While continuous performance is preferred for any assay, trans-fusion services are particularly susceptible to interruptions that may interfere with testing. If these interruptions occur after the red blood cell (RBC) suspension is made or after the last wash is decanted, direct antiglobulin test (DAT) results may be affected. This study aimed to assess if delays at these two procedural steps compromise DAT results. Samples with positive DATs (up to 2+) using anti-IgG (IgG-DAT) were included. RBC suspensions (2-5%) were prepared from unwashed RBCs or RBCs washed once. These suspensions were used to perform tube IgGDATs without delays to obtain baseline results. IgG-DATs were repeated with delays after preparation of the RBC suspension (1, 5, 15, 30, and 60 minutes) or after the last wash decant (1, 5, 15, or 30 minutes). Three anti-IgG reagents were used: Anti-IgG (Murine Monoclonal) Gamma-clone; Werfen, Barcelona, Spain; Anti-IgG (Rabbit); QuidelOrtho, Raritan, NJ; and AHG Anti-IgG; Bio-Rad, Hercules, CA. Samples were considered impacted if their IgG-DAT strength differed by two or more reaction grades from baseline. The analysis included 25 samples. Samples with baseline IgG-DAT results of 1+s and 2+ were determined to not be impacted. Three samples were weakened by delays after RBC suspension preparation. One sample was weakened when the addition of anti-IgG was delayed. While most samples with weakly positive baseline IgG-DAT results were not impacted, the samples with the weakest baseline IgG-DAT results were impacted more frequently than those with stronger baseline IgG-DAT results. Because it is impossible to predict which samples will not be impacted by delays in these testing steps, DAT procedures should be performed with minimal interruption.
The Rhnull phenotype is the rarest of the Rh blood group system phenotypes and is characterized by the complete absence of all Rh antigens on red blood cells (RBCs). This phenotype carries major clinical relevance because of chronic hemolysis and profound transfusion incompatibility. We performed a scoping review to summarize the molecular basis, serologic features, clinical manifestations, and transfusion challenges associated with published Rhnull cases. A systematic literature search of PubMed/MEDLINE and Scopus was conducted from database inception to May 2025. Eligible reports included case reports, case series, or observational publications of individuals with the Rhnull phenotype. Data were extracted and synthesized narratively according to a scoping review method. Six eligible publications describing eight individuals with the Rhnull phenotype were identified. Common findings included chronic hemolytic anemia, stomatocy tosis or spherocytosis, and reticulocy tosis. Despite generally moderate intrinsic hematologic manifestations, the Rhnull phenotype imposes a disproportionate transfusion burden because of near-complete donor scarcity. Anti-Rh29 alloimmunization was reported after transfusion or pregnancy exposure, restricting patients to Rhnull RBC units from family members, frozen RBC inventories, or rare donor registries. Early recognition, molecular characterization, and proactive integration of eligible individuals with the Rhnull phenotype into rare donor programs are critical for access to compatible blood.
While the previous review encompassed the Rh blood group system (Chou ST, Westhoff CM. The Rh and RhAG blood group systems. Immunohematology. 2010;26:178-86), this update focusses on the RHCE gene and its variants. Four new antigens- PARG, CEVF, CEWA, and CETW (RH60 to RH63)-were reported since the last update. RHCE*cEMI (RHCE*03.31) was amended from a null allele to an allele encoding very weak antigen expression. The following topics are discussed: cross-reactive alleles [such as RHCE*ceHAR (*01.22.01) and RHCE*ceCF (*01.20.06) which may type D+ with some monoclonal anti-D reagents], issues with hybrid alleles and allele dropout, common haplotypes (association between RHCE alleles and specific RHD alleles), and clinical considerations. While the detailed description of new Rh antigens has become rare, many RHCE alleles have been reported since the previous review, a result of increased adoption of DNAbased testing for red blood cell antigens in immunohematology laboratories. The Rh blood group system has fascinated generations of immunohematologists and is likely to continue to do so for decades to come.
A 52-year-old, first-time blood donor presented with panreactivity during routine antibody screening. Current serologic evaluation showed uniform 2+ reactivity by the gel-indirect antiglobulin test and the conventional tube low-ionic-strength saline-indirect antiglobulin test. The molecular analyses revealed a homozygous 17-base pair deletion spanning nucleotides 64-80 in exon 3 of SMIM1 (VEL*01N.01/VEL*01N.01), confirming the Vel-null phenotype. The antibody was identified as anti-Vel IgG3, reacting in tube testing, exclusively in the anti-human globulin phase. We present the first case of a Vel-null alloimmunized donor identified in Uruguay and describe the serologic, molecular, and functional characteristics of the associated antibody.
The Japanese Red Cross Blood Services archives donor blood samples from all donations for look-back surveys. These samples, approximately 5 million per year, are stored frozen at -30°C for 11 years and are subsequently discarded. Because such samples may also be valuable for antibody-related research and other purposes after the mandated storage period, we evaluated their potential applicability, with a particular focus on the preservation of anti- body levels. Fresh samples were compared with frozen-stored samples (-30°C for 14 years: 11 years of mandatory good manufacturing practice storage period + 3 years until measurement) for immunoglobulin levels (IgM, IgG, IgA, and IgE) as a general indicator of humoral immunity, tetanus toxin antibody titers as a long-lived vaccine-induced antibody, and ABO antibody titers (saline-direct agglutination test and saline-indirect antiglobulin test [saline-IAT]), which are factors in blood group typing and hemolytic transfusion reactions. For immunoglobulin levels, comparisons of non-paired fresh and frozen-stored samples were made from a random donor population (IgM, IgG, IgA: 105 cases each, IgE: 32 cases). For tetanus toxin antibody and ABO antibody, comparisons were conducted from the same donors with fresh and frozen-stored samples (tetanus toxin antibody: 47 cases, ABO antibody: 12 cases). No significant differences were observed in immunoglobulin levels, tetanus toxin antibody titers, or ABO antibody titers measured by the saline-direct agglutination method. Saline-IAT-measured ABO antibody titers were significantly lower in frozen-stored samples. Detailed results were as follows: (1) Median immunoglobulin levels (fresh vs. frozen-stored): IgM, 78.5 mg/dL versus 86.7 mg/dL; IgG, 1240.2 mg/dL versus 1280.2 mg/dL; IgA, 241.0 mg/dL versus 245.3 mg/dL; and IgE, 0.24 μg/mL versus 0.12 μg/mL. (2) Tetanus toxin antibody titers-geometric mean titer (GMT) (95% confidence interval [CI]): fresh 0.23 (0.12-0.43) IU/mL versus frozen-stored 0.28 (0.17-0.46) IU/mL. (3) ABO antibody titers-GMT (95% CI): saline-direct agglutination, fresh 65.4 (33.3-128.5) versus frozen-stored 51.7 (24.9-107.4); saline-IAT, fresh 391.0 (160.3-953.6) versus frozen-stored 248.7 (107.8-573.6). The evaluated immunoglobulin levels were largely preserved, suggesting that archived blood samples retain sufficient stability for antibody research. However, careful study design is necessary to minimize potential sample deterioration.
The JK blood group system encodes the antithetical antigens Jka and Jkb as well as Jk3.1 SLC14A1 on chromosome 18 spans approximately 28 kb and has 10 exons. The diallelic single nucleotide variant (SNV) c.838G>A p.(Asp280Asn) gives rise to the antithetical antigens Jka or Jkb, respectively. At the time of this publication and including this novel allele, there are 12 JK*01 alleles encoding Jk(a+w) and 29 JK*01 null alleles, and 6 JK*02 alleles encoding Jk(b+w) and 30 JK*02 null alleles.2 We describe two African American donors whose red blood cells (RBCs) tested Jk(a-b+) with polyclonal and monoclonal antisera. The RBC genotyping panel HemoID DQS (Agena Bioscience, San Diego, CA) predicted the sample to type Jk(a+b+). This discrepancy was investigated using Sanger sequencing of the coding exons and exon/intron junctions of SLC14A1.
Antibodies to antigens of the GE (Gerbich) blood group system are not encountered in transfusion center blood banks on a daily basis but are regular guests at reference laboratories. In this review, we present an update on the information on GE antigens and alleles that have been published since the release of the previous system review in 2010. Additionally, information on the clinical relevance of GE antibodies for both transfusion and hemolytic disease of the fetus and newborn and recent discoveries related to functional aspects of the glycophorin C and glycophorin D proteins have been included.
This article addresses the diagnostic challenges encountered and resolution of a neonate with blocked D phenomenon, where maternal anti-D had masked the D sites on the neonate's red blood cells (RBCs), initially resulting in false-negative D typing. This article emphasizes the importance of integrating serologic and molecular methods for accurate blood type determination, which is crucial for managing hemolytic disease of the fetus and newborn. A mother, whose RBCs typed as D- and with a history of D isoimmunization, gave birth to a neonate who presented with anemia and jaundice. Initial D typing revealed the neonate had D- RBCs. The mother's blood sample tested as group B, D- with a high anti-D titer (512). The neonate's direct antiglobulin test (DAT) was strongly positive for IgG, and eluate testing confirmed anti-D specificity, indicating that maternal anti-D was coating the neonate's RBCs. Treatment of the neonate's RBCs with a commercial ZZAP reagent removed bound IgG antibodies, converting the DAT to negative and revealing the neonate's RBCs to be group B, D+, thereby resolving the initial serologic dilemma. Further polymerase chain reaction-based molecular testing of the neonate's RBCs confirmed the D+ phenotype (R1R1), demonstrating the utility of molecular methods in complex cases.
This update on the RHAG blood group system (ISBT 030) (Chou ST, Westhoff CM. The Rh and RhAG blood group systems. Immunohematology 2010;26:178–86) reports the addition of three new low-prevalence antigens carried on the Rh-associated glycoprotein (RhAG). Kg (previously 700045; now RHAG5) has been demonstrated to be antithetical to the previously described high-prevalence DSLK (RHAG3). Two further low-prevalence antigens (RHAG6 and RHAG7) are described, both resulting from rare missense RHAG mutations encoding amino acid changes predicted to be externally located. All three new low-prevalence antigens have been implicated in hemolytic disease of the fetus and newborn. The RHAG system now comprises six antigens, two of high prevalence and four of low prevalence, including one antithetical pair. RHAG4 has been made obsolete.
Drug-induced immune hemolytic anemia (DIIHA) represents a rare but dangerous medical condition that more often affects children who are administered cephalosporins. A 10-year-old girl developed severe hemolysis after administration of cefuroxime and ceftriaxone for treating an infection linked to parotid lymphangioma. The patient had previously tolerated ceftriaxone and clindamycin but developed dizziness, pallor, and tachycardia, and her hemoglobin (Hb) levels decreased from 12.9 to 2.6 g/dL during her fourth day of treatment, which led to hypovolemic shock and transient renal dysfunction. Our objective is to establish cefuroxime and ceftriaxone as the responsible drugs for DIIHA through immunohematologic testing. The laboratory tests included the direct antiglobulin test (DAT) and the indirect antiglobulin test (IAT) along with drug-dependent antibody testing using the patient's serum (1) to react against group O red blood cells (RBCs) treated with cefuroxime and (2) to react separately against group O RBCs not treated with, but in the presence of, ceftriaxone, with and without complement, at both room temperature and by the IAT. The DAT showed positivity for IgG and C3d. The patient's serum reacted with cefuroxime-treated RBCs by the IAT and reacted with untreated RBCs when ceftriaxone and complement were present. The laboratory results showed that drug-dependent antibodies were present to target RBCs. The patient's condition improved rapidly after stopping cephalosporins and starting ciprofloxacin/levofloxacin and clindamycin, which resulted in an Hb increase to 9.1 g/dL within 48 hours and the absence of hemoglobinuria. This case shows why health care professionals must identify DIIHA early through diagnostic testing even when patients have shown previous tolerance to antibiotics. This case report also shows that both cefuroxime and ceftriaxone were the drugs responsible for the DIIHA.
This update of the Indian (IN 023) blood group system (Xu Q. The Indian blood group system. Immunohematology 2011; 27:89-93) focuses on the discovery and clinical significance of new antigens, antibodies, and genetics since that review. The system now comprises six antigens, of which the more recently identified antigens are high-prevalence antigens (HPAs) IN:005 (INRA) and IN:006 (INSL); rare individuals lacking these antigens are found among the people from the Indian subcontinent. The Indian (IN) antigens are located on CD44, a single-pass transmembrane glycoprotein encoded by the CD44 gene on chromosome 11 at position p13. The absence of these HPAs is associated with homozygous missense mutations in CD44: 255C>G in exon 3, c.449G>A in exon 5 (for IN:-5, p.Arg150His), and c.276C>A in exon 3 (for IN:-6, p.His92GIn).
SC2 is a low-prevalence antigen of the Scianna blood group system, historically associated with hemolytic disease of the fetus and newborn and only one prior case of hemolytic transfusion reaction (HTR). We report a second case of anti-Sc2-mediated HTR in a 33-year-old woman with β-thalassemia major and a history of anti-Sc2. She presented for routine transfusion and received 1 group O, D-E-K-S-Jk(a-) red blood cell (RBC) unit that was crossmatch compatible by the antihuman globulin (AHG)-polyethylene glycol testing method. Shortly after the transfusion, she developed chills and back pain that resolved with meperidine. Several hours later, she experienced jaundice, dark urine, and fatigue. Laboratory evaluation revealed a hemoglobin drop below the pre-transfusion baseline, elevated bilirubin (8.0 mg/dL, reference range ≤1.2 mg/dL), and a 2+ incompatibility between the post-transfusion sample and the donor RBC unit segment. Although the direct antiglobulin test and the antibody screen remained negative, reference testing confirmed anti-Sc2 in the post-transfusion plasma, and the donor RBC unit was Sc2+. This case reinforces the clinical relevance of anti-Sc2, highlights limitations of conventional antibody screening and the AHG crossmatch in detecting low-prevalence antigens, and supports the need for heightened clinical suspicion and individualized transfusion strategies, including additional targeted pre-transf usion testing, early consultation with reference laboratories, and sourcing of antigen-negative units in patients with known rare alloantibodies.
We evaluated the impact of extended red blood cell (RBC) molecular matching on alloimmunization and transf usion-related outcomes in patients with sickle cell disease (SCD) and assessed the frequency and clinical significance of genotype- phenotype discrepancies. We conducted a retrospective analysis of 108 transfused patients with SCD who underwent phenotyping and molecular genotyping for clinically relevant RBC antigens. Patients were divided into two groups: those who received extended serologically matched RBC units (n = 55) and those who received extended molecularly matched RBC units (n = 53). Primary outcomes included the rate of alloimmunization, incidence of delayed hemolytic transfusion reactions (DHTRs), and the identification of antigen mismatches or discrepancies between genotype and phenotype. Molecular testing revealed clinically significant antigen mismatches in 42 percent of patients. Partial RH alleles were identified in 17 percent of patients. Discrepancies between genotype and phenotype were observed in 21.3 percent of patients. Alloimmunized patients were significantly more likely to have undetected mismatches. DHTRs after transfusion with RBC units that were serologically matched, but not molecularly compatible, were observed in two patients. In conclusion, extended RBC molecular matching improves the detection of clinically relevant antigen mismatches not identified by routine serologic methods and is associated with a lower risk of alloimmunization and transfusion-related complications.
Alloantibodies may develop after exposure to foreign red blood cell (RBC) antigens. Evanescence occurs when an antibody falls below the sensitivity threshold of methods used in pretransfusion testing. An alloantibody that has evanesced may go undetected, resulting in possible delayed hemolytic transfusion reactions, which lead to increased morbidity and mortality. A survey was conducted to analyze evanescence of alloantibodies over time. A total of 544 patients with 656 alloantibodies were evaluated. Median follow-up was 294 days (range 3-3852 days). Analysis showed that patient age at detection of alloantibody (p = 0.037), sex (p < 0.001), results of initial RBC antibody screen (p < 0.001), RBC transfusion (p < 0.001), length of follow-up period (p < 0.001), and alloantibody specificity (p = 0.004) significantly influenced the time of evanescence. Evanescence rate was the highest for anti-Jka, anti-C, and anti-M and the lowest for anti-Fya and anti-D specificities. Evanescence of alloantibodies represents a significant problem in routine pretransfusion testing. Beyond improving testing by implementing more sensitive methods, there is a place for preventive usage of extended antigen-matched RBC units or the application of post-transfusion protocols. Sharing of antibody information across centers can also improve transfusion safety in these centers.
We report a case of a 61-year-old male patient with a complex hematologic history including pre-B acute lymphocytic leukemia, allogeneic stem cell transplant, and newly treated colon cancer followed by diagnosis of high-risk myelodysplastic syndrome (MDS), who exhibited abolishment of D antigen production. The patient's red blood cells (RBCs), which originally typed as group A, D+, again typed as group A, D+, after receiving stem cells from a female donor with the same blood type. The reactivity of the patient's RBCs was strong when tested with anti-D reagent until he was treated for colon cancer. Within 6 months of diagnosis of cancer, he developed a mixed-field D typing result followed by a complete D- phenotype over the course of a few weeks, coinciding with the new diagnosis of MDS and initiation of immunosuppressive therapy. Polymerase chain reaction (PCR) testing revealed no weak or partial D variants, and subsequent Sanger sequencing confirmed the presence of a conventional RHD gene. A more focused analysis by chromosomal microarray identified deletions involving the RHD locus, supporting the hypothesis that active MDS disrupted gene expression. The patient received another stem cell transplant, this time from a group AB, D+ male donor. In a short period of time, MDS re-emerged along with re-identification of microarray mutations encompassing RHD in female cells (from the first donor) even after the second stem cell transplant. However, D expression remained strong, underscoring the presence of enough male donor RHD expression to maintain the D+ phenotype.
Irregular antibodies with the potential for causing transfusion reactions can be detected in healthy donors. The prevalence and characteristics of irregular antibodies in many populations are well known; however, the data of Vietnamese blood donors have not yet been reported. Our study was performed to assess the frequency and specificities of irregular antibodies among healthy blood donors in the north of Vietnam. A total of 199,281 blood donor samples were screened for irregular antibodies from 2021 to 2023. Antibody screening was performed by both microplate and tube methods. Positive tests were further confirmed using a gel card method. Subsequently, antibody identification was performed. The occurrence of a positive antibody detection test was 0.37 percent. Most cases had only one antibody (99.1%); the proportion of donors who had two and three antibodies accounted for 0.8 and 0.1 percent, respectively. The most frequently identified antibodies were of the MNS blood group system, with anti-Mia being the highest (72.7%) and then anti-M (12.9%), followed by the Lewis blood group system with anti-Lea (6.5%) and anti-Leb (2.4%). The results show the rate and characteristics of irregular antibodies in northern Vietnamese blood donors. These findings provide essential data to support recommendations for implementing antibody detection in donor testing nationwide. Moreover, the results underscore the importance of selecting appropriate reagent panels.
This review updates knowledge on the Kell (International Society of Blood Transfusion [ISBT] 006) and Kx (ISBT 019) blood group systems since the last review published in Immunohematology in 2015. It highlights new insights into the relationship between Kell glycoprotein and red blood cell (RBC) membrane stability, including recent discoveries of new antigens and alleles, and reporting of the first diagnosis of McLeod syndrome in an infant. The Kell and Kx blood group systems welcome an increasing number of antigens and/or alleles to their systems. Kell has a total of 38 antigens as of January 2025; a further 25 novel alleles encode Kmod phenotypes, and an additional 71 nucleotide changes are associated with the K0 (null) phenotype. XK follows a similar theme with an ever-increasing number of new alleles, all encoding the Kx- (null) phenotype. The review emphasizes the role of molecular diagnostics in resolving serologic ambiguities in the blood bank or assisting the diagnosis of neurodegenerative syndromes. The monitoring and management of anti-K in pregnancy is evolving. Emerging technologies such as single-cell sequencing and multi-omics analysis workflows, gene editing, and cellular therapeutics may unlock the inner workings of Kell and Kx protein mechanics and elucidate the function of Kell protein biology, structural immunogenicity, and explain why alloanti-K is capable of suppressing erythroid growth.
Automated pre-transfusion testing provides significant improvements in efficiency and productivity along with a reduction of the potential for errors. With reflex test capability and bidirectional Laboratory Information System interfacing, enhanced levels of effectiveness can be achieved in delivery of test results. To improve efficiency and productivity in our laboratory related to antibody identification (AbID) on our automated testing analyzer, we conducted a study that would allow for extended on-board utilization of our AbID reagent red blood cells (RRBCs). Our current process requires loading the 0.8 percent AbID RRBC panel onto the analyzer at the time of antibody detection and then removing and returning it to refrigerated storage once the AbID test has been completed. Our study was conducted at two hospital sites with an initial pilot study to determine the feasibility of using the RRBC panel on board with evaporation caps over a 7-day timeframe upon initial use of the panel and at two different timeframes later in the panel shelf life. Once the initial pilot study was completed and the feasibility of use established, a secondary study was initiated to determine if stability of reactivity was maintained using a rotational approach of time on board the analyzer compared with time in standard refrigerated storage. A 12-hour rotation at hospital 1 over a 2-week period and a 24-hour rotation at hospital 2 over a 3-week period were evaluated. Anti-c and anti-Fya were used at one site while the other site used anti-E and anti-K. Respective negative controls were tested at both sites. Results of the pilot study demonstrated that the reactivity of the antibodies tested over the 7-day timeframe was maintained along with antibody specificity. The secondary study demonstrated sustained reactivity strength when using the rotational approach but showed occasional yet inconsistent results with respect to specific RRBC deterioration, fibrin in patient's plasma, or indeterminate occurrence. Based on the results of the study, a 7-day on-board utilization protocol was established for routine use. The new extended on-board protocol offers enhanced performance, efficiency, and safety for our transfusion medicine operations.
When antibody screening results are positive in a patient"s sample, the next step is to identify the specificity of the antibody and plan for transfusion or treatment needs. Most often, the antibody can be identified at the transfusing facility; samples that are not resolved may be referred to an immunohematology reference laboratory. A portion of these referred samples may still not be resolved, and the antibodies in these cases have been termed "antibodies of unidentified or undetermined specificity". In this publication, we selected the term "unknown" for such antibodies. Local medical staff should be involved in the clinical management and transfusion recommendations for these patients. The flow charts described in this article are designed to guide the serologist through steps that may result in a defined antibody specificity, or they may not resolve the specificity, and thus, the antibody remains an "antibody of unknown specificity".
The Rh blood group system was last reviewed in Immunohematology in 2010 (Chou ST, Westhoff CM. The Rh and RhAG blood group systems. Immunohematology 2010;26:178-86). This update focuses on RHD, RhD structure, alterations in D expression, anti-D alloimmunization, and applications of RHD genotyping for weak and discrepant D phenotypes; identification of RHD genotypes that encode partial D phenotypes; and prevention and management of anti-D in pregnancy. Updates to the RHAG system and to RHCE and its encoded antigens are in recent or upcoming publications of Immunohematology, respectively.
