High Rate of Passenger Lymphocyte Syndrome after ABO Minor Incompatible Lung Transplantation.

Rationale: Passenger lymphocyte syndrome (PLS) may complicate minor ABO mismatched lung transplantation (LuTX) via donor-derived red cell antibody-induced hemolysis.Objectives: To ascertain the incidence and specificity of PLS-relevant antibodies among the study population as well as the dynamics of hemolysis parameters and the transfusion requirement of patients with or without PLS.Methods: In this cohort study, 1,011 patients who received LuTX between January 2010 and June 2019 were studied retrospectively. Prospectively, 87 LuTX (July 2019 to June 2021) were analyzed. Postoperative ABO antibody and hemolytic marker determinations, transfusion requirement, and duration of postoperative hospital care were analyzed. Retrospectively, blood group A recipients of O grafts with PLS were compared with those without.Measurements and Main Results: PLS affected 18.18% (retrospective) and 30.77% (prospective) of A recipients receiving O grafts, 5.13% of B recipients of O grafts, and 20% of AB patients receiving O transplants. Anti-A and anti-A1 were the predominant PLS-inducing antibodies, followed by anti-B and anti-A,B. Significantly lower hemoglobin values (median, 7.4 vs. 8.3 g/dl; P = 0.0063) and an approximately twice as high percentage of patients requiring blood transfusions were seen in PLS. No significant differences in other laboratory markers, duration of hospital stay, or other complications after LuTX were registered.Conclusions: Minor ABO incompatible LuTX recipients are at considerable risk of developing clinically significant PLS. Post-transplant monitoring combining red cell serology and hemolysis marker determination appears advisable so as not to overlook hemolytic episodes that necessitate antigen-negative transfusion therapy.

Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies.

Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed-field reactions. Such blood group discrepancies can either result from a haematopoiesis-confined or body-wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three-quarters were chimaeras (two same-sex females, four same-sex males, one sex-mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body-wide involvement in all instances. Moreover, genome-wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy-neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion- or transplantation-associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make-up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre-existent or age-related from disease-predisposing and disease-indicating cell clones.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Immunoglobulin Class Profiles of ABO Antibodies in Saliva and Serum of Healthy Individuals.

Introduction: The coronavirus disease (COVID-19) pandemic gave rise to studies investigating the association of ABO blood group with COVID-19 susceptibility. It is hypothesized that ABO antibodies might play a role in neutralizing SARS-CoV-2. However, ABO antibodies were exclusively analyzed in blood samples. Investigation of ABO antibodies in saliva, an easy-to-obtain surrogate for respiratory secretions, may provide novel insights into mucosal immunity crucial in early defense against respiratory pathogens. Methods: In this study, saliva and serum samples from healthy individuals with known blood groups were investigated using a flow cytometric method for separate anti-A/anti-B IgA, IgM, and IgG class antibody detection. Saliva samples were additionally tested using hemagglutination-based neutral and indirect anti-human globulin test gel cards. This method comparison was complemented by dilution experiments with a high-titer anti-A/anti-B WHO standard. Results: In saliva, IgA was the most abundant ABO antibody class, followed by IgM; IgG was detected only in low levels in all non-AB blood types. In serum, IgM was the predominant ABO antibody class in all non-AB blood types, followed by IgA and IgG, the latter mainly detected in group O individuals. Saliva and serum samples of group O individuals yielded the highest variability of ABO-specific antibody levels. Regardless of sample material and blood type, major interindividual differences in ABO antibody reactivities were recorded. Antibody levels correlated moderately between these two body fluids. There were no significant sex and age-group differences in ABO antibody levels in both serum and saliva. WHO standard dilution experiments yielded technique-specific limits of detection, illustrating the inherent differences of immunofluorescence versus agglutination. Conclusion: For the first time, salivary ABO antibodies were investigated by separate detection of the three most relevant antibody classes IgA, IgM, and IgG in a healthy cohort. This study opens new perspectives regarding mucosal ABO antibody class profiles and their potential influence on respiratory infections.

Leukocyte-Reactive Antibodies in Female Blood Donors: The Austrian Experience.

INTRODUCTION: Antibody-mediated transfusion-related acute lung injury (TRALI) is caused by antibodies against human leukocyte antigens (HLAs) or human neutrophil antigens (HNAs), and is one of the most serious complications associated with transfusion medicine. Prevention strategies like testing allo-exposed female blood donors have not yet been introduced nationwide in Austria. To assess the need and feasibility of routine leukocyte antibody testing, the prevalence of leukocyte-reactive antibodies in an Austrian female donor population was been determined using classical cell-based methods which were compared with a high-throughput bead-based method. METHODS: Sera from 1,022 female blood donors were screened using a granulocyte aggregation test (GAT) and a white blood cell immunofluorescence test (WIFT) after retesting and specification of positive samples by granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA). Potential HLA reactivities were confirmed using the microbeads assay LabScreenTM Mixed. The results in 142 donor sera and 38 well-defined reference sera were investigated by the microbeads assay LabScreenTM Multi and compared with classical cell-based methods. RESULTS: Reactivity with either granulocytes and/or lymphocytes was detected in 79 sera (7.7%), with the majority being HLA-specific. Antibodies against HNA were obtained in 7 samples (0.7%). The aggregating potential of the detected antibodies was observed in 9 cases (0.9%). Most of the leukocyte-reactive antibodies occurred at a donor age of between 35 and 59 years (n = 61). LabScreen Multi showed good agreement (κ = 0.767) for HNA antibody detection by cell-based assays, but double/multiple specificities (100% of 7 anti-HNA-1b sera) as well as false-negative results (40% of 15 HNA-3-specific sera) occurred. DISCUSSION: Leukocyte-reactive antibody screening is advised in Austrian female donors for safe blood transfusion, including single-donor convalescent plasma treatment of COVID-19 that may be implemented soon. For the introduction of LabScreen Multi, the combination with GAT should be considered to ensure correct anti-HNA-3a detection.

Ethical Issues and Management of Fetal Hemolytic Anemia Caused by Anti-Rh17 in a Multipara with Rare -D- Phenotype.

BACKGROUND: The development of allo-anti-Rh17 (anti-Hr0) in a -D- phenotype whose red blood cells (RBCs) lack CcEe antigens is most likely triggered by transfusion, transplantation, or pregnancy. Gene conversion is the predominating factor in generating RHD-CE-D and RHCE-D-CE hybrids like -D-. METHODS: We report here immunohematological and obstetrical data from 2 of the 5 pregnancies of a 24-year-old woman presenting with the -D- phenotype with anti-Rh17. Blood group typing, antibody screening, antibody differentiation, direct antiglobulin test (DAT), and antibody titers were performed by routine gel technology and tube testing. Additionally, molecular genetic analysis was performed. Fetal surveillance was done by sonographic evaluation of the fetal middle cerebral artery peak systolic velocity (MCA-PSV). RESULTS: Blood group typing showed O, C-c-D+E-e- and the DAT was negative. DNA sequencing revealed homozygosity for an RHCE-D(3-9)-CE null allele. Anti-Rh17 titers in the fourth pregnancy remained between 1:8 and 1:128, and no signs for a fetal anemia were observed. However, in the fifth pregnancy, the antibody titers increased up to 1:4,096. Signs of moderate fetal anemia were detected and cesarean section was performed at 34 + 6 weeks of gestation. The newborn presented with hemolytic anemia (cord blood hemoglobin [Hb] = 8.5 mg/dL). She received 2 compatible (small) packed RBC concentrates, phototherapy, and intravenous immunoglobulins. CONCLUSION: Our case shows that the risk for hemolytic complications increases with the number of pregnancies of sensitized women. Only people who also lack CcEe antigens are compatible as donors. The role of such rare donors as lifesavers, their freedom, and voluntariness conflict with the urgent need for compatible blood.

Aberrant ABO B Phenotype with Irregular Anti-B Caused by a Para-Bombay FUT1 Mutation.

BACKGROUND: Routine ABO blood group typing for pre-transfusion testing of a male Austrian patient of Far Eastern origin showed discrepant results with an apparently weak blood group B phenotype and irregular anti-B. MATERIALS AND METHODS: ABH phenotyping and cross-matching was done by standard serologic techniques and levels of H expression were determined by flow cytometry. ABO gene sequencing including regulatory regions as well as analysis of FUT1 (H), FUT2 (Secretor), and FUT3 (Lewis) were carried out. RESULTS: While monoclonal ABO antigen typing indicated blood group O, weak agglutination reactions using polyclonal human anti-B and anti-AB were seen. In reverse typing at room temperature, the plasma was reactive with A1 and A2 RBCs and negative with B and O cells, whereas at 4°C, anti-B reactivity was found. The indirect anti-globulin cross-match of the patient's plasma was positive with group B RBCs and negative with group O RBCs. Sequencing analysis showed the presence of ABO*B.01 (B114) allele and homozygosity for the FUT1 mutation c.551_552delAG. Flow cytometry demonstrated trace amounts of H antigen on the patient's RBCs. CONCLUSION: While a functional B allele was found, analysis of FUT1 and FUT2 genes revealed the presence of a rare para-Bombay genotype OhB. Interestingly, no anti-H but irregular anti-B was found in the patient's plasma, responsible for the positive cross-match with group B RBCs. Even though very rare and not reported for the European population, the presence of an H-deficient phenotype should be considered when investigating individuals with an unusual ABO blood group type.

Multicenter Study on Differential Human Neutrophil Antigen 2 Expression and Underlying Molecular Mechanisms.

BACKGROUND: The human neutrophil antigen 2 (HNA-2), which is expressed on CD177, is undetectable in 3-5% of the normal population. Exposure of these HNA-2null individuals to HNA-2-positive cells can cause immunization and pro-duction of HNA-2 antibodies, which can induce immune neutropenia and transfusion-related acute lung injury. In HNA-2-positive individuals, neutrophils are divided into a CD177pos. and a CD177neg. subpopulation. The molecular background of HNA-2 deficiency and the bimodal expression pattern, however, are not completely decoded. STUDY DESIGN: An international collaboration was conducted on the genetic analysis of HNA-2-phenotyped blood samples, including HNA-2-deficient individuals, mothers, and the respective children with neonatal immune neutropenia and regular blood donors. RESULTS: From a total of 54 HNA-2null individuals, 43 were homozygous for the CD177 *787A>T substitution. Six carried the CD177 *c.1291G>A single nucleotide polymorphism. All HNA-2-positive samples with >40% CD177pos. neutrophils carried the *787A wild-type allele, whereas a lower rate of CD177pos. neutrophils was preferentially associated with *c.787AT heterozygosity. Interestingly, only the *c.787A allele sequence was detected in complementary DNA (cDNA) sequence analysis carried out on all *c.787AT heterozygous individuals. However, cDNA analysis after sorting of CD177pos. and CD177neg. neutrophil subsets from HNA-2-positive individuals showed identical sequences, which makes regulatory elements within the promoter unlikely to affect CD177 gene transcription in different CD177 neutrophil subsets. CONCLUSION: This comprehensive study clearly demonstrates the impact of single nucleotide polymorphisms on the expression of HNA-2 on the neutrophil surface but challenges the hypothesis of regulatory epigenetic effects being implicated in the bimodal CD177 expression pattern.

Somatic mosaicisms of chromosome 1 at two different stages of ontogenetic development detected by Rh blood group discrepancies.

Spontaneous Rh blood group changes are a striking sign, reported to occur mainly in patients with hematologic disorders. Upon routine blood grouping, 2 unrelated individuals showed unexplained mixed red cell phenotype regarding the highly immunogenic c antigen (RH4), clinically relevant for blood transfusion and fetomaternal incompatibility. About half of their red cells were c-positive, whereas the other half were c-negative. These apparently hematologically healthy females had no history of transfusion or transplantation, and they tested negative for chimerism. Genotyping of flanking chromosome 1 microsatellites in blood, finger nails, hair, leukocyte subpopulations, and erythroid progenitor cells showed partial loss of heterozygosity encompassing the RHD/RHCE loci, spanning a 1p region of 26.7 or 42.4 Mb, respectively. Remarkably, in one case this was detected in all investigated tissues, whereas in the other, exclusively myeloid cells showed loss of heterozygosity. Both carried the RhD-positive haplotypes CDe and the RhD-negative haplotype cde RHD/RHCE genotypes of single erythroid colonies and dual-color fluorescent in situ hybridization analyses indicated loss of the cde haplotype and duplication of the CDe haplotype in the altered cell line. Accordingly, red cell C antigen (RH2) levels of both propositae were higher than those of heterozygous controls. Taken together, the Rhc phenotype splitting appeared to be caused by deletion of a part of 1p followed by duplication of homologous stretches of the sister chromosome. In one case, this phenomenon was confined to myeloid stem cells, while in the other, a pluripotent stem cell line was affected, demonstrating somatic mosaicism at different stages of ontogenesis.

Characterization of Novel RHD Allele Variants and Their Implications for Routine Blood Group Diagnostics.

The Rh system, including the highly immunogenic D antigen, is one of the clinically most important blood group systems in transfusion medicine. Numerous alleles of the RHD gene are associated with variant RhD phenotypes. In case of Rh incompatibility, some of them can induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Thus, accurate blood group diagnostics are critical for safe transfusion therapy. We characterized phenotypes of four individuals revealing weakened D expression during routine pre-transfusion testing. Standard gel card matrix techniques with monoclonal and polyclonal anti-D antibodies were used for serological typing, complemented using D epitope and antigen density analysis. Genotyping employing PCR with sequence-specific primers, genomic and allele-specific Sanger sequencing and in silico protein analysis were performed. Four novel RHD alleles associated with weak D or partial D phenotypes were identified. One of the mutations is predicted to disrupt the terminal stop codon and result in an elongated translation of the mutant D protein that phenotypically exhibits a loss of D epitopes. Furthermore, a hybrid gene formed with the homologue RHCE gene is described. The presented data enhances the understanding of the Rh system and may contribute to continued advances in blood group diagnostics.

Polyomavirus Nephropathy in ABO Blood Group-Incompatible Kidney Transplantation: Torque Teno Virus and Immunosuppressive Burden as an Approximation to the Problem.

Introduction: Earlier reports suggest that patients after ABO-incompatible kidney transplantation (ABOi) are at enhanced risk of developing BK-virus (BKV, also known as BK polyomavirus [BKPyV]) nephropathy (BKPyVAN). It remains elusive whether this is a result of more intense immunosuppression or an ABOi-associated "intrinsic attribute." To address this question, we measured Torque Teno virus (TTV) loads as a quantitative proxy for immunosuppressive depth in ABOi recipients and compared them to human leukocyte antigen-incompatible (HLAi, i.e. pretransplant donor-specific antibody-positive) and standard-risk transplant recipients. Methods: Our retrospective study screened 2256 consecutive kidney transplantations performed between 2007 and 2020 at the Medical University of Vienna. Out of 629 in-principle eligible transplantations, we were able to include 465 patients: 42 ABOi, 106 HLAi, and 317 control recipients. Longitudinal TTV- polymerase chain reaction (PCR) and BKV-PCR was carried out at predefined timepoints and ranged from pretransplant until month 24 posttransplantation. TTV loads and immunosuppression were evaluated in the context of BKV-associated complications. Results: ABOi recipients had a higher TTV load compared to HLAi and controls both at month 3 (median 1.5 × 109 vs. 2.4 × 108 vs. 9.1 × 107; P = 0.010) and at month 6 (3.1 × 109 vs. 1.4 × 107 vs. 6.4 × 107; P = 0.014) posttransplantation. Tacrolimus exposure was significantly higher in ABOi patients compared to HLAi and control patients (ABOi vs. HLAi: P = 0.007; ABOi vs. controls: P < 0.0001). Biopsy-proven BKPyVAN was more frequent in ABOi recipients when compared to HLAi and control recipients (11.9% vs. 2.8% vs. 4.1%; P = 0.046). Conclusion: Our data support the assumption that ABOi patients are indeed at higher risk to develop BKPyVAN. A higher TTV load and immunosuppressive burden suggest that intense immunosuppression, rather than an "intrinsic attribute" conferred by ABOi, may contribute to this finding.

Lower Levels of ABO Anti-A and Anti-B of IgM, IgG and IgA Isotypes in the Serum but Not the Saliva of COVID-19 Convalescents.

Individuals with ABO type O, naturally possessing anti-A and anti-B antibodies in their serum, are underrepresented among patients infected with SARS-CoV-2 compared with healthy controls. The ABO antibodies might play a role in the viral transmission. Therefore, we aimed to quantify anti-A/anti-B, including their subclasses IgM, IgG and IgA, in the serum and saliva of Caucasians (n = 187) after mild COVID-19 to compare them with individuals who had never been infected with SARS-CoV-2. Two samples were collected within two months after the diagnosis (median days: 44) and two months later. ABO antibodies were determined by flow cytometry. Additionally, total IgA in saliva and antibodies specific to SARS-CoV-2 were tested by ELISA. COVID-19 convalescents had significantly lower levels of anti-A/anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). Interestingly, no significant differences were observed in saliva. ABO antibody levels remained stable over the period considered. No relation of ABO to the level of SARS-CoV-2-specific antibodies was observed. Total IgA was lower in convalescents than in controls (p = 0.038). Whereas ABO antibodies in the saliva may not contribute to the pathogenesis of COVID-19, individual pre-existing high serum concentrations of anti-A/anti-B may have a protective effect against SARS-CoV-2 infection.

Time from venipuncture to cell isolation: Impact on granulocyte-reactive antibody testing.

BACKGROUND AND OBJECTIVES: Classical neutrophil-reactive antibody testing depends on the quick isolation of neutrophils from freshly taken whole blood. To allow a better logistic preparation before testing, the influence of time interval between venipuncture and cell isolation has been evaluated in this study. MATERIALS AND METHODS: Neutrophils and whole leukocytes were isolated from EDTA whole blood immediately (T0) as well as 4, 8 and 24 h after blood donation (T4, T8 and T24). These cells were tested against reference sera containing antibodies against HNA-1b, -2, -3a and HLA class I using granulocyte aggregation test (GAT), microscopic granulocyte immunofluorescence test (GIFT) and flow-cytometric white blood cell immunofluorescence test (Flow-GIFT/WIFT). RESULTS: GAT was the most error-prone test displaying overall weaker aggregation strengths already at T4 (overall accuracy OA = 0.72, κ = 0.58). GIFT results showed good agreement at T4 (OA = 0.86, κ = 0.79) and remained stable until T8, while test results were slightly impaired at T24 (OA = 0.71, κ = 0.55). Flow-GIFT/WIFT was identified as the most robust screening method, remaining stable even at T24. Calculated ratios (sample/negative control) decreased non-significantly and remained highly above the cut-off in all samples. CONCLUSION: Acceptable time limits for cell isolation are different for each screening method investigated. For GAT, cell isolation should be performed within 4 h, while GIFT tolerates a neutrophil isolation delay of 8 h. Flow-GIFT/WIFT isolation can be performed even after 24 h without impairment of the results. Using the latter test as a stand-alone pre-screening test, whole blood can be used from donors who are not directly accessible.