Red Cell Antigens Research Articles

Neonatal and Obstetrical Outcomes of Pregnancies Complicated by Alloimmunization.

Despite advances in the prevention of rhesus (Rh)(D) alloimmunization, alloantibodies to Rh(D) and non-Rh(D) red blood cell antigens continue to be detected in ∼4% of US pregnancies and can result in hemolytic disease of the fetus and newborn (HDFN). Recent reports on HDFN lack granularity and are unable to provide antibody-specific outcomes. The objective of this study was to calculate the frequency of alloimmunization in our large hospital system and summarize the outcomes based on antibody specificity, titer, and other clinical factors. We identified all births in a 6-year period after a positive red blood cell antibody screen result during pregnancy and summarized their characteristics and outcomes. A total of 707 neonates were born after a positive maternal antibody screen result (3.0/1000 live births). In 31 (4%), the positive screen result was due to rhesus immune globulin alone. Of the 676 neonates exposed to alloantibodies, the direct antibody test (DAT) result was positive, showing antigen-positivity and evidence of HDFN in 37% of those tested. Neonatal disease was most severe with DAT-positive anti-Rh antibodies (c, C, D, e, E). All neonatal red blood cell transfusions (15) and exchange transfusions (6) were due to anti-Rh alloimmunization. No neonates born to mothers with anti-M, anti-S, anti-Duffy, anti-Kidd A, or anti-Lewis required NICU admission for hyperbilirubinemia or transfusion. Alloimmunization to Rh-group antibodies continues to cause a majority of the severe HDFN cases in our hospital system. In neonates born to alloimmunized mothers, a positive DAT result revealing antigen-positivity is the best predictor of anemia and hyperbilirubinemia.

Management of Fetal Hemolytic Disease Due to Anti-Rh17 With the D−Phenotype [ID 2683500

INTRODUCTION: The rare blood D− haplotype produces multiple Rh antibodies against red blood cell antigens such as anti-Rh17 or anti-Hr0 antibodies if a patient is sensitized to Rh antigens. METHODS: This report discusses successful management of a pregnancy in a 28-year-old woman with high anti-Rh17 titers leading to hemolytic disease of the fetus (HDFN). The combination of a D− allele and the weak D 45 allele has not been reported previously, and it may be that the hybrid allele expresses D antigen epitopes such that the D antigen is not weakened. During the studied pregnancy, the patient received plasmapheresis and intravenous immunoglobulin (IVIG) infusions along with intrauterine transfusions (IUTs) when absolutely necessary. She underwent uncomplicated repeat cesarean delivery at 32 5/7 weeks of gestation resulting in a female infant weighing 2,020 g. Patient informed consent was obtained for this case report. CONCLUSION: This patient with the D−/D− blood phenotype leading to anti-Rh17 antibodies after her first pregnancy was managed successfully. Her baby with HDFN and several instances of hydrops fetalis, was successfully managed and treated. The patient’s blood donations for IUTs and maternal plasma exchange therapies promoted efficiency and favorable outcomes for the baby, but did put the mother in an anemic blood range.

Generation of a human induced pluripotent stem cell line (YUCMi020-A) from peripheral blood mononuclear cells derived from a female with the Jr(a−) blood type

The Jra antigen, the only antigen within the JR blood group system, is a high-prevalence red blood cell (RBC) antigen found in over 99 % of the global population. An induced pluripotent stem cell line (YUCMi020-A) was generated from peripheral blood drawn from a Jr(a−) phenotype individual, who was homozygous for a null mutation of ABCG2*01N.01 (rs72552713, c.376C>T; p.Gln126*). The generated line exhibited pluripotent characteristics and no chromosomal aberrations. This cell line will serve as a cell source, enabling us to produce RBCs with the Jr(a−) phenotype in vitro, which can be used for transfusing individuals with anti-Jra antibodies.

Maternal red blood cell alloimmunization prevalence in the United States

AbstractHemolytic disease of fetus and newborn (HDFN) is a life-threatening disease mediated by maternal alloimmunization to red blood cell (RBC) antigens. Studies of maternal alloimmunization prevalence in the United States lack national data. This study describes prevalence and trends in alloimmunization in pregnancy in the United States. RBC antibodies (abs) were identified in a large, nationwide, commercial laboratory database from 2010 through 2021. The cohort comprised pregnancies for which the year of laboratory collection and patient’s state of residence were available. Data were normalized based on US Centers for Disease Control and Prevention estimates of live births and weighted by year and US Census Division. Cochrane-Armitage tests assessed temporal trends of alloimmunization. Of 9 876 196 pregnancies, 147 262 (1.5%) screened positive for RBC abs, corresponding to an estimated prevalence of 1518 of 100 000 pregnancies. Of identified RBC abs, anti-D comprised 64.1% pregnancies (586/100 000). Prevalence of other high-risk RBC abs for HDFN included anti-K (68/100 000) and anti-c (29/100 000). Incidence of all 3 high-risk abs increased from 2010 to 2021 (all P < .001). Among almost 10 million pregnancies in the United States, comprising an estimated 14.4% of all pregnancies, 1.5% screened positive for RBC abs. Almost three-quarters (679/100 000 [74.3%]) of RBC abs identified were high risk for HDFN. Although prevalence of anti-D is difficult to interpret without the ability to distinguish alloimmunization from passive immunity, it remains problematic in HDFN, ranking second only to anti-K in critical titers. Given the sequelae of HDFN, new initiatives are required to reduce the incidence of alloimmunization in patients of reproductive potential.

Naturally Occurring Anti-D in a Male Blood Donor, Unresolved Mystery.

In accordance with numerous national and international criteria, screening donated blood for abnormal antibodies against red cell antigens is crucial for patient safety. Antibodies can be formed by three mechanisms: by immune responses, naturally occurring, or passively acquired. In accordance with departmental policy, ICT was performed on a 30-yearold male blood donor's sample in addition to ABO Rh grouping. He tested A Rh Dnegative and had a positive ICT (+ 2). Anti-D was identified by using the antibody screening and identification panel. Retesting revealed similar results, so we called the donor. We were unable to find a source for the Rh isoimmunization during the course of his history. On further work-up, we found that antibodies reacted only in the antiglobulin phase with no reaction in the saline phase, suggesting an IgG type of antibody. Also, the anti-D titers (IgG) of the donor were up to 1:32. All potential sources of Rh isoimmunization have been ruled out for a male donor. Therefore, we assume that this donor has naturally occurring anti-D, perhaps created by certain environmental factors, bacteria, or components of the COVID vaccine that have cross-reactivity with the D antigen. This instance emphasizes how crucial the ICT test on the donor blood is. In our case, all potential causes of alloimmunization were ruled out, leaving only exposure to antigens with antigenic similarity to D antigens as a viable explanation.

Association of Secretor And Nonsecretor Status with Lewis Phenotyping Among Blood Donors: A Prospective Observational Study from Tertiary Care Hospital.

ABO (ABH) antigen expression on red cell antigens and secretion of ABH substances in body fluids are governed by different genes. The presence or absence of the Secretor gene is the basis of secretor status positivity and negativity, respectively. Determination of the secretor status has an essential role in the medical field. It was a prospective observational study to determine the association of secretor and nonsecretor status with Lewis phenotyping among voluntary blood donors. It was conducted from Jan 2020 to December 2021 at the Department of Transfusion Medicine in a tertiary care hospital in South India. Among 318 donors, secretors were 229 (72%), and nonsecretors were 89 (28%). The Lewis phenotype was consistent with secretor status, i.e., All Le (a-b +) were secretors accounting for 213 (66.9%), and All Le(a + b-) were nonsecretors accounting for 80(25.1%)donors. In the Le(a-b-) phenotype, 16(5.0%) were secretors with Se positivity but defective Lewis expression, and 9(2.8%) were nonsecretors with sese. Detection of Lewis phenotype indirectly reflects the blood group secretor status. Hence, in case of discrepancies in ABO blood groups, Bombay and Para Bombay groups, testing for Le(b) confirmation will be useful.

Comparison of the solid-phase red cell adherence assay and tube method for detection and identification of red blood cell antibodies.

Identifying antibodies to red blood cell antigens is one of transfusion medicine's critical responsibilities. The International Society of Blood Transfusion recognizes 354 red blood cell antigens. Accurate identification of clinically significant alloantibodies is imperative for preventing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and solid-phase red cell adherence assay techniques. We performed a retrospective study on all antibody screens performed between 2007 and 2021 at Stanford Transfusion Services. Initially, 631,535 antibody screens were performed using a solid-phase technique. Subsequent antibody identifications were performed using a combination of tube testing and solid-phase techniques. Antibody screening resulted in 28,316 (4.5%) positive samples. Antibody identification performed on both platforms identified 50 discordant samples. The anti-E antibody had the lowest sensitivity (98.99%) in the automated solid-phase technique, while anti-Jkb had the lowest sensitivity (98.78%) with the PEG-IAT method. To our knowledge, this is the first robust, 15-year study comparing methodologic sensitivity to detect clinically significant alloantibodies. The incidence of discordant results between PEG-IAT and the solid-phase technique was low. Among discordant samples, anti-Jka was commonly detected using the solid-phase method but not with the PEG-IAT. In contrast, anti-E was commonly detected by PEG-IAT but not by the solid-phase method.

The use of intracellular dyes to create a multiplexed flow cytometry-based red blood cell phenotyping assay.

Flow cytometry can be used to phenotype red blood cell antigens, allowing for high-throughput testing while using low reagent volumes. This article utilizes intracellular dyes to pre-label red blood cells to further multiplex flow cytometry-based red blood cell antigen phenotyping. Red blood cells were pre-labelled using the intracellular dyes V450 and Oregon Green. These dyes are detected fluorescently via flow cytometry. Four combinations of intracellular staining were used to allow four patient or donor red blood cells to be analysed in a single test well. Antigen phenotyping was then performed via flow cytometry using a previously described method. The intracellular dyes showed uniform staining when measured in mean fluorescence intensity and allowed the red blood cells to be clearly distinguished from one another. The presence or absence of red blood cell antigens was determined with 100% accuracy. The use of intracellular dyes allowed a fourfold increase in the throughput of our previously described flow cytometry-based red blood cell antigen phenotyping method. The described method allows up to 48 patients to be simultaneously phenotyped using a single 96-well microplate. Furthermore, additional fluorescent dyes could potentially increase the throughput exponentially.

Correlation Between the Rh/ABO System and Infection Due to SARS Cov2 in Morocco*

The association between COVID-19, ABO blood group system and Rhesus needs to be explored in order to suggest a model of the mechanism linked to SARS CoV2 infection. A retrospective case-control association study was performed on the different regions of Morocco and carried out on a population of size n = 5039 including a total of 4268 COVID-19 patients and 771 control groups. The differences in the Rh/ABO blood group distribution between COVID-19 patients and the control group were analyzed. The relationship between ABO blood type, sexes, Rhesus and COVID-19 characteristics was analyzed. The analyzes of association between the blood group and the infection related to SARS CoV2 showed a statistically significant difference in the individuals of groups B, O, AB) respectively, but not blood group A. In addition, patients with the Rh+ phenotype are less vulnerable to infection therefore the latter are considered to have a protective effect, however the Rh- categories are more vulnerable to infection so they are risk factors. Blood type was related to some clinical characteristics of patients with COVID-19. The presence of the A and B antigens increase the risk of infection with SARS COV2. While the presence of anti A and anti B antibodies makes group O protector against infection. Patients with the Rh+ phenotype are less vulnerable to infection than Rh-therefore these are considered to have a protective effect which is the presence of the D antigen in red blood cells.

A novel frameshift mutation in RHAG leads to Rhnull phenotype in a Chinese individual.

We recently encountered a Rhnull phenotype proband within one family in the Chinese population. Rhnull is a rare autosomal recessive disorder characterized by the absence of the Rh antigens on the erythrocyte membrane, resulting in chronic hemolytic anemia. This study described the serological and molecular analysis of a Chinese Rhnull proband and his immediate family. Red blood cells antigen phenotyping and antibody screening/identification were conducted. RHD, RHCE, and RHAG were analyzed using genomic DNA by polymerase chain reaction and sequence analysis. Serologic tests showed a D-C-E-c-e- phenotype in the proband associated with the suspicion of anti-Rh29 (titer 16). Molecular analyses showed a new mutation (c.406dupA) in exon 3 of RHAG. This duplication introduced a reading frameshift (p.Thr136AsnfsTer21). The RHAG mutation was found in the homozygous state for the proband and heterozygous state for his parents. We identified a novel RHAG mutation resulting in the Rhnull phenotype of the regulator type. Inheritance of the novel allele was shown by family study.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Meeting the transfusion needs of a patient with anti-Ena requires an international effort.

This extraordinary case showcases the identification of a rare anti-Ena specificity that was assisted by DNA-based red blood cell antigen typing and collaboration between the hospital blood bank in the United States, the home blood center in Qatar, the blood center Immunohematology Reference Laboratory, as well as the American Rare Donor Program (ARDP) and the International Society for Blood Transfusion (ISBT) International Rare Donor Panel. Ena is a high-prevalence antigen, and blood samples from over 200 individuals of the extended family in Qatar were crossmatched against the patient's plasma with one compatible En(a-) individual identified. The ISBT International Rare Donor Panel identified an additional donor in Canada, resulting in a total of two En(a-) individuals available to donate blood for the patient.

Transfusion Support in Hematopoietic Stem Cell Transplantation: A Contemporary Narrative Review.

Hematopoietic stem cell transplantation (HSCT) is a cornerstone of modern medical practice, and can only be performed safely and effectively with appropriate transfusion medicine support. Patients undergoing HSCT often develop therapy-related cytopenia, necessitating differing blood product requirements in the pre-, peri-, and post-transplant periods. Moreover, ensuring optimal management for patients alloimmunized to human leukocyte antigens (HLA) and/or red blood cell (RBC) antigens, as well as for patients receiving ABO-incompatible transplants, requires close collaboration with transfusion medicine and blood bank professionals. Finally, as updated transfusion guidelines and novel blood product modifications emerge, the options available to the transplant practitioner continue to expand. Herein, we detail contemporary blood transfusion and transfusion medicine practices for patients undergoing HSCT.

A machine-learning method for biobank-scale genetic prediction of blood group antigens.

A key element for successful blood transfusion is compatibility of the patient and donor red blood cell (RBC) antigens. Precise antigen matching reduces the risk for immunization and other adverse transfusion outcomes. RBC antigens are encoded by specific genes, which allows developing computational methods for determining antigens from genomic data. We describe here a classification method for determining RBC antigens from genotyping array data. Random forest models for 39 RBC antigens in 14 blood group systems and for human platelet antigen (HPA)-1 were trained and tested using genotype and RBC antigen and HPA-1 typing data available for 1,192 blood donors in the Finnish Blood Service Biobank. The algorithm and models were further evaluated using a validation cohort of 111,667 Danish blood donors. In the Finnish test data set, the median (interquartile range [IQR]) balanced accuracy for 39 models was 99.9 (98.9-100)%. We were able to replicate 34 out of 39 Finnish models in the Danish cohort and the median (IQR) balanced accuracy for classifications was 97.1 (90.1-99.4)%. When applying models trained with the Danish cohort, the median (IQR) balanced accuracy for the 40 Danish models in the Danish test data set was 99.3 (95.1-99.8)%. The RBC antigen and HPA-1 prediction models demonstrated high overall accuracies suitable for probabilistic determination of blood groups and HPA-1 at biobank-scale. Furthermore, population-specific training cohort increased the accuracies of the models. This stand-alone and freely available method is applicable for research and screening for antigen-negative blood donors.

Current challenges of blood transfusions in patients with thalassemia in India and future perspectives

The introduction of regular red blood cell transfusions transformed thalassemia major from a fatal childhood disease into a chronic disorder. Thalassemia is highly prevalent in South Asia, including the Indian subcontinent, and blood transfusion remains the cornerstone of management for these patients. But safe blood transfusions still remain a major problem in India. Difficulties in maintaining adequate blood inventory, a lack of a national blood act, and fragmented blood transfusion services are some of the major contributing factors for the delay in blood supply. In most of the blood centers, alloantibody detection facilities and extended red cell antigen typing are unavailable. Awareness is the key to reducing alloimmunization, which limits the effectiveness of transfusions and the potential availability of blood. Patients with thalassemia are also at high risk of transfusion-transmitted infections unless appropriate blood screening is in place. Hence, many patients remain under-transfused, resulting in decreased health and quality-of-life outcomes. Facilities such as leucoreduction and immunohematological monitoring following a blood transfusion are often lacking in India, especially at the sub-district level. Continuous efforts to raise community awareness, regular training of health-care workers, and proper utilization of available resources are essential to ensuring safe blood transfusions for patients with thalassemia. Access to the new treatments at an affordable cost may reduce the blood transfusion burden for thalassemia patients in India.

Non-RhD alloimmunization in pregnancy: an updated review.

RhD alloimmunization in pregnancy is still the main cause of hemolytic disease of the fetus and neonate (HDFN). Nevertheless, there are other antigens that may be associated with the occurrence of this phenomenon and that have been growing in proportion, given that current prevention strategies focus only on anti-RhD antibodies. Although not widespread, the screening and diagnostic management of the disease caused by these antibodies has recommendations in the literature. For this reason, the following review was carried out with the objective of listing the main red blood cell antigen groups described - such as Rh, ABO, Kell, MNS, Duffy, Kidd, among others - addressing the clinical importance of each one, prevalence in different countries, and recommended management when detecting such antibodies during pregnancy.

Immune stimulatory effect of Nigella sativa in healthy animal models: A systematic review and meta-analysis

The immune-modulatory effects of black seeds (Nigella sativa seeds, NSS) are well documented, but the overall in vivo impact of this important natural medicinal product on immune system function has yet to be established. Here we systematically reviewed and meta-analyzed the effects of NSS on humoral [serum titers of immunoglobulins including IgG, IgM, anti-Newcastle virus disease (anti-NDV), and sheep red blood cell antigen (anti-SRBC)] and cellular immunity [total white blood cell (WBC) count and percentages of monocytes, lymphocytes, basophils, neutrophils, and eosinophils] in healthy animals. The PubMed, ScienceDirect, Web of Science, and Scopus databases were searched according to predefined eligibility criteria. Meta-analyses were performed to estimate the final effect size using RevMan software. Seventeen animal studies were eligible for analysis. For humoral immunity, the overall pooled effect size (ES) of NSS on serum titers of IgM and anti-NVD antibodies was not significantly different [mean difference (MD) 75.27, 95% CI: −44.76 to 195.30, p = 0.22 (I2 = 89%, p = 0.003), and −0.01, 95% CI: −0.27 to 0.25, p = 0.94 (I2 = 74%, p = 0.02), respectively]. However, NSS significantly increased serum titers of IgG and anti-SRBC antibodies [MD 3.30, 95% CI: 2.27 to 4.32, p = 0.00001 (I2 = 0%, p = 0.97), and 1.15, 95% CI: 0.74 to 1.56, p = 0.00001 (I2 = 0%, p = 0.43), respectively]. For cellular immunity, the ES of NSS on WBCs, monocytes, and lymphocytes were not significantly different [MD 0.29, 95% CI: −0.55 to 1.13, p = 0.50, (I2 = 14%, p = 0.32), - 0.01, 95% CI: −0.45 to 0.44, p = 0.97 (I2 = 0%, p = 0.77), and 4.73, 95% CI: −7.13 to 16.59, p = 0.43, (I2 = 99%, p = 0.00001), respectively]. In conclusion, black seeds enhance humoral immunity in healthy animals but do not affect cellular immunity.

Evaluating the TaqMan Jra-Genotyping Method for Rapidly Predicting the Presence of Anti-Jra Antibodies.

The Jra antigen is a high-prevalence red blood cell (RBC) antigen. Reports on cases of fatal hemolytic disease of the fetus and newborn and acute hemolytic transfusion reactions suggest that antibodies against Jra (anti-Jra) have potential clinical significance. Identifying anti-Jra is challenging owing to a lack of commercially available antisera. We developed an alternative approach to rapidly predict the presence of anti-Jra using the TaqMan single-nucleotide polymorphism (SNP)-genotyping method. Residual peripheral blood samples from 10 patients suspected of having the anti-Jra were collected. Two samples with confirmed Jr(a-) RBCs and anti-Jra were used to validate the TaqMan genotyping assay by comparing the genotyping results with direct sequencing. The accuracy of the assay in predicting the presence of anti-Jra was verified through crossmatching with in-house Jr(a-) O+ RBCs. The TaqMan-genotyping method was validated with two Jr(a-) RBC- and anti-Jra-confirmed samples that showed concordant Jra genotyping and direct sequencing results. Jra genotyping for the remaining samples and crossmatching the serum samples with inhouse Jr(a-) O+ RBCs showed consistent results. We validated a rapid, simple, accurate, and cost-effective method for predicting the presence of anti-Jra using a TaqMan-based SNP-genotyping assay. Implementing this method in routine practice in clinical laboratories will assist in solving difficult problems regarding alloantibodies to high-prevalence RBC antigens and ultimately aid in providing safe and timely transfusions and proper patient care.

Phenotyping of Rh and Kell blood group antigen in thalassemia and its impact on alloimmunization in a tertiary care hospital

Objectives: Alloimmunization to red cell antigens is a dreaded complication in multitransfused patients, leading to difficulty in obtaining compatible red blood cell units and development of delayed hemolytic transfusion reactions. The objective of this study was to assess the impact of partial matched phenotype blood (for RhD, C, c, E, e, and Kell antigens) on alloimmunization in thalassemics versus non-phenotype matched blood (ABO &amp; RhD). Materials and Methods: This cross-sectional study was conducted over a period of two years where 250 patients with thalassemias were enrolled. They were divided into two groups, patients in Group I (n = 180) who received partial matched phenotype blood since initiation of transfusion therapy and those in Group II (n = 70) subjects who received usual matched blood. Statistical analysis: All statistical calculations were done using statistical package for the social sciences (SPSS) 21 version. Data were described in terms of range, median (interquartile range [IQR]), frequencies, etc. Results: The median (IQR) age of the study population was 12 (7–18) years (range 6 months–36 years). The most common Rh antibodies were anti-D (2.85%), anti-E (2.85%), anti-C (1.42%), and anti-c (1.42%), and Kell antibodies were (7.1%). It was seen that chances of developing autoantibodies (37% vs. 5%), alloantibodies 11 (15.7% vs. 0%), and transfusion reactions 25 (35.7% vs. 3.3%) were more in Group II subjects as compared to Group I. A significant difference was seen with febrile non-hemolytic transfusion reactions in between two groups 0.001 (95% confidence interval 2.98–65.73). Conclusions: Patients with thalassemia should be typed for RhD (C, c, E, and e) and Kell antigen before initiation of transfusion, which will help in reducing the rate of alloimmunization, autoimmunization, and frequency of transfusion and will improve the overall survival rate in thalassemia.

Emergence of additional anti-C with amplification of anti-D titer post-Rh-Kell phenotype-matched intrauterine transfusions in severe hemolytic disease of fetus

Abstract Transplacental ultrasound-guided intrauterine transfusion (IUT) acts as a lifesaving therapy to prevent fetal anemia or even to reverse fetal hydrops. IUTs are generally initiated after 22–24 weeks of gestation and repeated every 2–4-week period of gestation. Although Rh-Kell phenotype-matched, fresh irradiated leukoreduced donor-packed red cells help to increase fetal hemoglobin level, this invasive procedure can increase fetal complications by fetomaternal hemorrhage. Women receiving IUTs are noted to be high allo-responders to red cell antigens, which can cause enhanced antibody titer or the formation of additional antibodies which might complicate future pregnancies. Hereby, we are reporting the case of a multiparous woman who underwent three sessions of IUTs between 24 weeks and 31+-week period of gestation and developed an additional anti-C antibody which was incidentally detected during compatibility testing at 34 weeks, along with raised anti-D immunoglobulin G titer of 1024 from initial titer of 128.