomenon and its natural course. Disclosures: No relevant conflicts of interest to declare.

Background : ABO incompatibility in maternal-fetal relationship has been shown to cause hemolytic disease of the newborn (HDNB); a survey which is not yet done in this locality. Aim: Frequency of ABO blood group maternal-fetal incompatibility, maternal iso-agglutinins, and immune agglutinins quantitation was carried out in Osogbo, Osun State, South-West of Nigeria. Settings and Designs : A total of 260 subjects comprising 130 postpartum mothers within the age range of 22-35 years having good obstetrics history and normal delivery, with their 130 neonate babies were used for the study. Materials and Methods : ABO cell and serum groupings were carried out on the subjects using standard antisera and cells with appropriate controls. Direct Coomb's Test was carried out on neonate red cells. Antibody quantitation by double dilution on the maternal serum using red cells containing corresponding antigen to the antibody was determined. A titer, which is the reciprocal of the highest dilution showing agglutination by Indirect Coombs Test, was determined. Another batch of sera was pretreated with 2-mecarptoethanol before determining the titer. Statistical Analysis: The distribution study results obtained were compared in percentages, whereas the antibodies quantitation was expressed as titers using the mode of the titers for compariso-agglutininsn. Results and Conclusions : Thirty-eight percent (50) mothers were ABO incompatible with their babies, whereas 62% (80) mothers were compatible. The distribution of blood groups in the compatible population showed blood group O (45%); A (30%); B (20%); and AB (5%). Mothers O, A, and B carrying incompatible babies had a frequency of 24% each, whereas mothers AB had 28%. Serologist differences occur in maternal ABO antibodies of corresponding incompatible baby ABO antigens. A high incidence of ABO maternal-fetal incompatibility observed without detection of immune agglutinins is indicative of a rare incidence of HDNB due to ABO incompatibility in the population studied.

BACKGROUND Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D, but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS An IgG anti-D concentration of 61.5 +/- 4.8 microg per ampoule (mean +/- SD) was found by ELISA in IRP 68/419. This result was in agreement with previous determinations obtained by radioimmunoassay (60 microg/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 microg of IgG1 (78.6%), 3.0 microg of IgG2 (4.8%), 9.7 microg of IgG3 (15.8%), and 0.4 microg of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 microg per mL. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.

Objectives Anti-Rhc antibodies may be the reason for the hemolytic disease of the newborn, therefore, noninvasive Rhc determination is important for pregnancy monitoring. For this purpose, we decided to introduce real-time polymerase chain reaction (PCR) method. Methods Blood from 200 donors, plasma and whole-blood from 11 Rhc-negative mothers, as well as blood from fathers and newborns were examined. Rhc sensitivity and specificity were first determined by real-time PCR using genomic DNA from donors. The same Rhc genotyping method was used for fetal Rhc detection in maternal plasma. To confirm the fetal Rhc-negative result, plasma was tested with a panel of biallelic insertion/deletion polymorphisms for the presence of fetal DNA. Results The c allele assay showed full specificity. The mean Ct value for one copy of c allele diluted in C-negative DNA was determined from extrapolating the correlation curve as 39.9. Full concordance was observed between the fetal Rhc genotypes from maternal plasma and the newborn phenotypes. Conclusions Preliminary results show that it is possible to examine fetal c allele of RHCE gene in the plasma of pregnant women with anti-c by means of a noninvasive method. The diagnostic accuracy of the procedure, however, has yet to be confirmed in a larger group. Copyright © 2008 John Wiley & Sons, Ltd.

To investigate if the prediction of hemolytic disease of the newborn (HDN) in infants of RhD immunized women has been improved by introduction of AutoAnalyzer quantitation and may be further improved by the use of a functional assay.Manual antibody titration, AutoAnalyzer quantitation and chemiluminescence test (CLT) were compared by testing 42 sera from 38 RhD immunized mothers. The sera were also screened for the presence of monocyte-reactive antibodies which have the potential to protect the unborn.Among the 42 infants there were 19 unaffected by HDN and 23 affected by HDN. Manual titration correctly predicted the occurrence of HDN in 29/42 (69%), AutoAnalyzer was correct in 28/42 (67%) and CLT showed correct predictions with 30/42 (71%). In babies born without signs of HDN, maternal monocyte-reactive antibodies were found in 13/18 cases. The majority (9/13) were HLA class I-specific, the remaining four antibodies were either HLA class II or monocyte-specific. In affected HDN group, 8/16 monocyte-reactive anti- bodies were HLA class II or monocyte-specific.AutoAnalyzer and CLT improve the ability to discriminate unaffected babies from those affected by RhD HDN, when compared to manual titration. A protocol for the laboratory management of RhD immunized women is proposed that includes these tests to further improve the prediction of HDN. This study has also highlighted the need for more investigations into the protective role of monocyte-reactive antibodies in HDN.

Anti-Holley (Hy) has been reported as an IgG antibody occurring in previously transfused or multiparous black patients. In this case anti- Hy was identified in a 16-year-old black, primigravida female admitted at 32 weeks gestation because of premature rupture of the membranes. On admission, her blood type was determined to be A2B, D-positive and an antibody screen was negative. A second antibody screen, performed 4 days later, was positive in all three cells. Anti-Hy was subsequently identified. The antibody was reactive at room temperature, 37°C, and in the antiglobulin phase. IgG and IgM components of anti-Hy were demonstrated in the maternal serum, documenting a primary immune response. This resulted in serologic findings not previously described for anti-Hy. A direct antiglobulin test on the newborn red cells was negative and there was no clinical evidence of hemolytic disease of the newborn (HDN). A monocyte monolayer assay performed with maternal serum yielded negative results. Recent scientific information has resulted in the placement of Hy in the Dombrock blood group system. Alloantibodies to Dombrock system antigens have not been associated with severe HDN. Immunohematology 1996;12:62–65.

Monocyte ADCC assays are helpful indicators of the severity of hemolytic disease of the newborn (HDN) due to anti-D. It would be particularly useful if the assays also accurately predicted the ability of antibodies to high-frequency antigens (UFA) to cause HDN. To investigate this possibility, 14 antenatal sera containing antibodies to HFA were tested and the results correlated with the severity of HDN. Antibody titers were determined using an indirect antiglobulin test (IAT). Eight sera (anti-Hy, -Dib, -Jra -ScI, -Inb, -Wrb, and-U [n =2]) had low ADCC activity (< 28%) and did not cause HDN. The IAT titers ranged from 4 to 128. Two antibodies (anti-Ena and anti-Rh29) had high ADCC activity (≥ 72%) and high IAT titers (≥ 1,024), and both caused severe HDN. The remaining four antibodies (anti-Cra, -Inb, and -U [n = 2]) had ADCC activity ≥ 40 percent and titers ≥ 128, but they did not cause HDN. The inability of anti-Cra and anti-Inb to cause HDN is presumed to be related to fetal antigen expression and tissue distribution. The anti-Inb was further investigated using monocyte binding and inhibition studies. The absence of HDN in the presence of potent anti-U remains unexplained. In general, antibodies with low ADCC activity do not cause HDN, but high ADCC activity does not always indicate severe HDN. Immunohematology 1996;12:20-26.

Introduction Anti-erythroid antibodies are the classical marks of serologic and hemolytic transfusion reactions. These reactions can occur acutely or in a delayed timeframe, while the sensitizing antibody may derive from the host or be passively acquired. In those with concurrent hemolysis, the red blood cell (RBC) breakdown may be severe enough to command supportive care. However, in those with non-hemolytic delayed serologic transfusion reactions (NH-DSTRs), the threat applies more towards the future rather than the present time. Downstream hazards range from hemolytic disease of the newborn, to delays and difficulties sourcing antigen-negative blood (when the antibody is known), or an anamnestic response with higher odds of hemolysis on antigen re-exposure (when the antibody becomes unknown by evanescence and healthcare fragmentation). Data are lacking on inpatient outcomes associated with discovering a new NH-DSTR during a hospital admission. A review of NH-DSTRs was thus performed in a large academic hospital (34,000 RBC dispensations annually). Methods A retrospective review of a transfusion reaction database was undertaken at a large academic hospital in Toronto, Canada. Transfusion reactions (TRs) occurring during inpatient admissions (excluding emergency room and outpatient visits) from 1/1/2010-31/12/2015 were included. NH-DSTR was defined as the presence of a new antibody on repeat screen post transfusion with no evidence of hemolysis. Other anti-RBC antibody mediated TRs included acute hemolytic transfusion reactions (AHTR) (both host-derived and passively-acquired [from products such as intravenous immunoglobulin]), and delayed hemolytic transfusion reactions (DHTR) occurring with or without serologic findings. A comparison was also made against all inpatient TRs not due to RBC antibodies (non-anti-RBC TRs). Disturbances deemed unrelated to transfusion were excluded. Outcomes included length of stay (LOS), interval between TR recognition and discharge, severity of TR (as per the International Society of Blood Transfusion grading system), and death. Results A total of 783 inpatient TRs were reviewed. The distribution of TRs (Figure 1) included 562 (71.8%) non-anti-RBC TRs and 221 (28.2%) anti-RBC TRs. Within the anti-RBC TRs, 159 (71.9%) were classified as NH-DSTRs. The mean age of all patients was 57 (± 17) with 49.4% of reactions occurring in females. Compared with non-anti-RBC and other anti-RBC transfusion reactions, NH-DSTRs were significantly less frequently classified as severe (Table 1). The overall LOS and remaining days in hospital after TR were significantly longer in those with NH-DSTRs compared with the two other groups (Table 1). Post-reaction LOS was longer by a median of 5 or 7 days for NH-DSTR versus non-anti-RBC TRs and other anti-RBC TRs respectively. There was no significant difference between groups when evaluating inpatient mortality. Conclusions NH-DSTRs are associated with a longer LOS when compared with all other TRs. This relationship holds even in comparisons with other anti-RBC TRs. Causality is not established by this analysis, nor is there a biologic rationale for a NH-DSTR to directly impact LOS. However, the propensity to form a new anti-RBC antibody may reflect an underlying pro-inflammatory comorbid state that itself may be influencing LOS. Further studies are needed to confirm this association. Nevertheless, given any potential for additional/current impacts beyond future ramifications, the precautionary principle is strengthened for the value of curating the full extent of a recipient's antibody history, and prophylactically matching for minor antigens if resources permit. Table 1 Comparison of outcomes between NH-DSTRs versus non-anti-RBC TRs and other-anti-RBC TRs. Table 1. Comparison of outcomes between NH-DSTRs versus non-anti-RBC TRs and other-anti-RBC TRs. Figure 1 Frequency of transfusion reactions from January 1, 2010 to December 31, 2015. Abbreviations: allergic transfusion reaction (ATR), febrile non-hemolytic transfusion reaction (FNHTR), transfusion associated circulatory overload (TACO), transfusion associated dyspnea (TAD), bacterial contamination (BaCon), transfusion related acute lung injury (TRALI), inflammatory transfusion reaction (ITR), citrate reaction (CR), acute passive serologic/hemolytic transfusion reaction (APSHTR). Figure 1. Frequency of transfusion reactions from January 1, 2010 to December 31, 2015. Abbreviations: allergic transfusion reaction (ATR), febrile non-hemolytic transfusion reaction (FNHTR), transfusion associated circulatory overload (TACO), transfusion associated dyspnea (TAD), bacterial contamination (BaCon), transfusion related acute lung injury (TRALI), inflammatory transfusion reaction (ITR), citrate reaction (CR), acute passive serologic/hemolytic transfusion reaction (APSHTR). Disclosures No relevant conflicts of interest to declare.

Traditionally, IgG subclassing has been performed using qualitative assays. Quantitation of IgG subclasses may have prognostic value in evaluating alloimmunized pregnancies. A quantitative enzyme-linked immunosorbent assay (ELISA) was implemented for measuring IgG subclasses of red blood cell (RBC) antibodies (AB) isolated by adsorption/elution from the sera of alloimmunized pregnant women. The assay is a sandwich enzyme immunoassay using monoclonal antibodies specific for the relevant IgG subclasses and anti-human IgG peroxidase conjugate to quantitate the amount of bound IgG. The sensitivities of the assay for IgG1, 2, 3, and 4, respectively, were 4, 23, 4 and 2 micrograms/l. The results for each subclass for a given AB were expressed as a percentage of the total. In a series of pregnant mothers with ABs: E (4), Fya (2), Jka (1) and S (1), the mean percentage +/- 1SD of each subclass was: IgG1 61 +/- 34; IgG2 14 +/- 22; IgG3 18 +/- 28 and IgG4 4 +/- 17. IgG1 or IgG3 accounted for greater than 50% of the AB subclass distribution in 5 cases that resulted in hemolytic disease of the newborn (HDN). Although only a small number of samples was studied, changes in the concentrations of IgG1 or IgG3 during gestation suggest a correlation with the presence or absence of HDN. The ELISA may be used to quantitate the IgG subclasses of RBC ABs and may be valuable in predicting the severity of HDN in alloimmunized pregnancies.

We report on flow cytometric IgG subclass determinations of red cell antibodies using polyclonal FITC-labeled antibodies. The limit of detection of this method was 1 ng anti-D per 1 x 10(7) red cells. The inter- and intra-assay coefficients of variance were 8.2 and 2.3%, respectively. In 8 newborns with a positive direct antiglobulin test (DAT) in the gel centrifugation test (GCT), due to ABO antibodies, IgG1 was detected in all and IgG2 additionally in 4 of these cases. In 5 severe cases of hemolytic disease of the newborn (HDN) due to anti-D, large amounts of IgG1 were found, and in 3 of these 5, IgG3 in combination with IgG1. In 8 mild or moderate HDN cases (4 anti-D, 2 anti-E, 1 anti-Fya, 1 anti-Jka), phototherapy sufficed, and IgG1 was the only antibody. In 7 adult patients with malignant lymphoma and a positive DAT (GCT), only small amounts of IgG1 red cell autoantibodies could be demonstrated by flow cytometry. In 5 further patients with malignant lymphoma, a positive DAT, and severe hemolytic anemia, large amounts of IgG1 autoantibodies were found and IgG3 was also present in 3 of these cases. Flow-cytometric determination of IgG subclasses may be a useful tool in immunohematology, since subclass determinations were possible in all of these cases. This method is suited for clinical routine and offers the possibility of sufficient standardization.

Neonatal alloimmune thrombocytopenia (NAIT) is a clinical syndrome that resembles hemolytic disease of the newborn, affecting the platelets only. The thrombocytopenia results from the maternal alloantibodies reacting with specific human platelet antigens (HPAs) on the fetal platelets. Forty-four maternal plasma samples were screened for platelet alloantibodies using qualitative solid phase enzyme-linked immunosorbent assay (ELISA) commercial kit (LIFECODES Pakplus, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA), and both the maternal and the corresponding cord blood samples were genotyped (LIFECODES ThromboType, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA). HPA genotyping results correlated with the genetic frequencies in the Taiwan population. A total of 34 newborns (77.3%) had partial HPA genotyping mismatches with the corresponding mothers. The most common partial mismatches between mothers and neonates in HPA genotypes were 13 (29.5%) in both HPA-3b and HPA-15a, followed by 12 (27.3%) in HPA-15b, and 8 (18.2%) in HPA-3a. The frequencies of homozygotic mother with heterozygotic neonate were 15.9% in both HPA-3a and HPA-15b, 9.1% in HPA-15a, 6.8% in HPA-3b, and 2.3% in both HPA-2a and HPA-6a. In this study, maternal HPA antibodies were found in five samples, whereas HLA class I antibodies were found in seven maternal plasma samples from the antibody screen. The results from this study have demonstrated that HPA mismatch is not the main cause for the production of HPA alloantibodies.

Intravenous Immunoglobulin G (IVIG) therapy has been used as a component of the treatment of hemolytic disease of the newborn. There is still no consensus on its use in ABO hemolytic disease of the newborn routinely. The aim of this study is to determine whether administration of IVIG to newborns with ABO incompatibility is necessary. One hundred and seventeen patients with ABO hemolytic disease and positive Coombs test were enrolled into the study. The subjects were healthy except jaundice. Infants were divided into two groups: Group I (n = 71) received one dose of IVIG (1 g/kg) and LED phototherapy whereas Group II (n = 46) received only LED phototherapy. One patient received erythrocyte transfusion in Group I, no exchange transfusion was performed in both groups. Mean duration of phototherapy was 3.1 +/- A 1.3 days in Group I and 2.27 +/- A 0.7 days in Group II (p < 0.05). Mean duration of hospital stay was 5.34 +/- A 2.2 days in Group I and 3.53 +/- A 1.3 days in Group II (p < 0.05). Mean duration of phototherapy was 4.0 +/- A 1.5 days and 2.73 +/- A 1.1 days in double and single doses of IVIG respectively, and this was statistically significant (p < 0.05). IVIG therapy didn't decrease neither phototherapy nor hospitalization duration in infants with ABO hemolytic disease. Meticulus follow-up of infants with ABO hemolytic disease and LED phototherapy decreases morbidity. IVIG failed to show preventing hemolysis in ABO hemolytic disease.

Antibodies to red cell antigens that are found at low frequency in the general population are rare causes of hemolytic disease of the newborn. To understand how to detect these cases, we provide a basic review of routine antenatal maternal antibody testing and report a case of a neonate with severe HDN caused by anti-Wright (anti-Wra), successfully managed with transfusion, phototherapy, and high-dose intravenous immunoglobulin.When hemolysis in a newborn is suspected in the absence of major blood group incompatibility or commonly detected maternal red cell antibodies, a direct antiglobulin test should be performed. A positive DAT should alert the clinician to the presence of maternal antibodies against low-incidence antigens. Antibodies to the Wraantigen are one such rare cause of HDN.

The Rh blood group antigen e is of high incidence and has many epitopes. Partial expression may occur, more commonly in black persons. Individuals with e variant phenotypes can make antibodies to epitopes they lack. While some of these antibodies may be specific for an antigen, e.g., hrB, others, like anti-Rh17 (anti-Hro), show broader specificity, compatible only with D–– and Rhnull red blood cells (RBCs). Anti-Rh17 in persons of the D–– phenotype has been reported to cause mild to fatal HDN. We report an example of anti-Rh17 produced by a black female with an e variant RBC phenotype that caused moderate HDN. A panel of seven monoclonal anti-e demonstrated her RBCs carried a variant e antigen, and her genotype was RHD, RHce by PCR-RFLP analysis. Amniotic fluid with ΔOD450 values from 30 to 35 weeks’ gestation predicted moderate HDN probability by the Liley method. At 38+ weeks, a viable 3165 g female infant was delivered. The infant’s direct antiglobulin test was 2+ with anti-IgG. Total bilirubin rose to 14.2 mg/dL within 48 hours. Indirect bilirubin peaked at 14.7 mg/dL. The bilirubin responded to triple phototherapy. The infant was discharged on day 6. Potential for infant morbidity due to anti-Rh17-mediated HDN and the importance of specifying risks to women with this antibody if they contemplate pregnancy are discussed. Immunohematology 2002:18:40–42.

Hemolytic disease of the newborn (HDN) due to anti-Hr0 antibody is typically severe and often fatal. We report a case of moderate HDN due to anti-Hr0 in a woman with the D––/D–– phenotype. A 33-year-old woman delivered her second child who was mildly jaundiced. The highest level of bilirubin was 26.1 mg/dL on the third day postpartum and the hemoglobin concentration was 14.0 g/dL. The newborn recovered after phototherapy and no mental retardation was noticed after 1 year of follow up. An exchange transfusion was excluded due to the lack of a compatible donor and the physical condition of the mother precluded blood donation. The maternal RBCs were D+C–c–E–e–; only G and Rh29 of the Rh system were expressed. Thus, her probable phenotype was D––/D––. Her alloantibody was identified as anti-Hr0 (anti-Rh17) as it reacted with all red blood cells (RBCs) but not her own, other D-– RBCs, and Rhnull RBCs. The results of the antibody titer (64) and activity in a chemiluminescense test (CLT; 34%) were consistent with a moderate HDN. Family studies were negative for the D––/D–– phenotype and consanguinity was not proved. This is the first described case of moderate HDN due to anti-Hr0. The result of antibody activity in the CLT might be helpful in predicting the severity of HDN in other rare HDN cases. Immunohematology 2000;16:109–111.

The Kidd system antibodies, if undetected, can cause immediate or delayed hemolytic transfusion reactions as well as hemolytic disease of the newborn. There have been anecdotal reports about the inefficiency of the manual Polybrene test in detecting these antibodies. Here, we sought to determine the sensitivity of the manual Polybrene test in detecting anti-Jk(a) and anti-Jk(b) antibodies and Jk(a) and Jk(b) antigens.Ten archived anti-Jk(a)/Jk(b) antibody positive human sera were examined by both the manual Polybrene test and the indirect antiglobulin test using polyspecific antibodies, monospecific anti-IgG antibodies and anti-C3 antibodies. Furthermore, 40 randomly selected donor blood samples were collected and phenotyped for the frequencies of Jk(a) and Jk(b) antigens using the manual Polybrene test and the indirect antiglobulin test. The results from these tests were further confirmed by saline tube tests.The manual Polybrene test displayed an overall sensitivity of 60% in detecting anti-Jk(a) and anti-Jk(b) antibody. Specifically, it had a sensitivity of 57.14% for anti-Jk(a) antibody and a sensitivity of 66.7% for anti-Jk(b) antibody. Furthermore, the manual Polybrene test exhibited a sensitivity of 46.15% for Jk(a) antigen and a sensitivity of 77.42% for Jk(b) antigen.The manual Polybrene test has a very low sensitivity in detecting anti-Jk(a) and anti-Jk(b) antibody, especially anti-Jk(a) antibody. It is also a very insensitive test for detecting Jk(a) antigen.

Maternal antibody-mediated fetal red blood cell destruction secondary to non-D Rhesus (Rh) antibodies is a significant cause of hemolytic disease of the newborn (HDN). Here, we report a rare case of severe HDN associated with maternal antibody to Rh e. In addition to severe anemia, the infant developed thrombocytopenia, conjugated hyperbilirubinemia and cholelithiasis. Resolution of the infant's cholelithiasis occurred following treatment with ursodeoxycholic acid.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by transplacentally acquired maternal antibodies reactive with platelet (PLT)-specific alloantigens (HPA antigens) and occurs in about 1 of every 1000 births.1-4 Many cases are mild and remit spontaneously, but thrombocytopenia can be severe and numerous fatalities have been recorded.2-5 Unlike its red blood cell (RBC) counterpart, hemolytic disease of the newborn (HDN), almost two-thirds of NAIT cases occur in firstborn infants.5 Numerous human PLT alloantigens (HPAs) are capable of inducing maternal immunization during pregnancy and causing NAIT.5-7 Maternal-fetal incompatibility for the high-frequency alloantigen HPA-1a (PlA1, Zwa) accounts for 75 to 85 percent of the reported cases.3,4,7 A specific serologic diagnosis, however, is made in fewer than half of the suspected cases.7 Non-HPA antigens such as ABH and Class I HLA are shared by PLTs and other tissues. Although anecdotal reports have claimed that HLA-specific antibodies can cause NAIT,8-10 there is no consensus on this point because infants born to multiparous women immunized against Class I HLA antigens nearly always have a normal PLT count. Fetal-maternal incompatibility for blood group A or B has not previously been implicated as a cause of NAIT and one report actually suggested that incompatibility for ABH may protect against NAIT as it does against HDN associated with fetal-maternal Rh incompatibility.11 It is not surprising that a role for ABH incompatibility in NAIT has not been considered because A and B antigens are expressed very weakly on PLTs of most normal individuals.12,13 Recent studies have shown, however, that a subset of A and B antigen positive normal subjects has PLTs that carry many times more than the usual number of A or B determinants12-14 and that these determinants are located on various PLT membrane glycoproteins (GPs), especially GPIIb and PECAM-1 (CD31).12 Curtis and colleagues12 showed that group A1 individuals can be divided into three subgroups; approximately two-thirds have PLTs with low A antigen expression (fewer than 2000 epitopes per PLT). A second group constituting approximately 30 percent has moderately increased A antigen expression (2-6,000 epitopes per PLT). Approximately 1 to 2 percent, however, have PLTs that carry 10 to 20,000 copies of A or B per PLT. The latter two groups were shown by statistical analysis to be distinct subpopulations and were designated “Type I” and “Type II” high expressers (H-Exp), respectively.12 The distribution of B antigen expression on PLTs appears to be similar to that of A antigen, but has been less well studied.12,14 Ogasawara and coworkers14 showed that PLTs from a “high-expresser” of blood group B were rapidly destroyed upon being transfused to a group O patient. Except for this single observation, however, the clinical significance of the H-Exp trait has not been critically examined. Here we describe a family in which two group B infants who inherited the Type II H-Exp phenotype from their father were born with moderately severe thrombocytopenia and present evidence that this complication was caused by transplacentally acquired, high-titer maternal immunoglobulin G (IgG) antibodies specific for blood group B.

The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.

The Cromer blood group system consists of 7 high incidence and 3 low incidence antigens that are carried on the complement regulatory glycoprotein called decay-accelerating factor (DAF; CD55). Despite laboratory results that would predict clinical significance, antibodies with specificities in the Cromer blood group system have not been reported to cause hemolytic disease of the newborn. It is possible that strong expression of DAF on the apical surface of antigen-positive fetally derived placental trophoblasts may absorb maternal antibodies that are directed to antigens in the Cromer blood group system. We studied two cases where strongly reactive anti-Cra and anti-Dra became undetectable during second and third trimesters of pregnancies.

Hemolytic disease of the newborn (HDN) is characterized by the destruction of fetal red blood cells by maternal immunoglobulin G (IgG) directed against antigens present on fetal erythrocytes. These paternally inherited antigens are not present on maternal cells and can stimulate the maternal immune system to produce antibodies when antepartum or intrapartum fetomaternal hemorrhage occurs. Maternal sensitization often occurs during the first pregnancy with a fetus that expresses the erythrocyte antigens and although this fetus is at low risk of HDN, future antigen-positive fetuses are at substantial risk of developing the disease. Transplacental passage of the resulting antibodies into the fetal circulation often leads to hemolysis that can range from mild to extreme depending on several factors including, among others, the antibody concentration, the transfer rate of immunoglobulin from the maternal to the fetal circulation, the antibody specificity, the functional maturity of the fetal spleen, and the IgG subclass (Fig. 1) (1).

Accurate phenotyping of red blood cells (RBCs) can be difficult in transfusion-dependent patients such as those with thalassemia and sickle cells anemia because of the presence of previously transfused RBCs in the patient's circulation. Recently, the molecular basis associated with the expression of many blood group antigens was established. This allowed the development of a plethora of polymerase chain reaction-based tests for identification of the blood group antigens by testing DNA. The determination of blood group polymorphism at the genomic level facilitates the resolution of clinical problems that cannot be addressed by hemagglutination. They are useful to (a) determine antigen types for which currently available antibodies are weakly reactive; (b) type patients who have been recently transfused; (c) identify fetuses at risk for hemolytic disease of the newborn; and (d) to increase the reliability of repositories of antigen negative RBCs for transfusion. It is important to note that PCR based assays are prone to different types of errors that those observed with hemagglutination assays. For instance, contamination with amplified products may lead to false positive test results. In addition, the identification of a particular genotype does not necessarily mean that the antigen will be expressed on the RBC membrane.

The determination of the RhD phenotype is important in transfusion medicine. However, due to the complexity of D antigen expression, the routine serological method cannot differentiate all RhD variants. In addition, the induction of the anti‐D antibody is still the major cause of severe hemolytic disease of the newborn (HDN). Therefore, it is important to understand RHD gene profiles. To analyze the RHD gene profiles of Taiwanese RhD‐negative donors, the multiplex PCR method was applied to amplify RHD specific exons 3, 4, 5, 7, and 9. Based on the PCR results, the 156 RhD‐negative donors were divided into 12 groups according to the different expression patterns of the RHD gene. These 12 groups were further divided into three categories: type I=Rh D(el) (21.8%); type II = partial D, containing some exons (9.0%); and type III = true RhD‐negative (69.2%). The results indicated that 21.8% of RhD‐negative donors in Taiwan were RhD(el), and 9% carried a part of the RHD gene. Six defined RhD variants were found in this study: four R(O) (Har), one D(Va), and two D(IVb). However, no true RhD‐negative or RhD(el) donor with the CcdEe phenotype was found in this analysis. J. Clin. Lab. Anal. 17:80–84, 2003. © 2003 Wiley‐Liss, Inc.

The results from fetal-maternal hemorrhage (FMH) detection and quantitation external quality assessment surveys conducted in Ontario indicate that the rosette test had a sensitivity and specificity for an FMH of more than 10 mL of 1.0 and 0.75, respectively, compared with 0.96 and 0.92, respectively, for acid elution. With FMH quantitation, the percentage error of the mean from the target FMH was 20% or more in 7 of 8 surveys, and coefficients of variation ranged from 39.5% to 71.8%. Inadequate Rh o (D) immune globulin prophylaxis could have occurred in 19.4% of the challenges with an FMH of more than 10 mL. The rosette and acid elution techniques are both effective for the detection or exclusion of FMH, but acid elution lacks adequate accuracy and precision for reliable FMH quantitation. Furthermore, a strategy of prescribing an extra 1,500-IU Rh o (D) immune globulin dose, in addition to the dose required to treat the volume of fetal blood detected, is an effective strategy to overcome the limitations of FMH quantitation by acid elution. Hemolytic disease of the newborn (HDN) is the result of maternal antibodies causing destruction of antigen-positive erythrocytes in the fetus. The most serious form of HDN is caused by maternal alloantibodies directed against the D antigen of the Rh blood group system. In this scenario, Rh– mothers are alloimmunized during a previous pregnancy with an Rh+ fetus. HDN occurs in subsequent pregnancies involving an Rh+ fetus when maternal anti-D crosses the

A saline-reactive antibody, anti-Uz, that reacted stronger with S+ than with S– red blood cells (RBCs) and failed to react with U– or ficin-treated RBCs has been previously reported. We describe an antibody of similar specificity in the postpartum serum of an untransfused woman and the eluate from her fourth child’s cord RBCs. The mother’s RBCs typed S–s+U+, He+(weak), and appeared to have normal glycophorin A and B content, as determined by immunoblotting. The direct antiglobulin test (DAT) and the autocontrol were negative. Her serum reacted stronger with S + RBCs only in the antiglobulin phase, and failed to react with U– or ficin-treated RBCs. The antibody was adsorbed completely by S–s+U+ RBCs, proving that anti-S was not present. Monocyte monolayer assay results with S+s–U+ and S–s+U+ RBCs indicated that transfusion of incompatible blood would not result in significant hemolysis. The child’s cord RBCs typed S–s+. The DAT was 3+ with anti-IgG, and an eluate prepared from these RBCs had the same reactivity as the maternal serum. The child showed no clinical signs of hemolytic disease of the newborn. In contrast to previous reports, these results suggest an immune form of anti-Uz. Immunobematology 1993;9:47–49.