Ca s e R e p o r t B subgroup detection in a small hospital transfusion service

E. Elardo, N. Elbadri, C. Sanchez, V. Powell, M. Smaris, Y. Li, J. Jacobson, T. Hilbert, T. Hamilton, and D.W. Wu

The ABO blood group system includes phenotypes, or subgroups, of Dolichos biflorus seeds) to agglutinate A1 RBCs but not that differ in the amount of A and B antigens present on the red 2 RBCs of other A subgroups. There are no comparable B1 and blood cells (RBCs). These subgroups also differ in the A, B, or B subgroups, but examples of group B RBCs that react weakly H substances present in secretions (for individuals who have 2 3 the secretor phenotype). B subgroups are very rare and are less or not at all with anti-B have been described. B subgroups frequently reported than A subgroups. Usually, B subgroups are are very rare and are less frequently reported.3 Usually, B discovered during serologic testing when there is a discrepancy subgroups are discovered during serologic testing when there between RBC and serum grouping results. Subgroups of B are usually identified by a reference laboratory using molecular and is a discrepant result between RBC and serum grouping tests. adsorption-elution methods. This report details a case of a young, Subgroups of B are difficult to classify and include B3, Bx/Bweak healthy, pregnant woman with a B subgroup detected by a small (previously known as Bx), Bweak (previously known as Bm), transfusion service using adsorption-elution methods. Serology and B phenotypes, according to the International Society of and genotyping of the ABO gene was performed at a reference el 4,5 laboratory where the serology was consistent with a B subgroup, (ISBT). The B subgroups can be classified but no changes were identified in ABO gene sequencing. It is by their agglutination strength with anti-B, anti-A,B, and important to correctly identify B subgroups in donors and anti-H; the presence or absence of anti-B in the serum; the recipients to help resolve ABO discrepancies and potentially pre- presence of B substance in saliva; the results of adsorption- vent ABO incompatibility in blood transfusion, thus minimizing 2,6 transfusion reactions. Immunohematology 2021;37:89–94. elution testing; and molecular studies (Table 1). An up-to- DOI:10.21307/immunohematology-2021-0014. date listing of molecular variants recognized by ISBT that give rise to the observed serologic phenotypes can be found Key Words: blood group discrepancy, ABO blood group, B on their Web site in the table for ABO (ISBT 001) blood group subgroup, transfusion reaction, ABO mismatch v1.2.4 Definitive identification of ABO subgroups is usually performed at a reference laboratory using molecular The ABO blood group system is the most clinically and adsorption-elution methods, which often have long significant antigen system in . The turnaround times. ABO gene is located on chromosome 9 and consists of seven exons. Exons 6 and 7 encode for the catalytic domain of the Case Report ABO glycosyltransferases. The A blood group is formed by the addition of N-acetyl-galactosamine to the H antigen, the Here we report a case in which a B subgroup was B blood group is formed by the addition of galactose to the H detected by an observant technologist using both automated antigen, and blood group O results when no sugar is added gel and manual tube methods with conventional reagents to the H antigen.1 ABO blood groups have subgroups that are at a busy hospital transfusion service. The facility is a 450- often distinguished by decreased amounts of A or B antigens bed, small, community hospital and level 1 trauma center. on red blood cells (RBCs). A and B subgroup phenotypes The hospital transfuses an average of 5500–6000 total units can be identified based on the quantity of A or B antigens of blood components annually. The lab received an EDTA- carried on RBCs and A or B substances present in secretions anticoagulated blood specimen for a type and screen order (for individuals who have the secretor phenotype). The most from a young healthy pregnant woman. Discrepant results common subgroups are A1 and A2 (in Europeans, 80% of all (forward as group O and reverse as group B) were observed 2 group A and AB individuals are A1 and 20% are A2). Blood while performing routine blood ABO testing by the gel method group A appears to have more subgroup variations than group (see Methods and Results). Repeat testing by a conventional

B. In general, the serologic distinction between A1 and other tube method showed the same discrepancy. This 29-year- subgroups of A is based on the ability of anti-A1 lectin (an extract old African American woman presented to an obstetric clinic

IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 89 E. Elardo et al.

Table 1. Characteristics of some more frequent B phenotypes

Reagents Antibodies in serum Other Substances present in Presence of a B Phenotypes Anti-A Anti-B Anti-A,B Anti-H Common Unexpected saliva of secretors in serum B 0 4+ 4+ 2+ Anti-A None B, H Yes

B3 0 ++mf ++mf 3+ Anti-A None B, H Yes (wk pos)

Bx 0 wk wk 3+ Anti-A Weak anti-B H No

Bm* 0 0/wk 0/wk 3+ Anti-A None B, H Yes (wk pos)

Bel* 0 0 0 3+ Anti-A Sometimes a H No weak anti-B 2From Harmening.2 *B specificity demonstrated only by adsorption-elution methods. 0 = negative; mf = mixed field; wk = weak; pos = positive. for routine prenatal care at 28 weeks, 4 days, gestational blood grouping on an automated gel-method analyzer (ORTHO period for Rh(D) determination for possible Rh immune VISION; Ortho Clinical Diagnostics, Raritan, NJ); antibody globulin (RhIG) administration. At the time of this visit, the detection test results were negative. An attempt to resolve the patient had no previous history of transfusion, pregnancies, discrepancy observed on the automated gel analyzer led the or transplantation, with current diagnosis of nonimmune, technologist to repeat testing using the conventional manual mild anemia, and a history of varicella. Her hemoglobin and tube method, which showed the same discrepancy. hematocrit were 10.3 g/dL (normal range 11.9–16.0 g/dL) and The manual tube method demonstrated no agglutination 29.7 percent (normal range 35–47%), respectively. No blood with the patient’s RBCs and blood grouping reagents (Ortho transfusion was needed at this time. Her RBCs were typed Clinical Diagnostics), including anti-A, anti-B, and anti-A,B as D–, and she received RhIG during this visit. The patient (BioClone [Murine Monoclonal Blend], San Diego, CA) at had previously tested as group O, D– with a negative antibody immediate spin, or after 30 minutes at room temperature, but detection test at two other hospitals for the same pregnancy, weak reactivity was noted with anti-B and anti-A,B at 4°C. The including her most recent visit 1 month prior. About 3 months patient’s serum showed the presence of a strong anti-A, but later, the patient had a spontaneous vaginal delivery with no no anti-B was detected using pooled RBCs (AFFIRMAGEN; transfusion demands. Postpartum RhIG was administered to Ortho Clinical Diagnostics) (Table 2). her in a timely manner. Although decreased expression of A and B antigens has been observed in patients with hematologic malignancies, our Methods and Results patient was a healthy young woman, suggesting the possibility that the discrepant ABO typing results were due to a weak B Serologic-based ABO phenotypes were determined by subgroup. To explore the possibility of a B subgroup, direct agglutination and adsorption-elution testing according to antiglobulin tests (DATs) were first performed by conventional standard methods and procedures described in the AABB tube method with anti-IgG (Rabbit; Ortho Clinical Diagnostics) Technical Manual.1 Discrepant results (forward as group O and and anti-C3b/-C3d (Murine Monoclonal; BioClone, Ortho reverse as group B) were observed while performing routine Clinical Diagnostics) reagents; all DAT results were negative.

Table 2. Results of forward and reverse grouping and antibody detection tests

RBCs with reagent Serum with reagent Antibody detection test

Anti-A Anti-B Anti-A,B A1 RBCs B RBCs Autocontrol (gel method) IS RT 0 0 0 4+ 0 0 30 minutes RT 0 0 0 4+ 0 0 Negative 4°C 0 W+ W+ 4+ 0 0 RBCs = red blood cells; IS = immediate spin; RT = room temperature; W+ = weak reaction.

90 IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 B subgroup detection in a hospital transfusion service

After ensuring that the patient’s RBCs were not coated with Discussion IgG and/or C3b/C3d by the DAT, adsorption was performed using washed patient RBCs and reagent anti-B (BioClone, This case showed that conventional serologic testing can be Murine Monoclonal Blend; Ortho Clinical Diagnostics).1 After used to detect a weak B subgroup. The detection of weak ABO 1-hour incubation at 4°C, an acid elution was performed. subgroups is very important in clinical settings, as well as in The eluate and final wash supernatant were tested in parallel blood donor centers, because a person with weak B expression against A1 and B reagent RBCs (AFFIRMAGEN), and three may be wrongly typed as group O when anti-B is present in the group O reagent antibody screening RBCs (SURGISCREEN; patient/donor plasma. Rare hemolytic transfusion reactions Ortho Clinical Diagnostics). The eluate reacted only with group (HTRs) due to ABO incompatibility involving ABO subgroups B RBCs when incubated at 37°C for 15 minutes and tested have been reported. As Chun et al.7 reported, a patient with 1 using the indirect antiglobulin test (Table 3). Because the cis-A1B3 interpreted as typical group A experienced adverse patient’s saliva was not available for secretor testing, whether effects after transfusion of 4 units of group A RBCs and 4 the patient has a Bweak or Bel phenotype remains unknown. units of group A (FFP). Although reaction between the B antigen of the cis-AB patient and anti-B from Table 3. Adsorption-elution results the group A FFP is theoretically possible, the authors could 7 O cells* not draw a definitive conclusion for the cause of hemolysis. To our knowledge, an HTR involving B subgroup has not been Reagent RBCs A1 RBCs B RBCs Cell I Cell II Cell III reported. Eluate 0 2+ 0 0 0 Theoretically, an HTR (perhaps with mild clinical Last wash 0 0 0 0 0 effects or shortened RBC survival due to lower-level antigen *Reagent antibody screening red blood cells (RBCs). expression on the patient’s RBCs) may happen in transfusions involving ABO subgroups, such as when a B subgroup recipient Since the absorption-elution results were consistent with a mistyped as group O is transfused with a group O plasma– weak B subgroup, the patient’s specimen was sent to a reference containing blood component, or a RBC unit from a donor laboratory for further evaluation. The reference laboratory’s with weak B subgroup mislabeled as group O is transfused to serologic testing confirmed the discrepant forward and reverse a group O recipient. Chaurasia et al.8 emphasized that when results. Initial molecular testing revealed no changes in ABO individuals with B subgroups present as transfusion recipients, exons 6 and 7 known to explain the discrepancy. Full sequenc- they should be transfused with group O RBC components ing of the ABO gene exons 1–7 of the A and B transferase genes and group B–specific plasma and components. These was performed using Sanger DNA sequencing technology. authors report that RBCs from B subgroup donors transfused The polymerase chain reaction (PCR) restriction fragment– to group O recipients can result in an adverse HTR.8 length polymorphism (RFLP) was performed for nucleotide In the United States, as required for pre-transfusion testing positions c.261(O1), c.467(A2), c.703(B), and c.1065+31 (B and by the AABB Standards for Blood Banks and Transfusion O2). The PCR-RFLP testing indicated the presence of c.703A Services, the ABO group shall be determined by testing RBCs and c.1065+31A, characteristic of B alleles, and c.261delG, with anti-A and anti-B reagents and by testing the serum or characteristic of O 1 alleles. The genomic sequencing of plasma for expected antibodies with A and B reagent RBCs. If exons 1–7 showed the presence of an O 1 , specifically a discrepancy is detected and transfusion is necessary before ABO*O.01.09 (c.261delG, c.318T, and c.467T), on exon 6 and resolution, only group O RBCs shall be issued.1 confirmed the presence of a B allele, specificallyABO*B.01 This type of serologic discrepancy, with decreased (c.297G, c.526G, c.657T, c.703A, c.796A, c.803C, and c.930A), expression of A or B antigens on RBCs, can be observed in on exons 6 and 7. The patient was heterozygous for each of acquired ABO subgroup phenotypes such as seen in infants, the variants found. No other changes were found in exons individuals with hematologic disorders, and pregnant 1–5. The reference laboratory concluded that the patient was women;9 however, our patient was healthy, and, although ABO*B.01/ABO*O.01.09 with a predicted phenotype of a weak pregnant, had typed as group O in two hospitals she visited B subgroup. Additional investigation was recommended. previously. These reported results indicate that anti-B was presumably detected in her plasma, although this information was not confirmed with the testing facilities. The technologist

IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 91 E. Elardo et al.

at our transfusion service was able to “dig deep” into this a hybrid between an O 2 and a B allele and characterized the case and used serologic investigation to discover a weak B associated decrease in B antigen expression. Sano et al.17 subgroup. The reference laboratory’s serology tests confirmed identified a positive regulatory element in intron 1. This this finding. The detection of a B subgroup is important to element was shown to enhance ABO promoter activity in an prevent potential ABO-mismatched transfusion reactions. erythroid cell–specific manner, and a partial deletion in intron Weak B subgroups are rarely detected in a hospital transfusion 1 involving the erythroid cell–specific regulatory element service setting. The ability to confirm a weak ABO subgroup was associated with Bm phenotypes. The authors suggested phenotype generally requires molecular testing. DNA-based that deletion of this enhancer-like element in intron 1 of ABO tests are increasingly being used at immunohematology regulates transcription in the Bm allele, leading to reduction reference laboratories to predict blood group phenotype, which of B antigen expression on cells of erythroid lineage, but not helps to improve transfusion medicine practices.10,11 Reference in mucus-secreting cells.17 In the study of Ying et al.,14 75 laboratories use several methods for genotyping, including samples of ABO variants were considered to have an unclear PCR followed by RFLP analysis, sequence-specific primer molecular basis, because only normal sequences were detected PCR, DNA sequencing, and array-based approaches.11 A in their ABO coding regions. Among these samples, 32 were notable advantage of molecular testing is its ability to identify from individuals with infection, hematologic disorders, and variant alleles associated with antigens that are expressed other diseases. For the remaining 43 individuals with ABO weakly or have missing or altered epitopes, thus helping to subgroups, seven were associated with the erythroid cell– resolve discrepant or incomplete blood group phenotyping.12 specific regulatory element in the first intron of theABO gene14 The disadvantages of molecular testing are mainly the longer based on the analysis of the nucleotide sequences of the partial turnaround time (days to weeks11) and higher cost, compared intron 1 covering this +5.8-kb region. The other 36 samples with serologic typing. in this group obtained from apparently healthy individuals In our patient’s case, full ABO gene sequencing did not (including four from pregnant women) might have an unclear identify any changes in the B allele that would explain the molecular basis.14 undetectable B antigen expression, raising the possibility that For the B subgroup patient in this case, our reference the patient may have a gene regulator or a promoter mutation laboratory suggested that further investigation would be in the non-coding regions of the B allele. Given that most weak conducted. Most hospital transfusion service laboratories do B phenotypes are due to random missense mutations in the last not perform molecular-based testing. They are usually capable 13 ABO exon, the failure to identify a mutation makes this case of serologic testing to identify A1 and A2, the most common somewhat unusual. Nevertheless, uncommon novel mutations A subgroups, but can recognize the possibility of weaker are increasingly identified given the widespread availability of subgroups of A or B by characteristic discrepancies between molecular testing, and mutations outside of the coding region RBC and plasma grouping tests.3 Hospital transfusion service are not uncommon. laboratories rarely conduct a serologic investigation to detect Huang et al.13 reported the analysis of 1.4 million Chinese weak B subgroup. This case, however, demonstrated that blood donors; 351 cases of weak ABO subgroups were conventional serologic testing can be used to detect a weak B identified serologically. In the 326 samples subject to DNA subgroup. The approach for detection of a weak B subgroup analysis, 44 ABO mutations were identified, of which only in the hospital laboratory setting is particularly useful when 10 were novel. No mutations were observed in 99 samples. A access to molecular-based reference laboratory testing is similar recent analysis of 211 ABO subgroups in Chinese blood limited or if the patient needs urgent blood transfusion. donors detected by discrepant serologic typing revealed that 36 The much lower cost and faster turnaround time of the samples (approximately 17%) including four pregnant donors conventional serology tests, in comparison to molecular-based had discrepant results that were not explained by underlying genotyping tests, offer advantages. Serologic testing has been disease or initial sequencing and may have an unexplained used elsewhere to identify and characterize ABO subgroups in molecular basis.14 Mutations leading to weak subgroups may a large group of blood donors and patients18 and B subgroups also occur in areas outside the coding region of the ABO gene. in a group of three patients and donors.19 Seltsam et al.15 reported that weak B phenotypes can be caused B subgroup should be suspected when serologic testing by sequence variation in the CCATT-binding factor (CBF)/ results show a discrepancy of forward as group O and reverse NF-Y region of the ABO gene, and Thuresson et al.16 described as group B, with a negative antibody detection test at immediate

92 IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 B subgroup detection in a hospital transfusion service

spin. B subgroup phenotype can then be determined by using References adsorption-elution testing, secretor testing for B substance, 1. Cohn CS, Delaney M, Johnson ST, et al. Technical manual. 6 and serologic classification discussed in this report. Guidelines 20th ed. Bethesda, MD: AABB, 2020. exist and are summarized in Table 1.2 2. Harmening DM. The ABO blood group system. In: Modern blood banking & transfusion practices. 7th ed. Philadelphia, Individuals with some B subgroups, such as Bx/Bweak and PA: FA Davis Company Publications, 2018. B , can produce anti-B. Transfusion of group O RBCs and el 3. Klein HG, Anstee DJ. Mollison’s blood transfusion in clinical group B plasma–containing components to these individuals medicine. 17th ed. Boston, MA: Blackwell, 2014. can address this type of B subgroup incompatibility due to 4. International Society of Blood Transfusion. Names for ABO anti-B to prevent an HTR. In our opinion, it is not clinically (ISBT 001) blood group alleles. Available from http://www. isbtweb.org/fileadmin/user_upload/Working_parties/WP_ necessary to further differentiate between B or B subgroups weak el on_Red_Cell_Immunogenetics_and/001_ABO_Alleles_ in these recipients. For this young healthy woman in our case v1.2.pdf. Accessed 4 January 2021. with B subgroup but without identified variant alleles, further 5. Reid ME, Lomas-Francis C, Olsson ML. The blood group research could be conducted on the non-coding regulatory antigen factsbook. 3rd ed. London: Academic Press, 2012. elements, such as PCR amplification and sequencing for the 6. Daniels G. Human blood groups. 2nd ed. Oxford, U.K.: Blackwell Science, 2002. 5.8-kb enhancer region in the intron 1 enhancer region, and 7. Chun S, Choi S, Yu HB, Cho D. Cis-AB, the blood group of on the sequence variation in the (CBF)/NF-Y enhancer region many faces, is a conundrum to the novice eye. Ann Lab Med of the ABO gene. However, it is possible that the patient’s 2019;39:115–20. pregnancy caused the B subgroup phenotype, with other 8. Chaurasia R, Rout D, Dogra K, et al. Discrepancy in blood grouping: subgroups of B: challenges and dilemma. Indian J unknown or no molecular subgroup basis. Identification Hematol Blood Transfus 2017;33:628–9. of the patient’s B subgroup may help to prevent potential 9. Bianco-Miotto T, Hussey DJ, Day TK, O'Keefe DS, Dobrovic ABO incompatibility, if the patient needs blood transfusion. A. DNA methylation of the ABO promoter underlies loss of Nevertheless, further investigation to determine the molecular ABO allelic expression in a significant proportion of leukemic patients. PLoS One 2009;4:e4788. basis of the patient’s B subgroup phenotype at this small 10. Reid ME. Transfusion in the age of molecular diagnostics. community hospital transfusion service is neither feasible nor Hematology Am Soc Hematol Educ Program 2009;1:171–7. necessary. 11. Sandler SG, Horn T, Keller J, Langeberg A, Keller MA. A model for integrating molecular-based testing in transfusion services. Blood Transfus 2016;14:566–72. Conclusion 12. Hossini-Maaf B, Letts J, Persson M, et al. Structural basis for red cell phenotypic changes in newly identified, naturally The hospital transfusion service identified, in a timely occurring subgroup mutants of the human blood group B manner, a B subgroup using conventional serologic methods; glycosyltransferase. Transfusion 2007;47:864–75. 13. Huang H, Jin S, Liu X, et al. Molecular genetic analysis of weak these methods had the potential of preventing an ABO- ABO subgroups in the Chinese population reveals ten novel mismatch transfusion reaction. This approach is also ABO subgroup alleles. Blood Transfus 2019;17:217–22. particularly useful when molecular testing by reference 14. Ying Y, Hong X, Xu X, et al. Molecular basis of ABO variants laboratory resources is limited or if the patient’s urgent need including identification of 16 novel ABO subgroup alleles in Chinese Han population. Transfus Med Hemother of blood transfusion cannot wait for the reference laboratory’s 2020;47:160–6. results. Nevertheless, the serologic testing approach to detect 15. Seltsam A, Wagner FF, Gruger D, et al. Weak blood group B the weak B subgroup often requires a confirmation by genomic phenotypes may be caused by variations in the CCAAT-binding analysis because this information is crucial to donor centers factor NF-Y enhancer region of the ABO gene. Transfusion 2007;47:2330–5. and transfusion services. 16. Thuresson B, Hosseini-Maaf B, Hult AK, et al. A novel B (weak) hybrid allele lacks three enhancer repeats but generates normal Acknowledgments ABO transcript levels. Vox Sang 2012;102:55–64. 17. Sano R, Nakajima T, Takahashi K, et al. Expression of ABO blood-group genes is dependent upon an erythroid cell-specific The authors would like to thank the staff at regulatory element that is deleted in persons with the B(m) the New York University (NYU) Langone Hospital–Brooklyn phenotype. Blood 2012;119:5301–10. and the New York Blood Center Reference Laboratory for their contributions to this case.

IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021 93 E. Elardo et al.

18. Nair R, Gogri H, Kulkarni S, Gupta D. Detection of a rare and Laboratories, NYU Langone Hospital–Tisch, New York, NY; subgroup of A phenotype while resolving ABO discrepancy. Milena Smaris, MLS, Blood Bank Assistant Supervisor, Department Asian J Transfus Sci 2019;13:129–31. of Pathology and Laboratories, NYU Langone Hospital–Brooklyn, 19. Thakral B, Saluja K, Bajpai M, et al. Importance of weak ABO Brooklyn, NY; Yanhua Li, MD, MS, FCAP, Associate Professor, subgroups. Lab Med 2005;361:32–4. Medical Director, Clinical Laboratories, Perlmutter Cancer Center, Lake Success, Huntington, Rego Park, and NYU Langone Health, Edward Elardo, MLS (corresponding author), Blood Bank New York, NY; Jessica Jacobson, MD, Associate Professor, Medical Technologist, Department of Pathology and Laboratories, NYU Director, Department of Pathology and Laboratories, Bellevue Langone Hospital–Brooklyn, 150 55th Street, Brooklyn, NY 11220, Hospital, New York, NY; Timothy Hilbert, MD, PhD, JD, Assistant [email protected]; Naima Elbadri, MS, MLS, Professor, Medical Director, Transfusion Services, Department of SBB(ASCP), Blood Bank Assistant Supervisor, Department of Pathology and Laboratories, NYU Langone Hospital–Tisch, New Pathology and Laboratories, NYU Langone Hospital–Brooklyn, York, NY; Tanya Hamilton, SBB (ASCP), Administrative Director, Brooklyn, NY; Carlos Sanchez, MLS, Blood Bank Supervisor, Transfusion Services, Department of Pathology and Laboratories, Department of Pathology and Laboratories, NYU Langone NYU Langone Hospital–Tisch, New York, NY; and Ding Wen Wu, Hospital–Brooklyn, Brooklyn, NY; Vivien Powell, MLS, Blood MD, PhD, Professor, Medical Director, Department of Pathology and Bank Manager, Transfusion Services, Department of Pathology Laboratories, NYU Langone Hospital–Brooklyn, Brooklyn, NY.

For information concerning the National Reference Immunohematology has a new Web site!

Laboratory for Blood Group Serology, including the American https://www.exeley.com/journal/immunohematology Rare Donor Program, contact Sandra Nance, by phone at Visit the new Web site to view abstracts of all published articles, read a selected article in Open Access format, (215) 451-4362, by fax at (215) 451-2538, or by e-mail at review announcements of upcoming events, and view [email protected]. ongoing laboratory advertisements.

Free Classified Ads and Announcements Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.)without charge.

E-mail information to [email protected].

94 IMMUNOHEMATOLOGY, Volume 37, Number 2, 2021