Low-incidence Antigen Research Articles

Delayed Hemolytic Transfusion Reactions Could be Prevented by a National Alloimmunization Registry

Abstract Evanescence of alloantibodies to RBC antigens places patients at risk of delayed hemolytic transfusion reactions (DHTRs) unless the transfusion service is aware of their alloantibody history. In the past year, three patients with sickle cell disease (SCD) developed a DHTR at our institution because their pre-transfusion testing did not reflect their full alloimmunezation. We sought to determine how often the pre-transfusion antibody screen is negative or fails to show the full alloimmunization profile. We reviewed the electronic records of all current adult patients with SCD receiving chronic outpatient RBC transfusions (including red cell exchange) to collect demographic and laboratory results to answer this question. Our analysis included 162 patients of African-American descent: 80 (49%) males and 82 (51%) females, with an average age of 34 years. The majority, 92 or 56%, had a history of alloimmunization with a mean number of 3 antibodies (range: 1-10). The most common were to the C, E, and K antigens. The next two most common alloantibodies were nonspecific, representing alloimmunization to an antigen that could not be further determined, followed by the D antigen. In 81 patients, the most recent antibody screen did not reflect their history, with most being completely negative (n=69), and the rest (n=12) unable to detect all alloantibodies of which they had a history. The antibodies most often undetectable were: Anti-E, Anti-C, and Anti-D. These are simultaneously the second, first, and fifth most common antibodies in this population, all of which are considered clinically significant and capable of causing a hemolytic transfusion reaction. Of note, the alloantibodies implicated in the recent DHTRs we observed, were to the JKa, Jkb, Fya and the S antigens. In addition, 28 individuals (17% of the total) had at least one antibody to a low incidence antigen (LIA) such as Goa, V, Vs, Jsa, Kpa and Lua. Five (3%) patients were exclusively sensitized to one or more low incidence antigens. Considering that commercially available reagent red cells utilized to perform pre-transfusion testing may lack LIAs, knowing the history of alloimmunization is the only way to prevent hemolytic reactions. In conclusion, evanescence and limitations of the commercially available reagents to test patients with SCD prior to a transfusion place them at increased risk of transfusion reactions. One potential way to minimize risk from the latter is to perform serologic crossmatching of all units intended for patients with SCD, independent of the result of the antibody screen. This is the practice we have instituted several years ago in our transfusion service. However, the solution to both problems is a national repository of alloimmunization histories accessible to all transfusion services. Such informatics tools would help avoid DHTRs in patients with and without SCD.

Rare Anti-Cw Antibody

Introduction: Anti-Cw is a low-frequency antibody against Cw (Rh8) antigen, a low incidence antigen of the Rh system. Anti-Cw antibody may cause mild to moderate hemolytic disease of the fetus and newborn (HDFN) and mild to severe immediate or delayed hemolytic transfusion reactions (HTR). The antigenic frequency of Cw in the Indian population is 1.25%. Two cases of Anti-Cw antibody are being reported which were detected during routine antibody screening, one an oncology patient and the other a healthy blood donor. Case 1: A 30-year-old male, known case of acute myeloid leukemia, with previous history of induction chemotherapy and blood transfusion was admitted for further management and chemotherapy. When three cell antibody screening was performed using an automated platform, the screen was positive with +2 reactions. Antibody identification using 14 cells suggested the presence of an Anti-Cw antibody. On further testing, the patient red blood cell (RBCs) were negative for Cw antigen. Case 2: A 20-year-old male, first-time, voluntary blood donor, with no history of blood transfusion donated whole blood. On performing three cell antibody screening using automated platform, screen was found to be positive with +3 reactions. Antibody identification performed using 14 cell panel suggested the presence of Anti-Cw antibody. On further testing, the donor RBCs were negative for Cw antigen. Conclusion: Antibody screening and identification is recommended in pretransfusion testing, especially in multi-transfused patients and patients belonging to the reproductive age group, so that antibody to low-frequency antigen can be detected and its potential to cause HTR and HDFN can be assessed.

Cw Alloimmunization in Multitransfused Thalassemic Patients of North India

Background and Objectives: The C Willis or Cw antigen is a low-incidence antigen of Rh system. The antibody against the Cw antigen (anti-Cw) is an IgG antibody which may occur naturally or may be immune in nature. The identification of Cw antibody is important since it has the potential to cause hemolytic disease of the newborn as well as hemolytic transfusion reaction. This study was conducted with the aim of determining the prevalence of Cw antibody in multitransfused thalassemic patients enrolled in a Regional Blood Transfusion Center (RBTC) of North India. Methods: A retrospective descriptive observational study was conducted at the Department of Immunohematology and Blood Transfusion, LHMC and Associated Hospitals. All transfusion-dependent thalassemic (TDT) patients and non-TDT (NTDT) patients enrolled in the RBTC of the hospital till December 2018 were included in the study. Antibody screening was performed in all recipients before each transfusion. The prevalence of anti-Cw was estimated. Results: A total of 567 thalassemic patients (including TDT and NTDT) were registered in RBTC, LHMC till December 2018. On pretransfusion antibody screening and identification, 3 out of 567 thalassemic patients were found to have alloimmunization against Cw antigen. The prevalence of anti-Cw in multitransfused thalassemics was 0.53%. Conclusion: The prevalence of anti-Cw is variable in different populations and it is not a very commonly reported antibody in patients with thalassemia. One should be aware of the approach to transfusion in thalassemic patients who develop Cw alloimmunization.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed. DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast. The heterozygous GYP*Mur/GYPB (n= 101), homozygous GYP*Mur/GYP*Mur (n= 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n= 1) and GYP*Vw/GYPA (n= 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals. The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Blood group systems

Blood group systems

Novel hybrid genes and a splice site mutation encoding the Sta antigen among Japanese blood donors.

The low-incidence antigen Sta of the MNS system is usually associated with the GP(B-A) hybrid molecule, which carries the 'N' antigen at the N terminus. The GP(A-A) molecule with trypsin-resistant M antigen has been found in a few St(a+) individuals. Among Japanese blood donors, we screened 24292 individuals for the presence of St(a+) with trypsin-resistant 'N' antigen and 193009 individuals for the presence of St(a+) with trypsin-resistant M antigen. The breakpoints responsible for the Sta antigen were analysed by sequencing the genomic DNAs. A total of 1001 (4·1%) individuals were identified as St(a+) with trypsin-resistant 'N' antigen. Out of 1001 individuals, 115 were selected randomly for sequencing. Two novel GYP*Sch (GYP*401) variants with new intron 3 breakpoints of GYPA were detected in three cases. Twenty-five (0·013%) individuals were identified as St(a+) with trypsin-resistant M antigen. Five individuals had the GYP(A-ψB-A) hybrid allele; two of these five individuals were GYP*Zan (GYP*101.01), and the remaining three had a novel GYP(A-ψB-A) allele with the first breakpoint in GYPA exon A3 between c.178 and c.203. Nine individuals had a novel GYP(A-E-A) allele with GYPE exon E2 and pseudoexon E3 instead of GYPA exon A2 and A3. The 11 remaining individuals had a novel GYP(A-A) allele with a 9-bp deletion that included the donor splice site of intron 3 of GYPA. Our finding on diversity of glycophorin genes responsible for Sta antigen provides evidence for further complexity in the MNS system.

Immunohistochemical Validation of Rare Tissues and Antigens With Low Frequency of Occurrence: Recommendations From The Anatomic Pathology Patient Interest Association (APPIA).

Laboratories worldwide find it challenging to identify enough tissues and cases for verification and validation studies of low-incidence, rare antigens. These antigens have a low frequency of occurrence in the population, or have little or no expression in normal tissues. Validation studies are essential to assure testing standardization before introducing a new instrument, product, or test into the clinical laboratory. The College of American Pathologists has published comprehensive guidelines for the verification and validation of new immunohistochemical tests introduced into the laboratory menu. Within the guidelines, varied numbers of cases are required for nonpredictive versus predictive markers. However, regarding low-incidence antigens, the laboratory medical director determines the extent of validation required. Recommended practical solutions available to clinical laboratories for low-incidence validation include developing internal resources using the laboratory information system with retrospective and prospective search(s) of archival material and purchase of tissue microarray blocks, slides, or cell lines from external resources. Utilization of homemade multitissue blocks has proved to be extremely valuable in biomarker research and demonstrated great utility in clinical immunohistochemistry laboratories. Participation in External Quality Assessment program(s) may provide insufficient numbers or the ability to calculate concordance rates. However, supplementation with in-house tissues can allow a laboratory to reach the optimal number of cases needed for verification and/or validation schemes. An alternative approach is conducting a thorough literature search and correlating staining patterns of the new test to the expected results. These solutions may be used uniquely or together to assure consistent standardized testing.

Clinical significance of antibodies to antigens in the International Society of Blood Transfusion collections, 700 series of low-incidence antigens, and 901 series of high-incidence antigens

This article reviews information regarding the clinical significance of antibodies to antigens in the blood group collections, the 700 series of low-incidence antigens, and the 901 series of high-incidence antigens. Antibodies to many of the antigens in these groups are rarely encountered, meaning that available information is limited. For a few, the clinical significance-the potential to cause reduced survival of transfused antigen-positive red blood cells, a hemolytic transfusion reaction (e.g., anti-AnWj, anti-Emm), or hemolytic disease of the fetus and newborn (e.g., anti-Kg, anti-HJK)-has been documented. Many other specificities have so far been benign (e.g., anti-Csa, anti-M1).

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

BackgroundThe identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.MethodsWe generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.ResultsTransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.ConclusionsBoth stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

MNSs genotyping by MALDI‐TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles

Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs.

Frequency of Wr(a) antigen and anti-Wr(a) in Brazilian blood donors.

BackgroundWra is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wrb. Anti-Wra is a naturally occurring antibody that is found in approximately 1–2% of blood donors. The aim of this study was to determine the frequency of Wra and anti-Wra in Brazilian blood donors.MethodsA total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wra antigen. To detect the anti-Wra, samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wra.ResultsOf the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wra was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wra were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wra, four were IgG1, two were IgG3 and three anti-Wra were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wra might be of clinical significance.ConclusionThis study shows a very low frequency (0.06%) of the Wra antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wra in this population is higher than previously reported.

Acute extravascular hemolytic transfusion reaction due to anti-Kpa antibody missed by electronic crossmatch

BackgroundKpa antigen is a low incidence red blood cell antigen within the Kell system. Anti-Kpa alloantibody may be associated with acute and delayed hemolytic transfusion reactions. Case StudyWe report a case of a clinically significant acute extravascular hemolytic transfusion reaction mediated by previously unrecognized (and undetected) anti-Kpa alloantibody. This reaction occurred in a patient who met all criteria for electronic crossmatch, resulting in the transfusion of an incompatible red cell unit. ResultsPost-transfusion investigation showed the transfused red cell unit was crossmatch compatible at the immediate spin phase but was 3 + incompatible at the antiglobulin phase. No evidence of intravascular hemolysis was observed upon visual comparison of the pre- and post-transfusion peripheral blood plasma. Further testing showed the presence of anti-Kpa antibody. The clinical course of the patient included acute febrile and systemic reaction. ConclusionAcute extravascular hemolytic transfusion reaction may occur due to undetected anti-Kpa alloantibody. Various strategies for crossmatching are discussed in the context of antibodies to low incidence antigens.

Molecular Diagnostics in Transfusion Medicine: MALDI-TOF MS for Blood Bankers- The Alternative Approach for Genotyping

Serology, defined as antibody-based diagnostics, has been held as the analytical gold standard in transfusion medicine. However, at the present time, the influence of molecular diagnostics in transfusion medicine is rapidly growing. These methods can improve tissue typing (HLA typing), increase safety of blood products (NAT testing of infectious diseases), and enable blood group typing in difficult circumstances (prenatal non-invasive RhD typing and/or post-transfusion of blood products). Generally speaking, molecular testing involves determining the presence of single nucleotide polymorphisms (SNPs). Blood group antigens mostly result from single nucleotide differences in critical positions. However, most blood group systems cannot be determined by looking at a single SNP. To identify members of a blood group system, a number of critical SNPs have to be considered. Current platforms used to perform molecular diagnostics are predominantly gel-based, involving time-consuming multiple manual steps. To employ molecular methods in transfusion medicine in the future, the development of higher-throughput SNP genotyping, non-gel based platforms that allow rapid, costeffective screening are essential. Because of its potential for automation, high throughput and cost effectiveness, the special focus of this review is a relatively new technique: blood group genotyping by MALDI-TOF MS. Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has been previously reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This report is timely as promising preliminary results have recently been published from a large cooperative Swiss-German project that aimed to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. From this report, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method. Furthermore, Kell and RhD genotyping have been performed on foetal DNA from maternal plasma with excellent results. Additionally, current reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost efficiency when used in genetic analysis, clinical chemistry, microbiology, pharmaco-genetics, oncology and haematology. In summary, this paper examines a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS.

Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry–Based Blood Group Genotyping—The Alternative Approach

Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has previously been reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This review describes the development of a large cooperative Swiss-German project, aiming to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. Recent reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost-efficiency when used in genetic analysis, including forensics, pharmacogenetics, oncology and hematology. Furthermore, Kell and RhD genotyping have been performed on fetal DNA from maternal plasma with excellent results. In summary, this article introduces a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS. Although all data presented are preliminary, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method.

Hemolytic Disease of the Newborn Caused by Anti-Wright (Anti-Wra): Case Report and Review of Literature

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 Wra antigen are one such rare cause of HDN.

Occurrence of antibodies to low-incidence antigens among a cohort of multiply transfused patients with sickle cell disease

Abstract Data from an immunohematology reference laboratory were compiled retrospectively to determine the occurrence of the formation of alloantibodies to low-incidence antigens associated with the African American population (AA-LIAs) among patients with sickle cell disease (SCD). The AA-LIAs under study were V, VS, Jsa, and Goa. The records from 137 recurrently transfused patients with SCD were selected on the basis of transfusion activity from the 2009 calendar year. We found that 13 patients (9.4996) exhibited one or more antibodies to AA-LIAs, and a total of 17 antibodies to these AA-LIAs have been developed by these patients. The occurrence of antibody formation to AA-LIAs is significantly greater than that of alloantibodies to low-incidence antigens in the general population. Considering the possibility of the presence of antibodies to AA-LIAs in multiply transfused patients with SCD is warranted. Immunohematology 2011;27:143–145.

The ISBT 700 series of low-incidence and 901 series of high-incidence blood group antigens

Abstract The International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens developed a terminology that brought order to the chaos of antigen names. They classified antigens into three categories: systems, collections, and series. This review summarizes the early decisions of the Working Party with an emphasis on the 700 series of low-incidence antigens and 901 series of high-incidence antigens. Immunohematology 2011;27:131–135.

Antibodies to low-incidence antigens

Low-incidence antigens can be defined as those which are known to occur in less than 1% of individuals in most populations. The red cells of almost all blood recipients or prenatal patients therefore lack these rare antigens meaning a chance exposure to blood which does carry a lowincidence antigen almost always results in potential alloimmunization. However, since the antigens are so rare, the chance of exposure is very low. Incompatibility because of an antibody to a low-incidence antigen therefore does not happen very often, but when it does, can cause the same clinical problems, transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), as antibodies to more common specificities. Table 1 lists the low-incidence antigens currently recognized by the International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens. Most have been shown by biochemical and/or molecular methods to be rare variations in established blood group systems as shown. The antigens listed in the 700 series are recognized as distinct low-incidence antigens but there is no biochemical or genetic data that proves they belong to any of the blood group systems. Molecular studies have shown that most of the antigens are the result of a single nucleotide mutation in the gene which codes for a blood group protein, which in turn leads to a change of only one amino acid in the protein. In some cases the antigenic basis is more difficult to determine, particularly in the Rh, MNS and Gerbich blood group systems, where hybrid genes may be involved. For further information on the molecular basis of antigens the interested reader should consult a comprehensive textbook [1–3] Low-incidence antigens are categorized by their low frequency in most populations. Usually this means they are found to be rare or absent in all populations studied but in some cases the frequency can be much higher in some races or ethnic groups. One of the most striking examples of this is the Di antigen. It is rare on red blood cells from Caucasians and Africans but occurs in approxi-

The Redelberger antigen: a family study, a family story

Abstract The Redelberger antigen (Rba) was first discovered in 1974 on the RBCs of a blood donor who was an employee of the Community Blood Center in Dayton, Ohio. The discovery was made as a result of the investigation of a reagent contamination problem. Two examples of the Rba antigen were subsequently identified in the United Kingdom, but no “new”examples have been identified in the United States or Europe. Anti-Rba is a commonly occurring antibody, often found in combination with other antibody specificities, especially in combination with other antibodies to low-incidence antigens. Immunohematology 2006;22:48–51.

SERF: a new antigen in the Cromer blood group system.

The Cromer blood group system consists of eight high incidence and three low incidence antigens carried on decay-accelerating factor (DAF). This report describes the identification and characterization of a new Cromer high incidence antigen, named SERF. Sequence analyses of DNA from a Thai female whose serum contained the alloantibody to a high incidence antigen in the Cromer blood group system (anti-SERF) and from her two children were performed. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis on cDNA from the proband and PCR-restriction fragment length polymorphism analysis on DNA from Thais were also performed. To map the epitope, DAF deletion mutants were tested by immunoblotting with anti-SERF. Sequence analysis revealed a substitution of 647C>T in exon 5 DAF in the proband. The proband's two children and two of 100 Thais were heterozygotes 647C/T. Analysis using DAF deletion mutants revealed the antigenic determinant to be within short consensus repeat 3 (SCR3), which is encoded by exon 5. This study describes a novel high incidence antigen (SERF) in the Cromer blood group system characterized by the amino acid proline at position 182 in SCR3 of DAF. The SERF-negative proband has a substitution mutation that predicts for leucine at this position. SERF has been provisionally assigned the International Society of Blood Transfusion number 021.012 (CROM 12).