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Immune-deficient patients depend on the antibodies in pooled human immunoglobulin G (IgG) preparations to remain free from serious infections. The potency of IgG preparations is therefore an ongoing concern. The use of pooled IgG to prevent infection is based on the concept that healthy adults have recovered from infections earlier in life and maintain relatively high antibody titers. In general, vaccine-induced immunity is less robust or long-lasting than immunity after natural infection, and many infectious diseases which were formerly widely prevalent have become much less common due to improved hygiene and vaccines. This raises questions as to the adequacy of protective antibodies in current IgG preparations. This paper reviews available data on antibodies against selected bacterial and virus vaccine antigens in current IgG products. Most products contain sufficient antibody to yield levels above minimal protective concentrations to a broad range of pathogens and toxins. Illustrative examples of effects of vaccines on antibody content of IgG products are also discussed: antibody titers to hepatitis A virus in donor plasma pools in both the US and EU are dropping due to decreased natural infection, but they are still sufficient to provide robust protection. Increasing seroprevalence of hepatitis B virus as a result of immunization suggests that antibody titers against this virus may actually be increasing. Finally, serial studies suggest that pooled IgG provides protection against seasonal influenza viruses despite year-to-year antigenic drift, and is also likely to provide at least some protective antibody against potentially pandemic strains. [ABSTRACT FROM AUTHOR]

Background: The therapeutic value of the use of intravenous immunoglobulin (IVIG) to correct anemia and thrombocytopenia as a result of immunologic causes (hemolytic disease of the fetus and newborn and fetal or neonatal alloimmune thrombocytopenia) have been well established. Few published papers exist regarding the use of IVIG in adult settings. We report two patients with clinically significant antibodies against high-incidence antigens, who were successfully transfused with incompatible red blood cells (RBCs), in conjunction with IVIG plus steroids and IVIG.Case Reports: Case 1 was a 25-year-old patient (Hb SC) who was admitted with low hemoglobin (Hb) and low reticulocyte count. A diagnosis of parvovirus-induced RBC hypoplasia was made. The patient's sample contained anti-E, anti-N, and anti-U. The Hb decreased to 37 g/dL and urgent transfusion was provided with E-, N-, "least-incompatible" RBC units covered by IVIG and hydrocortisone. Case 2 was a 54-year-old patient who was admitted after a road traffic accident. Nonspecific weak antibody was detected. She received 6 units of least-incompatible RBCs. She was transferred to another hospital and received 2 least-incompatible units. Hb level decreased further and an additional unit was transfused. Samples were referred to the reference laboratory and strong anti-Jra detected. As there was clinical and laboratory evidence of hemolysis and Jr(a-) units were not available, IVIG was prescribed and least-incompatible units were transfused.Results: There were no hemolytic transfusion reactions, hemolysis ceased, and anemia improved in both cases.Conclusion: Prophylaxis with IVIG plus steroids and IVIG should be considered as a therapeutic option when transfusion of incompatible units is necessary. [ABSTRACT FROM AUTHOR]

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop. [ABSTRACT FROM AUTHOR]

Background: Immunoglobulin therapy including intravenous immunoglobulin (IVIg) has been used as an effective treatment for autoimmune/inflammatory conditions with few side effects. However, high-dose IVIg (1-2 g/kg) has been recognized as a cause of hemolytic anemia in non-blood group O patients. Hemolysis when observed has been due to anti-A/anti-B isoagglutinins contained in the IVIg. Recently, an isoagglutinin-reduced IVIg, whereby the anti-A and anti-B titers have been reduced by immunoaffinity chromatography, has been introduced; however, whether this new product is as efficacious as nonreduced immunoglobulin (Ig) or will result in less IVIg-associated hemolysis has not been resolved.Study Design and Methods: We used in vitro phagocytosis by monocytes and proinflammatory/anti-inflammatory macrophages, with isoagglutinin-reduced and -nonreduced Ig opsonized group A1 , B, and A1 B red blood cells, to estimate clinical significance of the IgG isoagglutinins. We also used immune thrombocytopenia (ITP) and rheumatoid arthritis (RA) mouse models to examine the in vivo efficacy of isoagglutinin-reduced versus -nonreduced Ig on the amelioration of the diseases.Results: In contrast to nonreduced Ig, phagocytosis was largely absent when isoagglutinin-reduced Ig was used at a concentration equivalent to a patient receiving 2 g/kg. The in vivo efficacy of isoagglutinin-reduced versus nonreduced Ig on the amelioration of experimental ITP and RA was similar, indicating no loss of efficacy due to the chromatographic removal of isoagglutinins.Conclusion: Isoagglutinin-reduced Ig should have efficacy similar to nonreduced Ig and result in less IVIg-associated hemolysis. [ABSTRACT FROM AUTHOR]

Background: Platelet refractoriness due to HLA immunization represents a problem in transfusion management of thrombocytopenic hematology patients. Refractory patients can be managed by HLA-selected platelet transfusions, but the optimal matching strategy is debated and how the degree of HLA mismatch influences transfusion outcome is poorly studied.Study Design and Methods: We studied 32 hematology patients who received 142 matched platelet units between 2007 and 2016. Four matching strategies were compared: 1) genomic HLA typing at the two digit level, performed using polymerase chain reaction-sequence-specific oligonucleotide probing; 2) serologic "eplet score" calculated using HLAMatchmaker; 3) cross-matching using lymphocyte cytotoxicity; and 4) matching based on donor-specific antibody (DSA) specificity, determined using Luminex. A 1-hour corrected count increment (CCI) of more than 7.5 × 109 /L was considered a successful response.Results: Selection of platelets with either a complete HLA match or an acceptable HLA mismatch based on genomic typing and DSA information, each predicted 86% successful transfusion responses. For HLA-mismatched transfusions, the eplet score correlated with CCI and the fraction of successful transfusions, but less well compared to DSA matching. Cytotoxic crossmatching was least predictive. For transfusions across one to four DSAs, the antibody reaction strength correlated with the 1-hour CCI, but many transfusions were successful despite the presence of DSA.Conclusion: A complete HLA-A and -B match or an acceptable mismatch based on DSA should guide identification of donors. Still, transfusions across DSAs are often successful, emphasizing that the presence of DSA is necessary but not sufficient for platelet clearance. [ABSTRACT FROM AUTHOR]

Background: A direct antiglobulin test (DAT) checks for antibody or complement on the surface of RBCs and is often done following a transfusion reaction. While passive anti-A and anti-B antibodies are known to cause positive DATs, the extent this occurs following transfusion is unknown.Study Design and Methods: DAT results, ABO type, eluate information, and blood product information were recorded on 1097 transfusion reactions at a large academic hospital over 8 years. The effect of patient blood type, product type, and plasma compatibility of blood product transfused on DAT results were determined. Statistical significance was determined using Chi-squared testing.Results: Patient ABO blood type was a strong predictor of a positive DAT, with type O patients having 6.7% positive rate and non-O patients having a positive rate of 20.6% (p < .0001). Plasma compatibility of the product was a strong predictor of a positive DAT, with plasma compatible transfusions having a 9.4% positive rate while plasma incompatible transfusions were positive 44% of the time (p < .0001). Elution studies found that anti-A/B antibodies were the most common antibody identified. Platelets were more likely to be associated with a positive DAT when compared with RBC transfusions (p < .05).Conclusions: These results demonstrate the patient ABO type and plasma incompatibility are strong predictors of positive DAT results following a transfusion reaction. Anti-A and anti-B antibodies are estimated to account for about 50% of positive DATs in this study. [ABSTRACT FROM AUTHOR]

Background: The association of the ABO blood group with COVID‐19 disease has been confirmed by several studies, with the blood group A patients being more susceptible and prone to a more severe clinical course of the disease. Additionally, several authors also addressed the association of ABO‐types and the levels of anti‐SARS‐CoV‐2 antibodies in convalescents, mostly supporting a theory that the non‐O blood group convalescents present with higher levels of anti‐SARS‐CoV‐2 antibodies. Study Design and Methods: Since previous findings were based on small convalescent cohorts, we quantified the anti‐SARS‐CoV‐2 antibody levels in a total of 3187 convalescent plasma donors with three commercial serological and one standard neutralizing antibody test. The majority of donors had undergone a mild form of the disease and the median time of sampling was 66 days after diagnosis. Results: None of the antibody quantitation results showed any significant association with the ABO blood group types. The same result was evident in the subgroup of vaccinated individuals (n = 370) and the subgroups when stratified according to post‐COVID‐19 periods (0–60, 60–120, and 120–180 days). Conclusion: In conclusion, we found no evidence to confirm that the ABO blood group types influence the level of SARS‐CoV‐2 antibody response in COVID‐19 convalescent plasma donors. [ABSTRACT FROM AUTHOR]

BACKGROUND: Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA-3a (R154) and three HNA-3b (Q154) peptides covering different parts of the first extracellular loop of CTL2 (aa55-231) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R154 peptides in the EIA nor with R154 or Q154 peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies. [ABSTRACT FROM AUTHOR]

Background: COVID‐19 high‐titer CCP selection is a concern, because neutralizing antibody (nAb) testing requires sophisticated labs and methods. Surrogate tests are an alternative for measuring nAb levels in plasma bags, including those that are pathogen‐reduced. Study design/methods: We studied a panel consisting of 191 samples from convalescent donors tested by nAb (CPE‐VNT), obtained from 180 CCP donations (collection: March 20–January 21) and 11 negative controls, with a total of 80 and 111 serum and plasma samples (71 amotosalen/UV treated), with nAb titers ranging from negative to 10,240. Samples were blindly tested for several surrogates: one anti‐RBD, two anti‐spike, and four anti‐nucleocapsid tests, either isolated or combined to improve their positive predictive values as predictors of the presence of high‐titer nAbs, defined as those with titers ≥160. Results: Except for combined and anti‐IgA/M tests, all isolated surrogate tests showed excellent performance for nAb detection: sensitivity (98.3%–100%), specificity (85.7%–100%), PPV (98.9%–100%), NPV (81.3%–100%), and AUC (0.93–0.96), with a variable decrease in sensitivity and considerably lower specificity when using FDA authorization and concomitant nAb titers ≥160. All surrogates had AUCs that were statistically different from CPE‐VNT if nAb≥160, including when using combined, orthogonal approaches. Conclusions: Surrogate tests (isolated or in combination) have an indirect good performance in detecting the presence of nAb, with lower sensitivity and specificity when high nAb titer samples are used, possibly accepting a considerable number of donors whose nAb titers are actually low, which should be evaluated by each laboratory responsible for CCP collection. [ABSTRACT FROM AUTHOR]

Editorial. Comments on the effect of HLA antibodies on hemolytic transfusion reactions. Presence of HLA antigens on red blood cells; Use of routine serology testing; Specificity of the antibody.

Background: Reliability of ABO-antibody measurement is important in the context of supplying low-titer ABO incompatible plasma-containing blood products. This study investigated the correlation of anti-A measurements between three different titer methodologies.Methods: Thirty-four blood group O individuals were included. IgM and IgG anti-A was measured by three different methods: (1) manual method (Bio-Rad ID-gel card), (2) automated method (Immucor NEO), (3) flow cytometry (FC) with calibration in molecules of equivalent fluorochrome (MEF). Data were log2 transformed to titer steps (TS) and log2 MEF, respectively. All three methods were benchmarked against the 14/300 WHO anti-A/anti-B standard reagent.Results: The correlation between the manual and automated methods was statistically significant for both IgM (Spearman's rs  = 0.89, p < .0001) and IgG (rs  = 0.95, p < .0001). The mean TS difference between the manual and automated methods was 0.61 for IgM (p = .0033) and 2.1 for IgG (p < .0001). The manual method yielded IgM titer results that were generally 1 titer step higher than the automated method, whereas for the IgG titrations the difference was generally a median of 2 TS higher. The FC median log2 MEF level was significantly correlated with TS of IgG and IgM for both manual and automated agglutination-based titer methods (0.69 ≤ r2  ≤ 0.91). With the WHO standard reagent, the manual method produced the expected results while the automated method's results were 1 TS lower for both IgM and IgG at all dilutions tested.Conclusion: These results indicate that all three methods are suitable for measuring anti-A in group O whole blood. [ABSTRACT FROM AUTHOR]

Introduction: Little is known about the neutralizing (nAb) and binding antibody kinetics in COVID‐19 convalescent plasma donors, especially during the first 100 days after disease onset. Materials and Methods: A cohort of previously RT‐PCR positive (detected by nasopharyngeal swab during the acute phase), male convalescent patients, all with mild symptoms, were enrolled in serial blood sample collection for a longitudinal nAb titers and anti‐nucleocapsid (NP) antibodies (IgM, IgG and IgA) evaluation. NAbs were detected by a cytopathic effect‐based virus neutralization test (CPE‐based VNT), carried out with SARS‐CoV‐2 (GenBank: MT350282). Results: A total of 78 male volunteers provided 316 samples, spanning a total of 4820 days of study. Although only 25% of donors kept nAb titers ≥160 within 100 days after the onset of disease, there was >75% probability of sustaining nAb titers ≥160 in volunteers whose initial nAb titer was ≥1280, weight ≥ 90 kg or obese, according to their body mass index (BMI), as evidenced by Kaplan–Meier analysis and Cox hazard regression (all p <.02). There was no correlation between the ABO group, ABO antibody titers and persistent high nAb titers. High IgG anti‐NP (S/CO ≥5.0) is a good surrogate for detecting nAb ≥ 160, defined by the ROC curve (sensitivity = 90.5%; CI95%: 84.5%–94.7%). Conclusion: Selection of CCP donors for multiple collections based on initial high nAb titers (≥1280) or BMI ≥ 30 kg/m2 provides a simple strategy to achieve higher quality in CCP programs. High IgG anti‐NP levels can also be used as surrogate markers for high nAb screening. [ABSTRACT FROM AUTHOR]

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets in the fetus or newborn by maternal platelet alloantibodies, mostly against human platelet antigen (HPA)-1a. Recent studies indicate that two anti-HPA subtypes exist: Type I reacts with epitopes residing on the plexin-semaphorin-integrin (PSI) and type II with plexin-semaphorin-integrin/integrin epidermal growth factor 1 (I-EGF1) domains of the β3 integrin. Here, we evaluated whether a Cys460Trp mutation in the I-EGF1 domain found in a patient with Glanzmann thrombasthenia can alter the binding of anti-HPA-1a.Methods: Stable HEK293 cell lines expressing wild-type and mutant αIIbβ3 and αvβ3 were generated to prove the reactivity of different antibodies against HPA-1a.Results: Flow cytometry analysis of wild-type (Cys460) and mutant (Trp460) expressed on HEK293 cells showed equal surface expression of αIIbβ3 and αvβ3. When tested with mutant αIIbβ3 cells, reduced binding was observed in Type II but not in Type I anti-HPA-1a. These results could be confirmed with platelets carrying Cys460Trp mutation. Interestingly, reduced binding of Type I antibodies was detected with mutant αvβ3 cells. Both antibody types were found in maternal sera from FNAIT cases by an antigen-capture assay with use of HEK293 transfected cells.Conclusions: These observations confirm the existence of Type I and Type II anti-HPA-1a. Furthermore, this study underlines different immunogenicity of HPA-1a antigen(s) residing on either αIIbβ3 or αvβ3. Further analysis of FNAIT cases from mothers having a fetus with and without intracranial bleedings with use of such an approach may highlight the functional relevance of different anti-HPA-1a subtypes. [ABSTRACT FROM AUTHOR]

Background: Intravenous immunoglobulins (IVIG) are derived from large human plasma pools. IVIG-associated hemolytic anemia (HA) is a known class effect, likely attributed to dose-dependent passive transfer of anti-A/B isoagglutinins. Two isoagglutinin reduction steps were implemented in the manufacturing process of Privigen (human 10% liquid IVIG): exclusion of high-anti-A-titer donors in 2013, replaced by specific immunoaffinity chromatography in 2015. We aim to estimate the clinical effectiveness of both measures.Study Design and Methods: Using the US hospital-based Premier Healthcare Database, three Privigen cohorts were generated based on calendar periods indicative of manufacturing changes: Period 1 (baseline) January 2008 to December 2012, Period 2 (high-anti-A-titer donor exclusion) October 2013 to December 2015, and Period 3 (immunoaffinity chromatography) October 2016 to April 2019. HA within a 10-day at-risk period after Privigen administrations was identified from review of patient record summaries. Incidence rate ratios (IRRs) were estimated from Poisson regression (Period 1 reference) adjusting for hospital setting, sex, age, Privigen indication, dose, and first use.Results: Crude incidence rates of HA were 1.49 per 10,000 person-days in Period 1 (38 HA, 9439 patients), 1.01 in Period 2 (20 HA, 7710 patients), and 0.14 in Period 3 (3 HA, 7759 patients). Adjusted IRR for HA in Period 2 was 0.71 (95% confidence interval [CI], 0.41-1.23), and in Period 3 was 0.10 (0.03-0.33) compared with Period 1. The IRR for HA in Period 3 compared with Period 2 was 0.14 (95% CI, 0.04-0.47).Conclusion: Implementation of immunoaffinity chromatography in Privigen manufacturing resulted in a significant 90% reduction of HA risk. HA has become a rare event in association with Privigen use. [ABSTRACT FROM AUTHOR]

Background: Red blood cell (RBC) alloimmunization is an important transfusion complication which is prevalent among sickle cell disease (SCD) patients. Autoimmune diseases are a known risk factor for RBC alloimmunization, suggesting that autoimmunity and post-transfusion alloantibody development occur through similar physiopathological pathways. Polymorphisms in the FcγR2B gene have already been associated with several autoimmune disorders and hypothetically could be associated with RBC alloimmunization. Our goal was to evaluate if important polymorphisms of FcγR2B have an impact on the risk of RBC alloimmunization among SCD patients.Study Design and Methods: This was a case-control study in which alloimmunized and non-alloimmunized SCD patients were compared in terms of the genotype frequency of the FcγR2B polymorphisms -386G/C, -120 T/A, and 695C/T, genotyped through direct Sanger sequencing.Results: A total of 237 patients met the eligibility criteria, 120 cases (alloimmunized) and 117 controls (non-alloimmunized). RBC alloimmunization was associated with female sex (p < 0.001), lifetime number of RBC units transfused (p = 0.002) and 120 T/A FcγR2B genotype (p = 0.031). The FcγR2B promoter region haplotype 2B.4 (386C120A) was positively associated with RBC alloimunization (p = 0.045). The logistic regression (LR) model identified female sex (OR 10.03, CI 95% 5.16-19.49; p < 0.001) and FcγR2B 2B.4 haplotype (OR 4.55, CI95% 1.1118.65; p = 0.035) as independent predictors of RBC alloimmunization in SCD patients.Conclusion: SCD patients with the FcγR2B 2B.4 haplotype had over a fourfold higher risk for RBC alloimmunization. This highlights the role played by FcγR2B on RBC alloimmunization and may be helpful in identifying the immune responders. [ABSTRACT FROM AUTHOR]

Background: Hemolysis is an infrequent but recognized and potentially serious adverse effect of intravenous immunoglobulin (IVIG). Relatively elevated hemolysis reporting rates were seen with some IVIG products with high anti-A/B isoagglutinin content, among which IgPro10 (Privigen, CSL Behring). For IgPro10, two isoagglutinin reduction measures were successively implemented: 1) anti-A donor screening and 2) immunoaffinity chromatography (IAC; Ig IsoLo)-based isoagglutinin reduction step included in the production process. The aim of this analysis was to investigate the effects of these isoagglutinin reduction measures on the reporting rates of IgPro10 hemolysis worldwide.Study Design and Methods: Between February 2008 and December 2018, hemolysis reports from the CSL Behring Global Safety Database were analyzed in relationship to changes in IVIG IgPro10 production methods. Further analysis classified hemolysis reports by indication and blood group.Results: Median (minimum-maximum) anti-A/anti-B titers were 32 (8-64)/16 (8-32) at baseline, 32 (8-64)/16 (8-32) after donor screening, and 8 (8-32)/4 (2-8) after implementation of IAC. The reporting rate of hemolytic reactions per 1000 kg IgPro10 sold was 4.05 cases at baseline, 2.00 after donor screening, and 0.50 after implementation of IAC. In 2018, there were seven reports of hemolytic reactions; representing 0.18 cases per 1000 kg IgPro10 sold, with a reduction of 95.6% versus baseline.Conclusion: Following implementation of the IAC isoagglutinin reduction step, spontaneous reports of hemolytic events with IgPro10 were significantly and consistently reduced versus IgPro10 without isoagglutinin reduction, offering patients a more favorable benefit-risk profile. [ABSTRACT FROM AUTHOR]

Background: Blood donors represent a healthy population, whose red blood cell (RBC) alloantibody persistence or evanescence kinetics may differ from those of immunocompromised patients. A better understanding of the biologic factors impacting antibody persistence is warranted, as the presence of alloantibodies may impact donor health and the fate of the donated blood product.Methods: Donor/donation data collected from four US blood centers from 2012 to 2016 as part of the Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) were analyzed. Clinically significant antibodies from blood donors with more than one donation who underwent at least one follow-up antibody screen after the initial antibody identification were included. Of 632,378 blood donors, 481 (128 males and 353 females) fit inclusion criteria.Results: Antibody screens detected 562 alloantibodies, with 368 of 562 (65%) of antibodies being persistently detected and with 194 of 562 (35%) becoming evanescent. Factors associated with antibody persistence included antibody specificity, detection at the first donation, reported history of transfusion, and detection of multiple antibodies concurrently. Anti-D, C, and Fya were most likely to persist, while anti-M, Jka , and S were most frequently evanescent.Conclusions: These data provide insight into variables impacting the duration of antibody detection, and they may also influence blood donor center policies regarding donor recruitment/acceptance. [ABSTRACT FROM AUTHOR]

Background: Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown.Study Design and Methods: Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay.Results: Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind.Conclusion: Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype. [ABSTRACT FROM AUTHOR]

Background: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT).Study Design and Methods: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages.Results: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients.Conclusion: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn. [ABSTRACT FROM AUTHOR]

Background: Patients with congenital haptoglobin deficiency can develop anti-haptoglobin antibodies after exposure to blood products, and they can suffer from life-threatening anaphylactic transfusion reactions. Here, we present a case of a 57-year-old Chinese male with myelodysplastic syndrome who manifested an anaphylactic transfusion reaction during the transfusion of platelets. The only abnormality detected during his reaction laboratory workup was an undetectable haptoglobin level in the absence of evidence of hemolysis.Study Design and Methods: Surface plasmon resonance (SPR) was explored as a method to be able to detect the presence of anti-haptoglobin antibodies in serum. First, haptoglobin was immobilized to the surface of an SPR sensor chip. The patient's serum sample was injected, and the binding response was monitored in real time. Serum samples from five healthy volunteers were used as negative controls. Binding specificity was assessed in competition experiments using soluble haptoglobin. Anti-IgG, -IgA, -IgM, -IgD and -IgE antibodies were used to identify the antibody isotype.Results: An IgG anti-haptoglobin antibody was detected in the patient's serum with SPR.Conclusion: SPR provided a rapid, readily available method for the detection of an IgG anti-haptoglobin antibody in an anhaptoglobinemic individual. This confirmed the underlying etiology of the anaphylactic nonhemolytic transfusion reaction and justified the necessity of stringently washed cellular products for all future transfusions and strong caution for future use of plasma-containing products. [ABSTRACT FROM AUTHOR]

Background: Recent advances in desensitization techniques and immunosuppressive therapy have led to improved outcomes after ABO-incompatible (ABO-i) kidney transplantation (KT). However, questions remain unanswered, particularly regarding which type of ABO isoagglutinin-immunoglobulin M (IgM) or immunoglobulin M (IgG)-is significantly involved in antibody-mediated rejection (AMR).Study Design and Methods: We retrospectively analyzed data from 120 patients who underwent ABO-i KT between 2012 and 2014. Preoperative plasma exchange was performed until the IgM isoagglutinin titer was 4 or less, regardless of the IgG titer. Clinical data were compared between patient groups with pre-KT IgG isoagglutinin titer 16 or greater (high IgG; titer range, 16-256; n = 39) and 8 or less (low IgG; titer range, -8; n = 81).Results: The median follow-up periods were 59 (high IgG) and 55 (low IgG) months. Patient survival at 5 years (p = 0.314) was 100% (high IgG) and 97.4% (low IgG). Graft survival at 5 years (p = 0.480) was 100% (high IgG) and 98.7% (low IgG). AMR by anti-ABO antibody occurred in only one patient in the low-IgG group.Conclusion: Patients with high pre-KT IgG isoagglutinin titers had equally successful outcomes as those with low IgG titers. ABO-i KT can be successfully performed by reducing the pre-KT IgM isoagglutinin titer to 4 or less, as determined by the immediate spin tube method. [ABSTRACT FROM AUTHOR]

Background: Blood transfusion is the first-line treatment for patients with thalassemia and many sickle cell patients. However, cases of unregulated blood transfusion are shown to carry a high risk of alloimmunization to red blood cells (RBCs), which can lead to a hemolytic transfusion reaction and be fatal to patients. Screening and identification of alloantibodies are, therefore, essential practice in blood transfusion services. Transfusion of phenotyped blood can minimize these risks to patients.Study Design and Methods: A prospective study was carried out on 1015 donors, and a prospective and retrospective study was carried out on 208 multiple transfused patients with β-thalassemia and sickle cell anemia. Donor and patient samples were subjected to Rh & K typing, and patient samples were also subjected to screening & identification of RBC antibodies. We aimed to determine the prevalence of RBC antigens in thalassemia and sickle cell patients, as well as blood donors, at King Abdulaziz University Hospital and the frequency of alloimmunization in the selected patients.Results: The most commonly detected Rh-phenotype in donors was R1r (32.02%), followed by R1R1 (23.25%). Only 9.16% of donors were positive for the K antigen. The prevalence of Rh and K blood group antigens was also reported: the highest detected Rh-phenotype was R1r (40.86%) followed by R1R2 (24.04%) with only (6.25%) positive patients for K antigen. The rate of alloimmunization among sickle cell anemia and thalassemia patients was 39.42% and 35.57%, respectively. The highest specificity rates of the alloantibodies were recorded for anti-E and anti-K in both patient groups.Conclusion: The rate of alloimmunization in transfused patients was high and particularly observed against the Rh and K antigens. This study emphasizes the clinical need for typing patient RBCs prior to transfusion so as to provide phenotyped matched blood units and minimize the risks and associated morbidities of alloimmunization. Keeping a database of phenotyped blood donors is essential for the clinically effective and safe management of transfusion patients. [ABSTRACT FROM AUTHOR]

Background: Pregnant women are routinely screened for red blood cell (RBC) antibodies early in pregnancy. If RBC-alloantibodies are detected, repeated laboratory testing is advised to timely identify pregnancies at high risk for severe hemolytic disease of the fetus and newborn (HDFN). We assessed for RBC alloantibodies, other than anti-D or anti-K, cut-offs for the titer and the antibody dependent cellular cytotoxicity (ADCC) test to select high-risk cases. To advise on test repeat intervals, and to avoid unnecessary testing, we evaluated the chance for exceeding the cut-offs for Rh antibodies other than anti-D, Jk, Fy, and S/s antibodies.Study Design and Methods: Diagnostic value of antibody titer and ADCC test was determined with data from a prospective index-cohort study, conducted in 2002-2004. Laboratory test outcomes were from a recent observational cohort (2015-2016).Results: A titer cut-off of ≥16 showed a sensitivity of 100% (95% CI:73-100%) and a positive predictive value (PPV) of 17% (95% CI:14%-20%). The percentage of pregnancies reaching a titer above the cut-off of ≥16 varied from 0% for anti-Jka /Jkb (n = 38) to 36% for anti-c (n = 97). The ADCC test showed no cut-off with a 100% sensitivity. However, in cases with a titer ≥16 and an ADCC test ≥30% a PPV of 38% was obtained to detect severe HDFN.Conclusion: A titer cut-off of ≥16 is adequate to detect all cases at risk for severe HDFN; the ADCC test may add a more accurate risk estimation. Repeated testing is recommended in pregnancies with anti-c. In pregnancies with other Rh antibodies a repeated test in the third trimester is recommended. [ABSTRACT FROM AUTHOR]

Background: Isohemagglutinins against ABO antigens absent on both recipient and donor red blood cells (RBCs) increase or decrease after ABO-incompatible hematopoietic stem cell transplantation (HSCT). However, few reports have described the changes in the isohemagglutinin titers and the characteristics in patients with recurrent hematologic conditions after ABO-incompatible HSCT.Case Report: A 59-year-old female with acute erythroid leukemia received a peripheral blood stem cell transplant from her HLA-haploidentical daughter. The patient was typed as group O with anti- A (4+) and B (4+) isohemagglutinins, while the donor was typed as group B. The bone marrow cells achieved complete donor cell chimerism on Day 13 after HSCT. On Day 120, the patient showed 97% B RBC type with persistent anti-A (3+) and without anti-B antibodies. On Day 375, her leukemia relapsed, and recipient type O RBCs and anti-B antibodies sequentially reemerged. However, clinicolaboratory hemolysis and erythroid aplasia were not detected in the patient.Results: The post-HSCT sera agglutinated the allo B RBCs, but not the donor B RBCs, while the pre-HSCT sera agglutinated both RBCs. The burst-forming/colony-forming units of erythroid formation from the donor peripheral blood stem cells were impaired by only the pre-HSCT sera and not by the post-HSCT sera.Conclusion: To our knowledge, this is the first report investigating the characteristic changes of isohemagglutinins between the pre- and post-HSCT sera in a patient with recurrent acute myeloid leukemia. The present study suggests that the plasma cells producing anti-donor B RBCs in the patient have been selectively eliminated or induced into an anergic state by the post-HSCT immunologic reconstruction. [ABSTRACT FROM AUTHOR]

Background: The ABO transplantation barrier can be breached if antibody is reduced to low levels. Current serologic methods involve testing natural RBCs against dilutions of plasma to determine antibody levels, but these methods are poorly standardized and inherently error prone with consequent large inter- and intra laboratory variation. We evaluated the feasibility of using antigen-standardized kodecytes and undiluted plasma as an alternative method for antibody measurement in patients preparing for ABO-incompatible kidney transplantation.Study Design and Methods: A panel of five kodecytes, bearing defined levels of synthetic blood group A type 2 antigen was developed (kodecyte assay) to show reaction patterns against undiluted plasma that were indicative of anti-A and anti-A,B levels. This panel was evaluated against the contemporary method of testing dilutions of plasma against A1 cells to determine titer (A1 cell assay) in both column agglutination and tube techniques. Evaluation samples included reference standards, 102 group O plus 23 group B donors, and 40 pre- and post-plasmapheresis samples from five prospective ABO-incompatible kidney transplant patients.Results: Comparisons between the kodecyte and A1 cell assays found greater than 90% correlation for all samples. Tube and column agglutination technology platform differences were observed with A1 cells and kodecytes. Discordant samples were generally found to have high ratios of IgG:IgM or vice versa.Conclusions: The kodecyte assay is a simple method that requires no sample dilution, and an optimized two-cell kodecyte panel is potentially capable of informing ABO-incompatible kidney transplantation decisions based on antibody levels. [ABSTRACT FROM AUTHOR]

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Background: Current national standards for pretransfusion testing do not address the frequency or optimal time interval to repeat antibody identification testing for patients in whom antibodies have been previously detected.Study Design and Methods: A retrospective review was performed of patients with existing red blood cell (RBC) antibodies who subsequently developed new antibody specificities. Data were drawn from a single institution where the antibody investigation was repeated if the screen suggested a new antibody or if 14 days had elapsed since the previous investigation. Clinically insignificant or drug-dependent antibodies were excluded. Among cases in which new antibodies were detected within 30 days of a previous sample that already demonstrated existing antibodies, the median and lower 95% confidence intervals for the number of days between the detection of the existing and new antibodies were determined.Results: Over a 9-year period, among 2114 patients with more than 1 antibody, 699 (33%) had serially detected antibodies from separate samples. Among 152 patients whose subsequent antibody was detected within 30 days of the existing antibodies, the median time interval to detection of the new antibody was 13 days. The lower 95% confidence interval was 1 day. By Day 3, 18% of the new antibodies had already appeared.Conclusion: In patients who form multiple antibodies, the serial emergence of clinically significant antibodies is common. In some patients, detection of a new specificity occurs in a sample drawn shortly after the sample that demonstrated the first antibody. These results have implications for the frequency of pretransfusion testing. [ABSTRACT FROM AUTHOR]

Background: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates.Study Design and Methods: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test.Results: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species.Conclusion: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products. [ABSTRACT FROM AUTHOR]

Background: Little information exists on red blood cell (RBC) alloimmunization in healthy US blood donors, despite the potential significance for donors themselves, blood recipients, and the blood center.Study Design and Methods: Donor/donation data were sourced from the Recipient Epidemiology and Donor Evaluation Study-III, which contains information from four US blood centers during 2012 through 2016. Multivariable logistic regression was used to assess prevalence of positive antibody screen by donor demographics, blood type, parity, and transfusion history.Results: More than 2 million units were collected from 632,378 donors, with 0.51% of donations antibody screen positive and 0.77% of donors having at least one positive antibody screen. The most common antibody specificities were D (26.4%), E (23.8%), and K (21.6%). Regression analysis indicated that increasing age, female sex, D-negative status, and history of transfusion and pregnancy were positively associated with a positive antibody screen. Prior transfusion history was most strongly associated with a positive antibody screen, with donors reporting a prior transfusion having a higher adjusted odds ratio (3.9) of having a positive antibody screen compared to donors reporting prior pregnancy (adjusted odds ratio, 2.0). Though transfusion was a more potent immune stimulus for RBC alloantibody formation than pregnancy, the sheer number of previously pregnant donors contributed to pregnancy being a risk factor for the majority of clinically significant RBC alloantibodies detected in females.Conclusion: These findings on prevalence of and risk factors for RBC antibodies may have implications for future medical care of donors and for operations at blood centers. [ABSTRACT FROM AUTHOR]

Background: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens.Study Design and Methods: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry.Results: Serological weak D and normal C + c- E- e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively.Conclusion: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression. [ABSTRACT FROM AUTHOR]

Background: Immunoglobulin replacement therapy is a cornerstone of the treatment of primary immunodeficiencies. Preparations used for replacement therapy are processed by purifying immunoglobulins from large pools of plasma, which were obtained from healthy donors. The constituent antibodies in these products depend on the immune history of the donor pool as well as manufacturing processes that differ among manufacturers. For these reasons various methods have been proposed to examine the levels and function of antibodies to organisms such as Streptococcus pneumoniae, which frequently causes infections in patients with immunodeficiencies. Pneumococcal antibody levels or antibody function can be measured with enzyme-linked immunosorbent assay (ELISA) or multiplexed opsonophagocytosis assay (MOPA). Although these assays were developed initially to assess the immunogenicity of pneumococcal vaccines, the techniques have been adapted to evaluate immunoglobulin products as well.Study Design and Methods: This article provides a concise review of the analytic techniques for measuring pneumococcal antibodies and prior studies of immunoglobulin products utilizing these methods.Results: Studies utilizing these assays have demonstrated that antibody levels of immunoglobulin products can vary with time, location, and manufacturer.Conclusions: We highlight current issues and future considerations concerning measurement of pneumococcal antibodies in immunoglobulin products, and the assays used for this purpose. [ABSTRACT FROM AUTHOR]

Background: Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied.Study Design and Methods: RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry.Results: RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors.Conclusion: The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway-mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage. [ABSTRACT FROM AUTHOR]

Background: The severe forms of thalassemia are the most common inherited anemias managed with regular blood transfusion therapy. Transfusion policies and complications are critical to quality of life and survival, but there is a lack of standardized care.Study Design and Methods: A survey of 58 items was completed in 2016 by 11 centers in California, Washington, Oregon, Nevada, and Arizona providing long-term care for thalassemia. The questionnaire addressed demographic information, transfusion practices and complications, and educational needs.Results: The centers followed 717 patients with β-thalassemia (314, 43.8%) or α-thalassemia (394, 55%). One-third (34.7%) of patients were transfusion-dependent. Indications and goals of transfusion therapy differed between centers. Prestorage leukoreduction was universal, while routine irradiation of units was limited to one site. Red blood cell antigen phenotype was determined before the first transfusion and patients received Rh/Kell-matched units. However, more than half of the transfused patients had received blood at multiple hospitals within or outside the United States. Alloantibodies were seen in 16.9% of transfused group, but management of such patients was variable. Unusual or emerging transfusion-transmitted pathogens were not observed. Multiple educational needs were recognized, with iron overload as the biggest challenge; the approach to iron chelation varied within the group.Conclusion: This study identified many patients not included in earlier surveys limited to major national centers, suggesting that the thalassemia population in the United States is vastly underestimated. Lack of evidence-based guidelines is a barrier to optimal care, which should be addressed through regional consortia of thalassemia centers. [ABSTRACT FROM AUTHOR]

Background: Antibody screening in pretransfusion tests is necessary to avoid critical complications of blood transfusion. Although red blood cells (RBCs) expressing relevant alloantigen(s) have been used for serologic antibody screening, little attention has been given to the use of cell lines, in which blood group antigen gene(s) are transduced, as reagent RBCs for antibody screening.Study Design and Methods: The use of an erythroid progenitor cell line for serologic tests was studied. The expression of blood group antigens of erythroid progenitor cells was analyzed by genotyping and flow cytometry. Serologic analysis including hemagglutination was performed using erythroid progenitor cells to evaluate their sensitivity for antibody detection. Overexpression of exogenous erythroid antigen by lentiviral transduction was carried out and investigated for antibody detection sensitivity.Results: Erythroid progenitor cells contained a substantial amount of hemoglobin and expressed sufficient levels of blood group antigens to detect corresponding monoclonal antibodies. Furthermore, the cell line could acquire an exogenous RBC antigen after lentiviral transduction and detected corresponding monoclonal and alloantibodies with equal sensitivity to antigen-positive RBCs.Conclusion: Application of erythroid progenitor cell lines for screening for unexpected antibodies could be helpful in solving issues such as reagent availability associated with the conventional RBC-based assay. The genetic expandability of erythroid progenitor cell lines by gene modification techniques could lead to the development of more convenient reagent RBCs. [ABSTRACT FROM AUTHOR]

Background: The FORS histo-blood group system was described in 2013 and much remains to be investigated regarding its genetic and immunohematologic characteristics, as well as its clinical importance. While presence of the c.887G>A-mutated GBGT1 gene, which results in FORS1 glycosphingolipid expression on human red blood cells (RBCs), is rare in the populations tested so far, naturally occurring anti-FORS1 in plasma appears common.Study Design and Methods: The Erythrogene database was utilized to probe genetic variation in GBGT1 among 2504 individuals in the 1000 Genomes Project. We screened 1108 Swedish blood donors for three principally important single-nucleotide polymorphisms (c.363C>A, c.886C>T, and c.887G>A) and selected samples were analyzed further. Screening for naturally occurring anti-FORS1 in plasma from 100 donors was performed using antigen-positive RBCs.Results: We identified 68 GBGT1 alleles, of which three were previously listed blood group alleles. Eight potential null alleles were observed, based on three different nonsense mutations. Four healthy donors were found homozygous for c.363C>A, which truncates the GBGT1-encoded Fs synthase prematurely. This is the first description of human knock-outs for GBGT1. The c.886C>T mutation that alters the same codon (p.Arg296Trp) changed by c.887G>A (p.Arg296Gln) was overexpressed to investigate if it induces the FORS1+ phenotype. However, c.886C>T did not result in synthesis of FORS1. We detected anti-FORS1 in 10% of all donors tested but none in the A1 or A1B groups.Conclusion: We have extended the knowledge of GBGT1 variants, allele frequencies, and the characteristics of naturally occurring antibodies in our newest carbohydrate blood group system, FORS. The finding of c.363C>A-homozygous donors indicates that GBGT1 is dispensable. [ABSTRACT FROM AUTHOR]

Background: Hemolytic disease of the fetus and newborn (HDFN) is caused by maternal antibodies against fetal red blood cell antigens, most often anti-D, -K, or -c. ABO incompatibility between mother and child and anti-D immunoprophylaxis (RhIG) are known to reduce the risk of D immunization and subsequent HDFN. However, no immunoprophylaxis has been developed to prevent non-D immunizations.Study Design and Methods: We evaluated whether ABO incompatibility has a preventive effect on formation of non-D alloantibodies, by performing a case-control study including pregnant women with newly detected non-D antibodies, identified within a nationwide data set, immunized during their first pregnancy and/or delivery. Subsequently, we assessed a possible protective effect of RhIG in a subgroup with non-Rh antibodies only. The proportions of previous ABO incompatibility and of RhIG administrations of these women were compared to the known rate of 19.4% ABO incompatibility and 9.9% RhIG administrations (D- women carrying a D+ child) in the general population of pregnant women.Results: A total of 11.9% of the 232 included immunized women had a possible ABO incompatibility in their first pregnancy (vs. expected 19.4%; 95% confidence interval [CI], 7.3-18.8; p = 0.036). Furthermore, 1.0% women with non-Rh antibodies were D-, delivered a D+ child, and had therefore received RhIG, whereas 9.9% was expected (95% CI, 0.18-5.50; p = 0.003).Conclusion: We found that ABO incompatibility and RhIG reduce the risks not only for D, but also for non-Rh immunizations, suggesting that antibody-mediated immune suppression in this condition is not antigen specific. [ABSTRACT FROM AUTHOR]

Background: Prevention of red blood cell (RBC) alloimmunization in patients with sickle cell disease (SCD) focuses on phenotypic RBC matching. We assessed alloimmunization among transfused patients with SCD after implementing leukoreduction and prophylactic antigen matching (PAM).Study Design and Methods: Retrospective review of transfusion and medical records for SCD patients 18 months to 81 years of age was performed covering two 5-year periods: Period 1, no PAM, occasional leukoreduction, and Period 2, consistent leukoreduction and extended PAM (Rh, Kell, S, Fy, Jk) for patients already alloimmunized. Patients transfused in Period 1 were excluded from Period 2.Results: A total of 293 patients were transfused in Period 1 and 183 in Period 2. Median time between first sample and last type and screen after transfusion was 2.12 years in Period 1 and 1.03 years in Period 2. Initial alloimmunization prevalence was lower in Period 2 (26.2%) versus Period 1 (37.5%) and after subsequent transfusions in Period 2 (23.8%) versus Period 1 (45.7%), although without significant difference after adjusting for number of units transfused, percentage of leukoreduced RBCs, sex, and age. Alloimmunized patients received more nonleukoreduced RBCs in Period 1 than nonalloimmunized. Patients transfused during inflammatory conditions were not significantly more likely to become alloimmunized.Conclusions: The prevalence of initial and subsequent RBC alloimmunization in Period 2 was lower than that in Period 1; however, overall prevalence remained high. We recommend leukoreduced, hemoglobin S-negative Rh and Kell PAM RBCs for transfusion of patients with SCD. Component and recipient factors affecting alloimmunization should be studied further. [ABSTRACT FROM AUTHOR]

Background: Sickle cell disease (SCD) patients undergo multiple red blood cell (RBC) transfusions and are regularly exposed to low-prevalence (LP) antigens specific to individuals of African descent. This study evaluated the prevalence of antibodies against LP antigens in SCD patients and the need to identify these antibodies in everyday practice.Study Design and Methods: Plasma from 211 SCD patients was tested with RBCs expressing the following LP antigens: RH10 (V), RH20 (VS), RH23 (DW ), RH30 (Goa ), KEL6 (Jsa ), and MNS6 (He).Results: Nine LP antibodies were found in eight patients (3.8%): five anti-RH23, two anti-RH30, and two anti-MNS6. The exposure risk, calculated for each LP antigen, was below 3% per RBC unit, for all antigens tested. Thus, in this cohort of transfused SCD patients, the prevalence of LP antibodies was similar to that of antibodies against antigens of the FY, JK, and MNS blood group systems. These findings also reveal the occurrence of anti-RH23 in SCD patients. No anti-RH20 or anti-KEL6 were found, despite the high frequency of mismatch situations.Conclusion: These results highlight the immunogenicity of these LP antigens, and the evanescence of antibodies against LP antigens. They also highlight the importance of appropriate pretransfusion testing for patients frequently transfused, who are likely to be exposed to multiple types of blood group antigens. [ABSTRACT FROM AUTHOR]

Background: Red blood cell (RBC) transfusions remain essential in the treatment of patients with sickle cell disease (SCD) and β-thalassemia. Alloimmunization, a well-documented complication of transfusion, increases the risk of delayed hemolytic transfusion reactions, complicates crossmatching and identifying compatible units, and delays provision of transfusions. Guidance is required to optimize the RBC product administered to these patients.Study Design and Methods: An international, multidisciplinary team conducted a systematic review and developed, following the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology, recommendations to assist treating physicians and transfusion specialists in their decision to select RBCs for these patients.Results: Eighteen studies (17 clinical studies and one cost-effectiveness study) were included in the systematic review. The overall quality of the studies was very low. In total, 3696 patients were included: 1680 with β-thalassemia and 2016 with SCD.Conclusion: The panel recommends that ABO D CcEe K-matched RBCs are selected for individuals with SCD and β-thalassemia, even in the absence of alloantibodies, to reduce the risk of alloimmunization. In patients with SCD and β-thalassemia who have developed clinically significant alloantibodies, selection of RBCs antigen negative to the alloantibody is recommended, if feasible. In these patients, selection of more extended phenotype-matched RBCs will likely reduce the risk of further alloimmunization. However, given the limited availability of extended phenotype-matched units, attention should be given to ensure that a delay in transfusion does not adversely affect patient care. [ABSTRACT FROM AUTHOR]

Background: D- individuals with previous D-incompatible pregnancies and/or blood transfusions, as well as those who are actively immunized with small-volume D+ red blood cells (RBCs), are stimulated to produce RhIG. Many factors could influence the stimulation of immunoglobulin production in response to foreign antigen (such as antigen immunogenicity and genetic factors), and it is unknown whether genetic markers could potentially identify responder anti-D donors.Study Design and Methods: Anti-D donors were assigned a responder profile based on their serum RhIG levels (n = 431). A subset of donors (n = 272) had DNA extracted for polymerase chain reaction genotyping assays for target genes in antigen presentation and pathogen recognition receptors (TLR2, TLR4, CD14, FcγRIIA, and the MHC Class II locus HLA-DRB1). Statistical tests for associations between anti-D donor responder profiles and genetic factors were performed.Results: A large proportion of our donors (38.7%) were classified as nonresponder donors, despite receiving multiple D+ RBC immunizations, whereas female sex was significantly associated with an all-responder profile (p < 0.001). The presence of the DRB1*15 allele and absence of the DRB1*04 allele were more likely to be associated with a responder anti-D donor, although not significantly after Bonferroni correction. A combination of the DRB1*15 allele and female sex was significantly associated with an anti-D donor responder profile.Conclusion: This study has identified female sex and the HLA-DRB1*15 allele as potentially useful markers that could be used to screen donors before entry into D immunization programs. [ABSTRACT FROM AUTHOR]

Background: Reported hepatitis E virus (HEV) antibody assay performance characteristics are variable. Using a subset of surplus US blood donation samples, we compared assays for detecting anti-HEV immunoglobulin M (Ig)M and IgG or total anti-HEV antibodies.Study Design and Methods: Samples from 5040 random blood donations, all HEV-RNA negative, collected primarily in the midwestern United States in 2015 were tested for anti-HEV IgM and IgG or total anti-HEV using assays manufactured by Diagnostic Systems, Wantai, and MP Biomedicals.Results: Overall, the percentage of detection for anti-HEV IgG and total anti-HEV was 11.4%, and for anti-HEV IgM was 1.8%. Nine samples were reactive for anti-HEV IgM by all assays, giving a recent infection rate of 0.18%. Anti-HEV IgG/total anti-HEV detection rates increased with age. Interassay agreement was higher among the IgG anti-HEV/total anti-HEV assays (84%) than the IgM assays (22%). Regression analyses of signal-to-cutoff ratios from IgG/total antibody assay were heteroskedastic, indicating no constant variance among these assays, suggesting they may detect different epitopes or were affected by waning or less avid antibodies in the US donor population.Conclusions: Although similar percentages of IgG anti-HEV/total anti-HEV detection were observed across the three commercial assays, each assay detected a unique sample subpopulation and was heteroskedastic when compared pairwise. Discordance was higher among anti-HEV IgM assays, but a recent HEV infection rate of at least 0.18% was estimated based on assay concordance. [ABSTRACT FROM AUTHOR]

Background: Although the subject of many previous studies, the importance of white blood cell (WBC) alloimmunization in granulocyte transfusion therapy has not been settled. In this study, we report the results of the effects of WBC antibodies in the RING (Resolving Infection in Neutropenia with Granulocytes) study, a randomized controlled trial comparing the efficacy of daily granulocyte transfusion therapy plus antimicrobials versus antimicrobials alone; the primary outcome results have been published previously.Study Design and Methods: One hundred fourteen subjects were enrolled in the study. Serum samples for WBC antibody determination were obtained from each subject at baseline and at 2 and 6 weeks. One hundred subjects had at least one antibody test result. Samples were tested for human leukocyte antigen (HLA) Class I and Class II antibodies as well as for granulocyte-specific antibodies using granulocyte agglutination and immunofluorescence techniques. All testing was performed at a central laboratory.Results: Baseline WBC alloimmunization was modest, depending somewhat on the assay. Seroconversion during the study was slightly higher in the granulocyte transfusion arm, but the differences were not statistically significant. There was no demonstrable effect of the presence of alloimmunization on the primary outcome (survival and microbial response at 42 days), the occurrence of transfusion reactions (either overall or pulmonary), or posttransfusion neutrophil increments.Conclusion: The presence or development of WBC antibodies had no demonstrable effect on any clinical aspect of granulocyte transfusion therapy. It appears that, at least in the patient population studied, there is no evidence suggesting need for concern about recipient WBC alloimmunization when prescribing granulocyte transfusions. [ABSTRACT FROM AUTHOR]

Background: Antibodies to Mia , MUT, and Mur are among the most frequently identified alloantibodies in Southeast Asia. Understanding the characteristics of these antibodies in terms of induction and evanescence would aid in optimizing methods for their detection.Study Design and Methods: Antibody testing results between the years 2013 and 2015 with relevant patient demographic data and red blood cell (RBC) transfusion history were retrieved. Cumulative alloimmunization incidence and evanescence to MUT and Mur were estimated by Kaplan-Meier analysis in relation to the number of RBC units transfused and time.Results: Of 70,543 selected patients, 6186 nonalloimmunized subjects with available antibody testing results posttransfusion were identified. Cumulative alloimmunization incidence for MUT increased from 0.12% (95% confidence interval [CI], 0.03-0.21) to 0.63% (95% CI, 0.25-1.01), while for Mur it increased from 0.04% (95% CI, 0-0.09) to 0.42% (95% CI, 0.05-0.79) when a patient was transfused 2 RBC units as compared to 12. Both antibodies had high evanescence rates and at 1 year, anti-MUT and -Mur will be detected in only 45% (95% CI, 35%-57%) and 27% (95% CI, 17%-43%), respectively, of previously positive patients. MUT and Mur immunogenicity was estimated to be 1.7 and 1.2 times higher than E when their rate of evanescence was taken into account.Conclusion: Antibodies to MUT and Mur develop following multiple RBC exposures. Immunogenicity of MUT/Mur and evanescence rates of the corresponding antibodies is higher compared to anti-E. Appropriate selection of antibody screening cells is needed in view of the high prevalence, immunogenicity, and evanescence of the antibodies. [ABSTRACT FROM AUTHOR]

Background: Influenza immune globulin, manufactured from plasma of convalescent or vaccinated donors has been proposed as a potential therapy for severe influenza. In 2009, a program was initiated to collect plasma from donors who self-identified as having had H1N1 influenza or having received the H1N1 pandemic vaccine. The goal of this study was to determine the efficiency of donor screening by self-identification without antibody testing, and to evaluate demographic predictors of high-titer donations.Study Design and Methods: Plasma samples from self-identified or control donors were randomly selected to evaluate hemagglutination inhibition (HAI) antibody responses. HAI titers were correlated with donor age, gender, location, and influenza exposure history.Results: Both self-identified vaccinated and convalescent donor groups had higher geometric mean titers (GMTs) against A/California/07/2009 (H1N1) virus compared to the control donors (39.9, 24, and 8.5, respectively). The proportion of samples with titers ≥64 in vaccinated, convalescent, and control donors were 54%, 37%, and 10%, respectively. Donations with titers ≤16 were predominant in control donors (80%) and substantial in convalescent (47%) and vaccinated (40%) donors. Titers did not correlate with donor age, gender, or geographical location. GMTs for vaccinated donors were significantly higher than for convalescent donors and in both groups significantly higher than in the control.Conclusion: Targeted collection of plasma containing high levels of anti-influenza antibodies from self-identified donors was effective, but could be further improved by reducing the number of low-titer donations. More selective donor screening and/or testing for influenza antibodies could increase the potency of an influenza antibody-rich immune globulin (FLUIGIV). [ABSTRACT FROM AUTHOR]

Background: Neonatal alloimmune neutropenia results from maternal alloimmunization to human neutrophil antigens. The alloantibodies involved in neonatal alloimmune neutropenia are against human neutrophil antigens HNA-1a, HNA-1b, HNA-1c, HNA-1d, HNA-2, HNA-3a, HNA-4a, HNA-4b, and HNA-5a; however, to date, antibodies specific to HNA-3b have not been reported.Study Design and Methods: Blood samples from 10,000 unselected neonates were analyzed, resulting in the selection of 88 neutropenic newborns (neutrophil count <1.5 × 109 /L) from 83 mothers (three pairs of twins and one triplet). HNA-3 genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism to identify the cases of maternal-fetal HNA-3 incompatibility. Serologic studies for detecting maternal HNA-3 alloantibodies were performed with the granulocyte agglutination test, the white blood cell immunofluorescence test, and a LABScreen Multi-HNA Kit.Results: Genotyping studies identified 13 of 88 (14.8%) instances of maternal-fetal HNA-3 incompatibility, with all mothers typed as HNA-3a/a and neonates typed as HNA-3a/b. Serologic studies revealed that five of 13 (38.5%) mothers carried anti-HNA-3b plus human leukocyte antigen antibodies and that three of 13 (23.1%) mothers had anti-HNA-3b without human leukocyte antigen antibodies.Conclusion: Here, we report the first three cases of neonatal alloimmune neutropenia associated with HNA-3b antibodies resulting in a neonatal alloimmune neutropenia incidence of one in 3333 live births. [ABSTRACT FROM AUTHOR]

Background: β-Thalassemia is a common hemoglobinopathy in the Arabian Peninsula. Red blood cell (RBC) transfusion is a cornerstone for its management, but can create significant challenges including RBC alloimmunization. Herein, we examine alloimmunization risk factors in Omani patients with transfusion-dependent β-thalassemia. Existing literature is summarized.Study Design and Methods: A retrospective review of all patients attending our center over 25 years was performed. Clinical and transfusion records were examined. Chi-square test was used to assess the association between the categorical variables. Nonparametric Mann-Whitney test was used to assess the association between transfusion and risk of alloimmunization.Results: A total of 268 patients were identified (168 adults and 100 pediatrics), of whom 226 are alive (84.3%). Males accounted for 53.4%. The cohort had a median age of 22 years (range, 2-43 years). The most common blood group was O+ (39%). The prevalence of alloimmunization was 9.3% with anti-E (24%) and anti-K (24%) being the commonest antibodies identified. There was a significant association between age and alloimmunization, with 68% of alloimmunized patients in the age group of 19 to 30 years (p < 0.01). Among adults, there was a significant association between alloimmunization and number of units transfused (p = 0.001). There was no association between alloimmunization and sex or history of splenectomy.Conclusion: Our study shows an association between alloimmunization and the age of the patients and number of units transfused. Transfusion support of this group of patients necessitates the availability of needed expertise and blood bank facilities. [ABSTRACT FROM AUTHOR]

Background: The successful introduction of prophylaxis with anti-RhD immunoglobulin has resulted in a significant decline of pregnancy-related RhD immunizations but also has decreased the availability of naturally immunized women as (new) anti-D donors. An influx of new donors is necessary to maintain a sufficient pool of anti-D donors. We investigated motivators, barriers, and predictors for anti-D donorship in RhD-immunized women.Study Design and Methods: A mixed-methods design was applied, including focus group discussions and questionnaires. Two focus groups (including 11 women) served as input for the questionnaire.Results: In total, 47.6% of 750 anti-D donors and potential donors completed the questionnaire (50.4% donors; 38% nondonors; 11.6% former donors). Almost 70% of the nondonors would have become donors if they had known about the possibility. Travel time investment was reported as a disadvantage; one-half of donors mentioned no disadvantages. Motivators for anti-D donorship were "doing something in return" (31.2%) and "preventing others having a sick child or losing a child" (33.9%). In multivariable analysis, living single (odds ratio, 5.8; p = 0.02) and living partnered without resident children (odds ratio, 7.9; p = 0.03), compared with living partnered with children, were predictors for anti-D donorship. Not being registered as an organ donor (odds ratio, 0.25; p < 0.001) predicted that the individual would not be an anti-D donor.Conclusion: The main barrier for anti-D donorship was a lack of knowledge. Positive predictors of anti-D donorship were living without resident children, altruism, and being registered as an organ donor. A blood bank should develop targeted recruitment strategies with a focus on spreading knowledge about anti-D donorship among RhD-immunized women. [ABSTRACT FROM AUTHOR]

Background: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder associated with multiple arteriovenous malformations. HHT patients may require red blood cell (RBC) transfusion due to spontaneous hemorrhage or surgical bleeding. Because HHT-associated hemorrhage often occurs in submucosa we hypothesized that RBC alloimmunization rates in HHT patients may be higher than those observed in other transfused patients and investigated this in a retrospective study.Study Design and Methods: Eighty-five patients with HHT who were transfused at our tertiary care facility were identified. A group of randomly selected, chronically transfused patients without HHT were used as controls (n = 207). RBC transfusion and alloantibody data were extracted from medical records.Results: Alloimmunization rates among patients with HHT were significantly higher than those of controls (15.29% vs. 2.42%; p < 0.001), while HHT patients received fewer RBC transfusions at our institution compared to controls (4.27 units vs. 8.84 units; p < 0.0001). Anti-E, -K, and -c were the three most common alloantibodies identified in HHT patients. There was a trend for HHT patients to make more antibodies per alloimmunized patient than controls (2.38 alloantibodies vs. 1.60 alloantibodies), although this difference was not significant (p = 0.37).Conclusion: To our knowledge, this is the first study to evaluate RBC alloimmunization rates in patients with HHT. It is unclear whether the high alloimmunization rates observed are due to lifetime transfusion burden, underlying disease pathophysiology, or other variables. Additional studies are needed to evaluate the generalizability of our findings to other HHT populations and to consider the utility of prophylactic antigen matching for C/E/K. [ABSTRACT FROM AUTHOR]

Background: In addition to titration by indirect antiglobulin test most widely used, anti-D quantitation by continuous-flow analysis (CFA) may be performed to assess severity of maternal immunization. Only five studies have reported its added value in the management of pregnancies complicated by anti-D immunization.Study Design and Methods: A retrospective study of 74 severe anti-D-immunized pregnancies was conducted from January 1, 2013, to December 31, 2014, in the Trousseau Hospital in Paris (France). Concentration of maternal anti-D was measured by titration and by CFA two-stages method (2SM; total amount of anti-D) and one-stage method (1SM; high-affinity IgG1 anti-D). These biologic data were analyzed according to the severity of the hemolytic disease of the fetus and the newborn.Results: The value of 5 IU anti-D/mL in maternal serum is validated as a threshold to trigger ultrasonographic and Doppler fetal close follow-up. A high 1SM/2SM ratio was associated with a higher risk of intrauterine transfusion (IUT). For pregnancies requiring IUT and without increasing titer, maternal 1SM anti-D concentration tends to correlate with the precocity of fetal anemia. In the "without-IUT" group 1SM and 2SM anti-D concentrations correlate significantly with cord bilirubin levels of the newborn at birth.Conclusion: Altogether our results underline the importance of anti-D quantitation by CFA to optimize the management of anti-D-alloimmunized pregnancies. [ABSTRACT FROM AUTHOR]