Alloimmune Disease Research Articles

Major reduction in occurrence of anti-c and anti-E in pregnancy after more than 10 years of preventive matched transfusion with most benefit for c-matching.

Extension with cE-matching of the transfusion policy for women under 45 years to prevent alloimmunization and hemolytic disease of the foetus and newborn (HDFN) was evaluated. After implementation of cEK-matching, anti-c occurrence decreased from 46.8 to 30.4 per 100 000 pregnancies (RR 0.65, 95% CI 0.54-0.79), while anti-E occurrence decreased from 122.1 to 89.9 per 100 000 pregnancies (RR 0.74, 95% CI 0.66-0.84). The c-negative women showed a higher anti-E occurrence before cEK-matching and a more pronounced decline with the new policy. This indicates that cEK-matched transfusion effectively reduces alloimmunization, and that a cK-matched approach could prevent most transfusion-related alloimmunization and HDFN.

A rare presentation of severe alloimmune hemolytic disease of newborn pertaining to minor blood group ‘c’ incompatibility: a case report and review of literature

Hyperbilirubinemia is one of the most widely seen causes of neonatal morbidity. Haemolytic disease of fetus and newborn is caused by maternal alloantibodies to the fetal RBCs. It is responsible for incompatibility between maternal and fetal blood groups, which results in destruction of fetal red blood cells causing hyperbilirubinemia. ABO and Rh incompatibility are the most common causes of severe indirect hyperbilirubinemia. Besides ABO and Rh isoimmunization, minor blood group incompatibilities such as anti-Kell, anti-C, anti-c, anti-E, anti MNS, Duffy, KIDD, P, Lutheran and Lewis have also been identified as causes of severe neonatal jaundice with an incidence of 385/1,00,000 live births in South-East Asia. We, hereby report a rare case of a full term 2.2 kg newborn presented with severe anemia with reticulocytosis and neonatal hyperbilirubinemia at second hour of life. In view of strongly positive DCT and no Rh negative or ABO setting, minor blood group incompatibility screening test was performed in the mother which revealed presence of multiple alloantibodies; however, the red cell phenotyping confirmed the presence of anti-c antibodies in maternal sera responsible for neonatal alloimmune haemolytic anemia. The baby was offered intensive phototherapy with intravenous immunoglobulin.

SFM Fetal Therapy Practice Guidelines: Intrauterine Blood Transfusion

AbstractDespite routine antenatal anti-D prophylaxis with immunoglobulin, Rh alloimmunization and hemolytic disease of the fetus and newborn continue to occur due to a myriad of reasons. Intrauterine intravascular transfusion (IUT) or fetal blood transfusion is a therapeutic prenatal procedure in which specifically prepared and treated unit of donor red blood cells is injected intravascularly into the umbilical vein under ultrasound guidance. Originally introduced in the year 1963, it continues to be the standard treatment for severe fetal anemia. The objective of this guideline is to provide an evidence-based update of IUT for the perinatal healthcare providers.

Alloimmune hemolytic disease of the fetus and newborn: genetics, structure, and function of the commonly involved erythrocyte antigens.

Hemolytic disease of the fetus and newborn (HDFN) can occur when a pregnant woman has antibody directed against an erythrocyte surface antigen expressed by her fetus. This alloimmune disorder is restricted to situations where transplacental transfer of maternal antibody to the fetus occurs, and binds to fetal erythrocytes, and significantly shortens the red cell lifespan. The pathogenesis of HDFN involves maternal sensitization to erythrocyte "non-self" antigens (those she does not express). Exposure of a woman to a non-self-erythrocyte antigen principally occurs through either a blood transfusion or a pregnancy where paternally derived erythrocyte antigens, expressed by her fetus, enter her circulation, and are immunologically recognized as foreign. This review focuses on the genetics, structure, and function of the erythrocyte antigens that are most frequently involved in the pathogenesis of alloimmune HDFN. By providing this information we aim to convey useful insights to clinicians caring for patients with this condition.

Characterization of ZG16b protein exocrine function and biomarker potential for salivary gland damage in chronic graft vs. host disease

Abstract The oral cavity is a frequent target of chronic Graft versus Host Disease (cGVHD) following allogenic hematopoietic stem cell transplant (allo-HSCT). Detection of early cGVHD-induced damage to exocrine tissues, such as salivary glands, is challenging to detect prior to irreversible tissue fibrosis. Recent work in our laboratory used shotgun proteomics to identify putative salivary biomarkers at salivary gland cGVHD onset. Saliva samples from patients affected with oral cGVHD post allo-HSCT had a drop in protein content of zymogen granule 16b (ZG16b), recapitulated in immunohistochemical staining of human minor salivary gland (MSG) biopsy sections. ZG16b loss correlated with CD45+ cells infiltration. This protein is poorly characterized, and immunofluorescence tissue staining suggested ZG16b protein localization to acinar cells. ZG16b cellular expression localized to serous and serous mucous acinar cell populations in single cell RNAseq (scRNA seq) data from healthy human labial MSG. Little is known about the function of this ZG16b in other exocrine tissues such as pancreas. scRNA seq analysis of repository data suggests ZG16b colonization in the pancreatic ductal cells. Similar to MSG, the pancreatic Zg16b gene was expressed in mucus secretion pathways and additional interactions with cell migration and homeostasis markers. Ongoing work is being done to further probe for binding partners for ZG16b (endogenous proteins, components of the oral microbiome and immune cell receptors) to distinguish the protein from its homologs. In parallel, the utility of salivary ZG16b as a cGVHD and general salivary gland damage biomarker is being tested. This work will bring clarity Zg16b’s role in exocrine tissues and alloimmune disease. NIH, NIDCR Intramural program

Cases of RhD variants RhD*DAU2/DAU6 and RhD*weak D type 4.1 in pregnant women in Saudi Arabia.

The D antigen is one of the most immunogenic and clinically significant antigens of the Rh blood group system due to its various genotypes that encode for more than 450 different variants. Accurate RhD typing and D variant identification is crucial specially in prenatal screening during pregnancy. Women with RhD -ve phenotype are eligible to Rh immune globulin (RhIG) prophylaxis for the prevention of anti-D alloimmunization and hemolytic disease of the fetus and newborn (HDFN). However, there are some women who possess RhD variant alleles, who are mistakenly grouped as RhD positive and considered not eligible for RhIG prophylaxis, putting them at risk of anti-D alloimmunization and consequently leading to HDFN during subsequent pregnancies. Here, we describe two cases of RhD variants DAU2/DAU6 and Weak D type 4.1 in obstetric patients who were grouped as RhD +ve with negative antibody screening during routine serologic testing. Weak/Partial D molecular analysis using genomic DNA Red Cell Genotyping (RCG) revealed that both patients had RhD variants, one of which DAU2/DAU6 allele associated with anti-D alloimmunization. According to routine testing neither patients received RhIG or transfusion. In this case report we document to our knowledge the first reported cases of RhD variants among pregnant women in Saudi Arabia.

Neonatal sepsis in alloimmune hemolytic disease of the fetus and newborn: A retrospective cohort study of 260 neonates.

Among neonates with hemolytic disease of the fetus and newborn (HDFN), we aimed to describe the frequency of central-line use, indications for insertion, and incidence of confirmed and suspected sepsis, including antibiotic treatment over a 10-year surveillance period. All neonates with HDFN admitted to our neonatal intensive care unit between January 2012 and December 2021 were included in this retrospective, cohort study. Annual proportions of infants with a central-line and central-line-associated bloodstream infection (CLABSI) rates (per 1000 central-line days and per 100 infants) were evaluated. Numbers of confirmed and suspected early- and late-onset sepsis episodes were assessed over the entire study period. Of the 260 included infants, 25 (9.6%) were evaluated for suspected sepsis, with 16 (6.2%) having ≥1 confirmed sepsis episode. A total of 123 central-lines were placed in 98 (37.7%) neonates, with impending exchange transfusion (ET) being the most frequent indication. Of the 34 (34.7%) neonates in whom a central-line was placed due to impending ET, 11 (32.4%) received no ET. Overall CLABSI incidence was 13.58 per 1000 central-line days. Neonates with a central-line had a higher risk for confirmed late-onset infection (RR 1.11, 95% CI: 1.04-1.20) and sepsis work-up (RR 1.10, 95% CI: 1.03-1.17) compared to infants without a central-line. Sepsis incidence among neonates with HDFN remains high, in particular in those with a central-line. Considering the substantial proportion of neonates with a central-line without eventual ET, central-line placement should be delayed until the likelihood of ET is high.

Intravenous immunoglobulin in hemolytic disease of the newborn: A moving target in time.

Alloimmune hemolytic disease of the newborn (AIHDN) results in hemolysis, anemia, hyperbilirubinemia with the potential for brain damage. Intravenous immunoglobulin (IVIG) has been investigated as an alternative low-risk procedure for the treatment of AIHDN in addition to traditional treatment methods such as phototherapy and exchange transfusion (ET). To evaluate the effectiveness of IVIG therapy in decreasing ET needs based on risk factors and clinical outcomes. Charts of neonates born >30 weeks of gestation who underwent phototherapy and were administered IVIG therapy due to AIHDN between January 2013 and July 2018 were retrospectively reviewed. Sixty-three neonates were included in our study. Forty-three of them (68.3) % were full-term infants. ABO incompatibility (n = 33, 52.4%) was the major cause of AIHDN (n = 63). Additional risk factors for jaundice were found to coexist in 95.2% (n = 60) of the infants. Fifteen infants (23.8%) required ET, mostly due to Rh incompatibility (n = 11, 73.3%). Mortality was observed in 3.2% (n = 2) of the patients, 1.6% (n = 1) of whom were related to ET. Serum albumin value was found to be negatively correlated with the requirement for ET (r = 0.713, P < 0.001), whereas serum bilirubin albumin ratio was positively correlated (r = 0.489, _P < 0.001). Nine (14.3%) infants needed a simple transfusion during the hospitalization period, whereas five (7.9%) infants had readmission for simple transfusion after discharge. Apnea was the only complication seen in one (1.6%) patient. IVIG treatment should be considered due to its relative benefits when compared to exchange transfusion. In addition to its safety, it is a less complicated treatment modality with low side effect rates. It may be justified for elective use in neonates suffering from AIHDN, who will require ET with a risk of mortality by decreasing the peak of total serum bilirubin levels.

P1741: EXAMINING THE BURDEN OF ILLNESS ASSOCIATED WITH TRANSFUSION-DEPENDENT ΒETA-THALASSEMIA FROM THE PATIENT’S PERSPECTIVE

Background: β-thalassemia is a rare genetic blood disorder affecting approximately 1 in 100,000 people globally. Typically diagnosed in childhood, β-thalassemia is marked by the inability to produce hemoglobin, which results in chronic anemia. Transfusion-dependent β-thalassemia (TDβT) is the most severe form of this disorder, with treatment requiring frequent red blood cell transfusions (RBCTs). Recognized complications of the frequent RBCTs include alloimmunization, iron overload, resultant cardiac and liver disease, and reported poor health-related quality of life (HRQoL). Aims: To examine the burden of illness and HRQoL in patients diagnosed with TDβT. Methods: Potential study participants were recruited with the assistance of the Cooley’s Anemia Foundation. Participants were ≥18 years of age, had a self-reported physician diagnosis of β-thalassemia, and had received ≥1 RBCT in the past 6 months. After providing informed consent, patients completed an institutional review board-approved 30-minute online survey that included both validated patient-reported outcome instruments, and other questions written expressly for the purpose of this study. Data were collected in May 2021. Patients who completed the survey earned an incentive payment of 75 US dollars. Results: A total of 100 patients completed the survey; mean age was 36.0 years (standard deviation [SD] 10.4) and 35% of patients were male. Patients had been diagnosed with TDβT for a mean of 34.4 years (SD 10.3) and experienced a mean of 9.6 (SD 4.3) RBCT events in the past 6 months. As an indication of severity, 66% of patients reported requiring >1 RBCT per month, 31% each month, and 3% every other month. Side effects of RBCT included iron overload (in 81% of patients), rashes/hives (74%), fatigue (61%), and pain/bruising at injection site (52%). Mean time required for an RBCT was 7.4 hours (SD 11.3), including transfusion preparation time. Travel and wait times were an additional 7.1 hours. Among all patients, 70% reported a “moderate” to “extremely high” burden of RBCT. On HRQoL, 71% of patients reported “wondering what life would be like without β-thalassemia,” 86% reported “feeling fatigued” leading up to RBCT, and 42% reported “not being as social with friends/family due to the time needed for RBCT.” On validated psychological instruments, 52% of patients reported mild-to-severe depression (Patient Health Questionnaire [PHQ-9]) and 53% reported mild-to-severe anxiety (Generalized Anxiety Disorder Questionnaire [GAD-7]), over the previous 2 weeks. Summary/Conclusion: The survey results suggest there is a substantial time, side-effect, and HRQoL burden associated with treatment by RBCTs in patients diagnosed with TDβT. This includes considerable time devoted to preparing for and receiving transfusions, transfusion-related side effects, and increases in depression and anxiety. These findings contribute to the understanding of the humanistic and psychological burden of RBCT dependency in patients with β-thalassemia. New treatment options that can improve outcomes in this population are needed.

Severe fetal and mild hemolytic disease of newborn

Maternal antibody production is stimulated when fetal red cells are positive for an antigen absent onmother's red cells. Alloimmune hemolytic disease of fetus and newborn due to anti E is uncommon. We report a case of anti-E hemolytic disease in a neonate who had severe fetal and mild neonatal hemolytic manifestations. The neonate was treated with phototherapy. He also received intravenous immunoglobulin and single PCV transfusion.

Anti-U hemolytic disease of the fetus and newborn managed by multiple intrauterine transfusions: a case report and review of the literature

Background: The U-antigen is part of the MNS blood group. Named for its almost universal expression, a U-negative phenotype is reported in ~1% of individuals of African descent. The spectrum of IgG-mediated anti-U hemolysis includes alloimmune disease of the fetus and newborn. The clinical presentation among affected newborns ranges from mild anemia to erythroblastosis fetalis.

Alemtuzumab-Induced Hypothyroidism Presenting in Pregnancy - the First 2 Reported Cases

Abstract Introduction: Alemtuzumab is a monoclonal antibody that improves outcomes in patients with relapsing-remitting multiple sclerosis. It binds to CD52 and causes a profound depletion of B and T lymphocytes. Autoreactive T cells often form as the immune system repopulates, causing autoimmune thyroid dysfunction in approximately 35% of patients. Alemtuzumab has been associated with new-onset hyperthyroidism during a subsequent pregnancy. Here, we present the first two cases of alemtuzumab-induced hypothyroidism diagnosed in pregnancy. Diagnosing and treating de novo thyroid dysfunction in pregnancy is essential for optimal fetal development and maternal wellbeing. Clinical Cases: Case A: A 28-year-old woman conceived 26 months after alemtuzumab therapy. Prior to pregnancy, routine TFTs showed euthyroidism. At 17 weeks’ gestation she presented with fatigue, and blood tests confirmed overt hypothyroidism (TSH &amp;gt;100mIU/L (&amp;lt;4IU/L), fT4 3.7pmol/L (9-15pmol/L)). TRABs were positive. She was treated with levothyroxine, and TFTs normalised by 38 weeks’ gestation. Serial fetal ultrasounds showed constant growth velocity and no fetal goitre or tachycardia. The baby was born at term with birthweight on the 30th centile. On day 5 postpartum, the baby had a normal TSH of 3.1mIU/l (0.27-4.20mIU/l) but elevated fT4 of 31.5pmol/L (12-22pmol/L), which fell spontaneously to 22.0pmol/L by day 11. Case B: A 37-year-old woman conceived 19 months after alemtuzumab therapy. Prior to pregnancy, routine TFTs had been within normal limits. However at 5 weeks’ gestation, blood tests showed overt hypothyroidism (TSH 47.9mIU/L (&amp;lt;4iU/L)), fT4 8.8pmol/l (10-16pmol/L)). Her only symptom was fatigue. TRABs were elevated. She was treated with levothyroxine, and TFTs normalised by 16 weeks’ gestation. Serial fetal ultrasound scans showed constant growth velocity and no fetal goitre or tachycardia. The baby was born at term with birthweight on the 25th centile. On day 1 postpartum, the baby had a normal fT4 (16.5pmol/l)but elevated TSH 13.02mIU/L, which normalised spontaneously by day 11 tp 1/64mIU/l. Conclusion: To optimise pregnancy outcomes, we recommend that women previously treated with alemtuzumab should be screened monthly for thyroid dysfunction during a subsequent pregnancy. The neonatal team should be alert to transient neonatal thyroid dysfunction, which, like spontaneous alloimmune neonatal thyroid disease, appears to settle spontaneously.

Exchange transfusions in severe Rh-mediated alloimmune haemolytic disease of the foetus and newborn: a 20-year overview on the incidence, associated risks and outcome.

Background and objectivesGuidelines and indications for exchange transfusion in haemolytic disease of the foetus and newborn (HDFN) have changed drastically in the past decades, causing a decline in exchange transfusion rate. This study aims to evaluate the incidence of exchange transfusions (ETs) in neonates with Rh‐mediated HDFN over the past 20 years at our centre, and report potentially ET‐related complications as well as indicators for bilirubin encephalopathy.Material and methodsIn this observational study, 438 neonates were included with HDFN, born ≥ 35 weeks gestational age at the Leiden University Medical Centre between January 2000 and July 2020. The incidence of ET and procedure‐related complications were assessed in three consecutive time periods determined by changes in guidelines and indications for ET.ResultsThe incidence of ET in our centre declined from (104/156) 67% (time period 2000–2005), to (39/181) 22% (2006–2015) and to (10/101) 10% (2015–2020, p < 0·001). The maximum bilirubin levels in neonates after birth increased from 13·6 mg/dL (or 233 μmol/L), to 15·0 mg/dL (257 μmol/L) and to 15·3 mg/dL (263 μmol/L). The incidence of complications associated with the use of ET (including sepsis, haematologic disorders and respiratory failure) remained stable throughout the years, and no neonates died during the study period.ConclusionExchange transfusion incidence declined significantly over the past two decades. Decrease in ET incidence, and concomitant decrease in exposure and expertise, was not associated with an increase in procedure‐related complications.

Red blood cell alloimmunization and sickle cell disease: a narrative review on antibody induction.

The high prevalence of red blood cell (RBC) alloantibodies in people with sickle cell disease (SCD) cannot be debated. Why people with SCD are so likely to form RBC alloantibodies, however, remains poorly understood. Over the past decade, a better understanding of non-ABO blood group antigen variants has emerged; RH genetic diversity and the role this diversity plays in RBC alloimmunization is discussed elsewhere. Outside of antigen variants, the immune systems of people with SCD are known to be different than those of people without SCD. Some of these differences are due to effects of free heme, whereas others are impacted by hyposplenism. Descriptive studies of differences in white blood cell (WBC) subsets, platelet counts and function, and complement activation between people with SCD and race-matched controls exist. Studies comparing the immune systems of alloimmunized people with SCD to non-alloimmunized people with SCD to race-matched controls without SCD have uncovered differences in T-cell subsets, monocytes, Fcγ receptor polymorphisms, and responses to free heme. Studies in murine models have documented the role that recipient inflammation plays in RBC alloantibody formation, with human studies reporting a similar association. Murine studies have also reported the importance of type 1 interferon (IFNα/β), known to play a pivotal role in autoimmunity, in RBC alloantibody formation. The goal of this manuscript is to review existing data on factors influencing RBC alloantibody induction in people with SCD with a focus on inflammation and other immune system considerations, from the bench to the bedside.

Maternal Red Blood Cell Alloimmunization Managed with Intrauterine Blood Transfusion: Predictors of Poor Outcome

Background: The rhesus (Rh) and Kell blood group systems are the most common of over 50 different antigens capable of causing maternal red blood cell (RBC) alloimmunization and severe fetal hemolytic disease. Anti-K and anti-D are responsible for a significant proportion of fetal anemia requiring intrauterine transfusion (IUT). Whilst IUT of packed RBCs improves neonatal survival and morbidity, clinical prognostic indicators are lacking. Our primary objective was to identify predictors of adverse outcome. Methods: We conducted a retrospective single-center study at Mount Sinai Hospital (MSH), Toronto, Canada. All pregnant patients alloimmunized with anti-K and anti-D as a single antibody, between 1991 and 2018 were included. Data were obtained from patient medical records, ultrasound reports and information from the transfusion medicine laboratory. Data included maternal demographics, antibody titers, pregnancy history, number of IUTs, hemoglobin (HB) concentration at the beginning and end of all IUTs. Neonatal outcomes included survival, mode of delivery, gestational age at delivery, birth weight, HB at birth and need for neonatal transfusion, phototherapy or intravenous immunoglobulins (IVIG). Our primary outcome was the composite outcome of stillbirth or neonatal death (SB/NND). We also constructed a secondary outcome consisting of top-up neonatal transfusion, exchange transfusion, phototherapy, or use of IVIG. Medians and interquartile ranges (IQR) or mean±SD were used as summary statistics and compared by Mann-Whitney or t-test; p&amp;lt;0.05 was statistically significant. Outcome predictors were identified using multivariable logistic or linear regression analysis; accounting for patient level clustering by marginal Generalized Estimating Equation did not significantly affect variables. Data were analyzed using SPSS. Results: 116 women with 128 pregnancies and 425 IUTs with anti-K or anti-D as a single antibody were identified. Median maternal age was 31 years (27.0-35.0) for anti-K and 32 years (23.6-40.6) for anti-D. The gestational age at 1st IUT differed significantly between anti-K and anti-D (24.3 vs 28.7 weeks respectively, p=0.004). Women with anti-K antibodies required more IUTs than women with anti-D (3.84 vs 3.12 IUTs, p=0.036) and HB at 1st IUT was significantly lower in the anti-K group (5.10 vs 7.05 g/dL, p=0.001) (Table 1). Following initiation of IUT, the time from 1st IUT to delivery was 69.6 days in the anti-K group and 54.6 in the anti-D group (p=0.06). The daily decrease of HB between 1st and 2nd IUT (as a marker of disease severity), development of fetal hydrops and severe preterm birth did not differ significantly between the two groups. Mean gestation age at delivery was 35.0 weeks in the anti-K and 36.0 weeks in the anti-D group (p=0.28), with 87.1% and 93.9% survival (p=0.37), respectively. The proportion of neonates requiring phototherapy, IVIG and exchange/top-up transfusion was comparable across the two antibody groups (Table 1). Regression analysis showed that delivery occurred sooner if HB dropped more rapidly between the first two IUTs (p=0.01). Each additional transfusion gained on average 22.5 days in utero (Table 2). In multivariable analysis, gestational age at 1st IUT was the only predictor of a SB/NND outcome (adjusted OR 0.79 [95%CI 0.67-0.93]; p=0.006). With 1st IUT at 23 weeks, the risk of SB/NND was 8%, but only 2.5% at 28 weeks and &amp;lt;0.01% at 36 weeks (Table 3). Gestational age at delivery (adjusted OR 1.11 [95%CI 1.00 to 1.23]; p=0.046) and HB at 1st IUT (adjusted OR 0.84 [95%CI 0.72 to 0.99]; p=0.038) were the only predictors of a composite adverse blood product requirement/intervention outcome (Table 4). The odds of blood product requirement/intervention postnatally at 1st IUT (median HB at 1st IUT of 6.6g/dL) at 28 weeks were 43% and increased by a further 38% for delivery at 34 weeks. Conclusion: The earlier in gestation that IUTs are implemented, the higher the odds of a SB/NND; however the later the gestation at delivery, the greater the odds of the neonate requiring blood products post-partum. The greater the HB drop between the 1st and 2nd IUT, the shorter the 'time between the first IUT and delivery', which increases the odds of a SB/NND outcome. Disclosures Garbowski: Vifor Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Imara: Consultancy. Shehata:Ferring: Honoraria.

20 Years of Follow-up Alloimmunization and Hemolytic Disease in Newborn: Has Anything Changed in the Field Over the Years?

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.

Distribution of maternal red cell antibodies and the risk of severe alloimmune haemolytic disease of the foetus in a Chinese population: a cohort study on prenatal management

BackgroundHaemolytic disease of the foetus and newborn (HDFN) is the most common aetiology of haemolytic anaemia and hyperbilirubinaemia in foetuses and neonates. Studies on the distribution of antibodies that cause haemolytic disease of the foetus (HDF) in China are limited, and the effects of multiple antibodies on the severity of HDF need further evaluation.MethodsAn observational cohort study from January 2005 to December 2019 was conducted in two hospitals affiliated with Sun Yat-sen University. Maternal red cell alloimmunization was identified by the Guangzhou Blood Centre. In total, 268 pregnant woman-foetus pairs were divided into four groups according to the type of maternal alloantibodies: anti-D, anti-D combined with other antibodies, other single-antibody and other multiple antibodies. The obstetric history, antibody characteristics, incidence of severe HDF and foetal outcomes were collected and compared. Logistic regression analysis of the risk factors for HDF and survival analysis of the severe HDF-free interval were conducted.ResultsAnti-D was the most common cause of HDF, followed by anti-M. No anti-K- or isolated anti-c-associated HDF was found. The incidence of severe HDF was higher in the group with anti-D combined with other antibodies than in the group with anti-D alone (P = 0.025), but no significant difference was found in haemoglobin level and reticulocyte count in the anaemic foetuses between these two groups. Foetuses in the other single-antibody group had a lower reticulocyte count (P = 0.007), more IUTs (P = 0.007) and an earlier onset of severe HDF (P = 0.012). The maximum antibody titre was significantly lower in the other single-antibody group than in the anti-D group (P < 0.001). A high maternal antibody titre (P < 0.001), multiple affected pregnancies (P < 0.001) and other single-antibody (P = 0.042) were independent risk factors for HDF. A higher reticulocyte count (P = 0.041) was an independent risk factor for severe HDF in anaemia foetuses affected by Rh(D) alloimmunization.ConclusionsThe distribution of HDF-associated antibodies in China is different from that in Western countries. Other single non-Rh(D) antibodies could increase the risk of HDF, and anti-D combined with other antibodies would not influence the severity of foetal anaemia compared with anti-D alone.

PS1236 COST-EFFECTIVENESS AND COST-UTILITY ANALYSIS OF MULTIPLE TREATMENT STRATEGIES USING ABVD AND/OR BEACOPP IN THE TREATMENT OF ADVANCED-STAGE HODGKIN LYMPHOMA

Background: Donor-derived red blood cells (RBCs) are the most common form of cellular therapy, coupled with donor-dependency, alloimmunization and blood borne disease risks. In vitro derivation of RBCs from an immortal source, as iPSCs provide an alternative solution to these issues. In addition, in vitro iPSC-RBCs allow genome editing for possible gene therapies, while large scale production would be beneficial in RBC drug delivery. The current limitation of iPSC-RBCs is their development immaturity, leading to low enucleation potential, which requires improvement prior clinical application. Aims: We aim to push iPSC-derived erythroid differentiation to definitive erythropoiesis yielding enucleated erythrocytes Methods: A minimum of three independent iPSC lines were used during the studies. These iPSC lines were single cell seeded and emerging colonies were differentiated towards erythroblasts using specific growth factor cocktails and media developed at Sanquin. Human mononuclear cells isolated from fetal liver, cord blood and adult blood were also differentiated to erythroblasts. Flow cytometry was used to assess culture purity, stage of erythropoiesis and enculeation. RT-PCR and HPLC was used to quantify the developmentally defined globins. RNA was isolated from the different erythroblasts and RNA-sequencing was performed and analysed. Single cell RNA-seq was performed on single cell FACS-sorted iPSC derived hematopoietic cells. Results: To further develop the process, we have established a GMP-compatible iPSC generation and differentiation protocol, that upon hematopoietic specification yielded erythroblasts (EBL). This method resulted in 2–6∗105 EBLs/iPSC with 100% CD71+/CD235+ (erythroid-specific markers). Further maturation yielded orthochromatic normoblasts containing a mix of primitive and definitive erythroid waves, leading to poor enucleation potential and/or reticulocyte stability. Total RNAseq analysis comparing iPSC to in vitro derived definitive erythroid cells from fetal liver, cord and adult revealed significant differences and identified specific genes that may be used to further augment iPSC-erythroid terminal differentiation to erythrocytes. For instance, iPSC-derived erythroblasts lack expression of specific erythroid regulators (e.g. MYB and KLF1 target genes SOX6, BCL11A and BCAM). Single-cell RNAseq of early iPSC differentiation was performed, aiming to recognize distinct waves of hematopoiesis. Five cell clusters were identified of which, one was HSPC-like, 3 lineage-committed populations and 1 of unknown origin. This data coupled with index sorting, defined the HSPCs as CD71+/CD235−, and the erythroid cells as CD71+/CD235+ expressing embryonic and fetal Hbs. In addition, we have found differentially expressed markers between the iPSC-RBCs and definitive erythroid cells that may be used to better define specific erythroid waves during iPSC differentiations and/or fetal development. We hypothesize, that the identified HSPCs potentially give rise to developmentally more mature cells, which we aim to expand. Currently we are pursuing different approaches among which forward programming, to support early iPSC-HSPCs that produce developmentally more mature definitive erythroid cells including proper enucleation potential. Summary/Conclusion: In conclusion, we have performed extensive RNA analysis of iPSC, cord, fetal and adult erythroblast and identified potential targets that may aid in proper iPSC-derived erythroid differentiation to enucleated red cells.

Predicting anaemia and transfusion dependency in severe alloimmune haemolytic disease of the fetus and newborn in the first 3months after birth.

Infants with haemolytic disease of the fetus and newborn (HDFN) often require erythrocyte transfusions in the first 3months of life. We aimed to evaluate the incidence, timing and potential predictors of transfusion-dependent anaemia. An observational cohort of 298 term and near-term infants with severe HDFN treated with or without intrauterine transfusion (IUT) was evaluated. Transfusions were administered to 88% (169/193) of infants with IUT and 60% (63/105) without IUT. The following potential predictors were associated with less anaemia: K compared to D immunisation [odds ratio (OR) 0·13, 95% confidence interval (CI): 0·03-0·55], higher reticulocyte count at birth [per 10 parts per thousand (‰) higher, OR 0·99, CI: 0·97-1·00] and exchange transfusion (OR 0·11, 95% CI: 0·03-0·50). Without IUT, these variables were: lower reticulocyte count at birth (per 10‰ lower, OR 1·02, 95% CI: 1·00-1·03), lower maximum bilirubin after birth (per 10μmol/l lower, OR 1·01, 95% CI: 1·01-1·02) and exchange transfusion (OR 0·07, 95% CI: 0·01-0·20). In conclusion, potential predictors for anaemia in infants with severe HDFN varied between infants treated with and without IUT and are useful for selecting subgroups of infants at increased risk of anaemia.

Granzyme A-producing helper T cells are critical for lethal acute graft-versus-host disease

Abstract Graft-versus-host disease (GVHD) is a deleterious allo-immune response that occurs in patients undergoing hematopoietic stem cell transplantation (HSCT) for treatment of blood-derived malignancies. CD4 T helper (Th) cells within the donor HSC pool recognize allogeneic HLA molecules and traffic to multiple organs, including the intestines where increased inflammation correlates with worsened clinical prognosis. Despite the known role of Th cells in GVHD, the means by which they promote disease remain unclear. In our studies, we have identified a novel class of Th cells that produce the serine protease granzyme A (GrA) within the intestines of mice succumbing to GVHD. GrA+ Th cells were distinct from other Th lineages and largely lacked co-expression of other Th lineage-associated cytokines or cytolytic mediators. Importantly, mice that received HSCT with GrA-deficient CD4 T cells exhibited significantly reduced morbidity and mortality as compared to controls, suggesting that Th-derived GrA is critical for GVHD progression. In vitro, differentiation of GrA+ Th cells was optimally induced with a combination of IL-6, IL-21 and IL-4 in a STAT3- and STAT6-dependent fashion. In vivo, STAT3- was also partially required for the differentiation of GrA+ Th cells during the GVHD response. In summary, GrA+ Th cells represent a novel STAT3-dependent Th cell type that is critical for allo-immune mediated disease.