Chimerism and mosaicism shape our physical constitution and impact medical conditions.

ABO blood group discrepancies in healthy individuals were caused by body-wide chimerism and mosaicism. They can be evaluated with new diagnostic options for disease-related cell clones that are typically associated with mosaicism. The observations raise the attention for sporadic mixed-field observations of any blood group antigen. Commentary on: Dauber et al. Body-wide chimerism and mosaicism are predominant causes of naturally occurring ABO discrepancies. Br J Haematol 2024 (Online ahead of print). doi:10.1111/bjh.19618.

Cumulative donor-specific antibody threshold predicts platelet transfusion response in HLA-alloimmunized patients.

ABSTRACT: Up to a third of patients with hemato-oncologic conditions who have received multiply transfusions develop immune-mediated platelet transfusion refractoriness. Yet factors that influence posttransfusion platelet corrected count increments (CCI) in patients with HLA-alloimmune platelet transfusion refractoriness remain less well elucidated. Recent advances in HLA antibody characterization using fluorescent bead-based platforms enable the study of donor-specific antibody (DSA) avidity (as measured by mean fluorescence intensity [MFI]) and its impact on HLA-alloimmune platelet transfusion refractoriness. In this large retrospective study of 2012 platelet transfusions among 73 HLA-alloimmunized patients, we evaluated the impact of cumulative HLA DSA-MFI alongside other donor, platelet component, and patient characteristics on CCI at 2 and 24 hours after transfusion. As part of a quality improvement initiative, we also developed and tested a computerized algorithm to optimize donor-recipient histocompatibility based on cumulative DSA-MFI and sought other actionable predictors of CCI. In multivariate analyses, cumulative HLA DSA-MFI of ≥10 000, major/bidirectional ABO-mismatch, splenomegaly, transfusion reactions, and platelet storage in additive solution negatively affected 2-hour but not 24-hour posttransfusion CCI. The DSA-MFI threshold of 10 000 was corroborated by greater antibody-mediated complement activation and significantly more CCI failures above this threshold, suggesting the usefulness of this value to inform "permissive platelet mismatching" and to optimize CCI. Furthermore, DSA-MFI decreases were deemed feasible by the computer-based algorithm for HLA-platelet selection in a pilot cohort of 8 patients (122 transfusions) evaluated before and after algorithm implementation. When HLA-selected platelets are unavailable, ABO-identical/minor-mismatched platelet concentrates may enhance 2-hour CCI in heavily HLA-alloimmunized patients with platelet transfusion refractoriness.

40 years of researching the Del phenotype results in a change of transfusion practice.

Anti-D cannot agglutinate red cells of any Del phenotype in routine serology. Many individuals with East Asian ancestry who type D-negative in serology harbor a Del phenotype. Almost all such individuals carry one distinct DEL variant, dubbed Asian-type DEL, known as RHD*01EL.01, RHD*DEL1, RHD:c.1227G>A, formerly known as RHD(K409K). Clinical evidence strongly suggests that Asian-type DEL individuals can safely be transfused with RhD-positive blood and do not need anti-D prophylaxis in pregnancy.

CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

BACKGROUND: CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. METHODS: We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. RESULTS: Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. DISCUSSION: We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

Consideration for alpha-gal syndrome in two critically ill persons with group O blood who received group B plasma.

BACKGROUND: The U.S. Centers for Disease Control and Prevention (CDC) has reported increasing rates of alpha-gal syndrome, an allergic response after meat ingestion (AGS). AGS has been associated with prior exposure to tick bites or other biologics characterized by a life-threatening immunoglobulin E (IgE)-mediated hypersensitivity to galactose-alpha-1,3-galactose (alpha-gal) an oligosaccharide structurally similar to the group B antigen on red blood cells (RBC) found in most non-primate mammalian meat and products derived from these mammals. In 2023, Transfusion reported 3 group O recipients of group B plasma in the Washington, D.C. metropolitan area with no history of meat allergy who had anaphylactic transfusion reactions compatible with AGS. AIMS: We investigated allergic reactions in 2 additional patients who received ABO minor-incompatible blood products at 2 hospitals in the D.C. area during fall 2023. METHODS: For both patients, a medical chart review was performed and IgE levels to alpha-gal were measured. RESULTS: The first patient, a 64-year-old, O-positive patient status post heart transplant with no known allergies, was admitted with acute COVID-19 induced antibody-mediated transplant rejection and placed on extracorporeal membrane oxygenation (ECMO). While undergoing plasma exchange (PLEX) (50% albumin/50% fresh frozen plasma (FFP)), the patient tolerated 2 units of group O FFP and 1 unit of group A FFP before becoming hemodynamically unstable during transfusion of 1 unit of B-positive FFP. PLEX was stopped. The patient later died of sepsis from underlying causes. The second patient, a 57-year-old O-positive man with a history of melanoma and neuro fibromatosis type 1, was undergoing an abdominal resection including transfusion of 3 units of O-positive RBC when he suffered hypotension and ventricular tachycardia requiring intraoperative code after receiving 2 units of group B FFP. Hiveswere noted after resuscitation. The patient had a history of tick bites but no known allergies. He is alive 5 months after the possible allergic event. Both patients had full transfusion reaction evaluations and immunology testing results above the positive cutoff for anti-alpha-gal IgE. DISCUSSION AND CONCLUSION: Two patients with O-positive blood and no known allergies experience danaphyl axis after transfusion with group B FFP. The symptoms cannot definitively be imputed to an allergic transfusion reaction, but the presence of IgE against alpha-gal supports an association. Medicating patients with antihistamines and IV steroids pre-transfusion may prevent allergic reactions. Restricting group B plasma-containing products (plasma, platelets, cryoprecipitate) for patients who experience AGS-like symptoms may be considered.

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

BACKGROUND: Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. STUDY DESIGN AND METHODS: A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. RESULTS: The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. DISCUSSION: The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

DNA Reference Reagents for Genotyping RH Variants.

Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation.

Clinical Features and Immunogenetic Risk Factors Associated With Additional Autoantibodies in Anti-Transcriptional Intermediary Factor 1γ Juvenile-Onset Dermatomyositis.

OBJECTIVE: Novel autoantibody specificities including anti-CCAR1 were recently discovered in adult patients with anti-transcriptional intermediary factor (TIF1)-positive dermatomyositis (DM) and were associated with attenuated cancer emergence. The aims of the present study were to examine whether these autoantibodies occur in patients with juvenile-onset DM (JDM) and to determine their associated features. METHODS: Sera from 150 patients with anti-TIF1γ autoantibody-positive JDM in a cross-sectional cohort and 90 juvenile healthy controls were assayed for anti-CCAR1, anti-C1Z1, anti-IMMT, anti-TBL1XR1, and anti-Sp4 autoantibodies. Demographics, myositis autoantibodies, clinical features, medications, outcomes, and HLA-DRB1 and HLA-DQA1 alleles were compared between those with and without these autoantibodies. RESULTS: Any one of the anti-TIF1γ-associated autoantibodies was present in 44 patients (29%) overall, including 25 (17%) with anti-Sp4, 22 (15%) with anti-TBL1XR1, 14 (9%) with anti-CCAR1, 2 (1%) with anti-C1Z1, and 2 (1%) with anti-IMMT autoantibodies. These anti-TIF1γ-associated autoantibodies frequently co-occurred. Patients with any of the anti-TIF1γ-associated autoantibodies had less frequent falling (34% [15] vs. 53% [56], P = 0.032) and lower peak muscle enzymes. None of the patients had cancer. Among White patients, HLA-DRB1*03 was protective against an anti-TIF1γ-associated autoantibody (odds ratio 0.20, 95% confidence interval 0.07-0.52). CONCLUSION: Autoantibodies associated with anti-TIF1γ were found in isolation and in combination among a subset of patients with JDM. Patients with these autoantibodies had less severe muscle disease and were not enriched for HLA-DRB1*03. Additional autoantibodies among patients with positive anti-TIF1γ with JDM likely contribute to the heterogeneity of the anti-TIF1γ serologic subgroup.

Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165.

BACKGROUND: An original methodology for determining the D antigen density on red cells was published in 2000 and has been applied in many publications since. This flow cytometry-based assay remained largely unrevised utilizing monoclonal anti-Ds that are not readily available anymore. We updated the methodology to quantify erythrocyte D antigen sites using microspheres and monoclonal anti-Ds that are commercially available today. METHODS: The absolute D antigen density of a frozen standard CcDEe cell, drawn in 2003, a fresh blood donation from the same individual, drawn in 2022, and an internal control CcDEe cell, was quantified by flow cytometry using fluorescence-labeled microspheres. The internal control CcDEe cell was used in conjunction with 9 commercial anti-Ds to determine D antigen densities of 7 normal D, 4 partial D, and 11 weak D type samples, including 2 novel alleles. RESULTS: The reproducibility of the updated assay was evaluated with red cells of published D antigen densities. The current results matched the known ones closely. The new weak D types 164 and 165 carried 4500 and 1505 D antigens/red cell, respectively. The absolute D antigen density decreased from 27,231 to 26,037 in an individual over 19 years. DISCUSSION: The updated assay gave highly reproducible results for the D antigen densities of Rh phenotypes. Readily available anti-Ds allowed for the determination of the D antigen densities of 7 weak D types. The assay is suitable to evaluate the effects of distinct amino acid substitutions on the RhD phenotype.

Breakpoint regions of an RHD-CE(4-9)-D allele and a rare JK allele in a Pacific Islander individual.

BACKGROUND: Among 710 RHD alleles, 3 alleles have been shown to express CcEe antigens and, among 67 hybrid alleles of the RHD gene, 2 alleles have evolved to include RHCE exons 4-9. No breakpoint region had been described for such RHD-CE(4-9)-D hybrid alleles. In the Kidd blood group system, the JK*02N.01 null allele is found with high prevalence in the Polynesian population. We investigated a self-identified Pacific Islander with discrepant serologic and molecular results for his C and Jkb antigens. Another 8 samples with genotype-phenotype discrepancies in the Kidd blood group system were assessed. MATERIALS AND METHODS: A combination of published molecular methods and commercial kits were applied to analyze the RHD, RHCE, and SLC14A1 gene sequences, as were hemagglutination tests to determine the serologic phenotypes. RESULTS: Nucleotide sequencing of the RHD gene in the index case, including relevant intronstretches, and cDNA identified an RHD-CE(4-9)-D hybrid allele. Nucleotide sequencing of his RHCE gene confirmed the presence of 2 RHCE*ce alleles despite expressing the C antigen. Sequencing of his SLC14A1 gene documented the JK*02N.01 null allele. In the other 8 samples, 5 previously known SLC14A1 nucleotide substitutions were identified. The JK*02N.17 allele was determined to be Jkb-positive. DISCUSSION: We determined the 2 breakpoint regions of his RHD-CE(4-9)-D hybrid allele, which was likely distinct from the 2 previously published hybrid alleles due to the differences in the linked RHCE allele. His RHD variant was shown to express the C antigen. An SLC14A1 substitution was underlying his unexpected Jkb-negative phenotype. In a quality improvement project, we resolved 8 samples with similarly discrepant results between Jk serology and red cell genotyping.

Asian-type DEL (RHD*DEL1) with an allo-anti-D: A paradoxical observation in a healthy multiparous woman.

BACKGROUND: The DEL phenotype is the D variant expressing the least amounts of D antigen per red cell. Asian-type DEL (RHD:c:1227G > A) is the most prevalent DEL in East Asia without any anti-D alloimmunization reported before. We investigated the first observation of an anti-D in any DEL phenotype, reported in the Japanese language at a 1987 conference, only 3 years after the discovery of DEL. METHODS: We contacted the proband 35 years after the initial report. Standard hemagglutination, adsorption/elution, and flow cytometry tests were performed, as was nucleotide sequencing for the RHD, RHCE, and HLA class I and class II genes. RESULTS: The healthy multiparous Japanese woman, a regular blood donor, still had the anti-D of titer 8 representing an alloantibody by standard serologic methods. Unexpectedly, she carried an Asian-type DEL without any additional RHD gene variation. All 12 HLA alleles identified were known in the Japanese population. Interestingly, one of her HLA-DRB1 and a variant of her HLA-DQB1 alleles had previously been associated with anti-D immunization. CONCLUSION: We described an allo-anti-D, maintained for more than three decades, in an Asian-type DEL. The combination of two implicated HLA alleles were rare and could have contributed to the anti-D immunization. Continued monitoring of anti-D immunization events in patients with DEL is warranted, and we discuss possible mechanisms for further study. As only this single observation has been recognized in the last 35 years, the current recommendation is affirmed: Individuals with Asian-type DEL should be treated as Rh D-positive for transfusion and Rh immune prophylaxis purposes.

Primary oxidative phosphorylation defects lead to perturbations in the human B cell repertoire.

Introduction: The majority of studies on oxidative phosphorylation in immune cells have been performed in mouse models, necessitating human translation. To understand the impact of oxidative phosphorylation (OXPHOS) deficiency on human immunity, we studied children with primary mitochondrial disease (MtD). Methods: scRNAseq analysis of peripheral blood mononuclear cells was performed on matched children with MtD (N = 4) and controls (N = 4). To define B cell function we performed phage display immunoprecipitation sequencing on a cohort of children with MtD (N = 19) and controls (N = 16). Results: Via scRNAseq, we found marked reductions in select populations involved in the humoral immune response, especially antigen presenting cells, B cell and plasma populations, with sparing of T cell populations. MTRNR2L8, a marker of bioenergetic stress, was significantly elevated in populations that were most depleted. mir4485, a miRNA contained in the intron of MTRNR2L8, was co-expressed. Knockdown studies of mir4485 demonstrated its role in promoting survival by modulating apoptosis. To determine the functional consequences of our findings on humoral immunity, we studied the antiviral antibody repertoire in children with MtD and controls using phage display and immunoprecipitation sequencing. Despite similar viral exposomes, MtD displayed antiviral antibodies with less robust fold changes and limited polyclonality. Discussion: Overall, we show that children with MtD display perturbations in the B cell repertoire which may impact humoral immunity and the ability to clear viral infections.

Association of anti-TPM4 autoantibodies with vasculopathic cutaneous manifestations in juvenile dermatomyositis.

OBJECTIVES: AECAs are detected in multiple forms of vasculitis or vasculopathy, including JDM. High levels of tropomyosin alpha-4 chain (TPM4) gene expression in cutaneous lesions and TPM4 protein expression in some endothelial cells (ECs) have been proven. Furthermore, the presence of autoantibodies to tropomyosin proteins have been discovered in DM. We therefore investigated whether anti-TPM4 autoantibodies are an AECA in JDM and are correlated with clinical features of JDM. METHODS: The expression of TPM4 protein in cultured normal human dermal microvascular ECs was investigated by Western blotting. Plasma samples from 63 children with JDM, 50 children with polyarticular JIA (pJIA) and 40 healthy children (HC) were tested for the presence of anti-TPM4 autoantibodies using an ELISA. Clinical features were compared between JDM patients with and without anti-TPM4 autoantibodies. RESULTS: Autoantibodies to TPM4 were detected in the plasma of 30% of JDM, 2% of pJIA (P < 0.0001) and 0% of HC (P < 0.0001). In JDM, anti-TPM4 autoantibodies were associated with the presence of cutaneous ulcers (53%; P = 0.02), shawl sign rash (47%; P = 0.03), mucous membrane lesions (84%; P = 0.04) and subcutaneous edema (42%; P < 0.05). Anti-TPM4 autoantibodies significantly correlated with the use of intravenous steroids and IVIG therapy in JDM (both P = 0.01). The total number of medications received was higher in patients with anti-TPM4 autoantibodies (P = 0.02). CONCLUSION: Anti-TPM4 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies. Their presence correlates with vasculopathic and other cutaneous manifestations of JDM that may be indicative of more refractory disease.

Autoantibodies Recognizing Specificity Protein 4 Co-occur With Anti-Transcription Intermediary Factor 1 and Are Associated With Distinct Clinical Features and Immunogenetic Risk Factors in Juvenile Myositis.

OBJECTIVE: Autoantibodies recognizing specificity protein 4 (Sp4) were recently discovered in adults with idiopathic inflammatory myopathies (IIM). Anti-Sp4 autoantibodies co-occurred in patients with anti-transcription intermediary factor 1 (anti-TIF1) autoantibody-positive dermatomyositis (DM) and were associated with a reduced risk of cancer. In the present study, the prevalence and clinical features associated with anti-Sp4 autoantibodies in juvenile-onset IIM were investigated. METHODS: Serum samples from 336 patients with juvenile myositis in a cross-sectional cohort and 91 healthy controls were screened for anti-Sp4 autoantibodies using enzyme-linked immunosorbent assay. Clinical characteristics, outcomes, and HLA alleles of those with and those without anti-Sp4 autoantibodies were compared. RESULTS: Anti-Sp4 autoantibodies were present in 23 patients (7%) with juvenile myositis and were not present in any of the controls. Anti-Sp4 autoantibodies were found among each clinical myositis subgroup. The frequency of TIF1 autoantibody positivity was significantly higher among those with anti-Sp4 autoantibodies (21 [91%] versus 92 [30%], P < 0.001). In the anti-TIF1 autoantibody-positive subgroup, Raynaud's phenomenon (8 [38%] versus 2 [2%], P < 0.001) was more common and peak aspartate aminotransferase was significantly lower in those with anti-Sp4 autoantibodies. None of the patients with anti-Sp4 autoantibodies required a wheelchair. Among White patients, DQA1*04 and DRB1*08 were associated with anti-Sp4 autoantibodies. CONCLUSION: Anti-Sp4 autoantibodies were found in patients with juvenile-onset IIM, predominantly those with coexisting anti-TIF1 autoantibodies. Patients with anti-Sp4 autoantibodies represent a phenotypic subset of anti-TIF1 autoantibody-positive myositis characterized by frequent Raynaud's phenomenon and less pronounced muscle involvement, similar to adults with these autoantibodies. Novel immunogenetic risk factors for White patients with IIM were identified among juveniles with anti-Sp4 autoantibodies.

RND Pump-Mediated Efflux of Amotosalen, a Compound Used in Pathogen Inactivation Technology to Enhance Safety of Blood Transfusion Products, May Compromise Its Gram-Negative Anti-Bacterial Activity.

Pathogen inactivation is a strategy to improve the safety of transfusion products. The only pathogen reduction technology for blood products currently approved in the US utilizes a psoralen compound, called amotosalen, in combination with UVA light to inactivate bacteria, viruses, and protozoa. Psoralens have structural similarity to bacterial multidrug efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through genetic, biophysical, and molecular modeling analysis. The main efflux systems in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa are tripartite resistance-nodulation-cell division (RND) systems, which span the inner and outer membranes of Gram-negative pathogens, and expel antibiotics from the bacterial cytoplasm into the extracellular space. We provide evidence that amotosalen is an efflux substrate for the E. coli AcrAB, Acinetobacter baumannii AdeABC, and P. aeruginosa MexXY RND efflux pumps. Furthermore, we show that the MICs for contemporary Gram-negative bacterial isolates for these species and others in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens. IMPORTANCE Pathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by major efflux pumps in E. coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the MICs for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies.

BACKGROUND: Idiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown. METHODS: We elucidated the gene copy number (GCN) variations of total C4, C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion. RESULTS: The large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T (C4T=2+3) and C4A deficiency (C4A=0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28-2.91), p=5.0×10-53 for C4T, and 2.82 (2.48-3.21), p=7.0×10-57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44-84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies. CONCLUSIONS: C4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis.

Anti-FHL1 autoantibodies in juvenile myositis are associated with anti-Ro52 autoantibodies but not with severe disease features.

OBJECTIVES: Four-and-a-half LIM domains 1 (FHL1) is a muscle-specific protein. Autoantibodies against FHL1 were recently discovered in adults with idiopathic inflammatory myopathies (IIMs) and were found to be associated with clinical features and outcomes indicative of increased disease severity. Anti-FHL1 autoantibodies have not been described in children. Here, the prevalence and clinical features associated with anti-FHL1 autoantibodies were examined in a large North American cohort of juvenile patients with IIM. METHODS: Sera from 338 juvenile IIM patients and 91 juvenile healthy controls were screened for anti-FHL1 autoantibodies by ELISA. Clinical characteristics and HLA alleles of those with and without anti-FHL1 autoantibodies were compared among those with juvenile IIM. RESULTS: Anti-FHL1 autoantibodies were present in 10.9% of juvenile IIM patients and 1.1% of controls. The frequency of anti-FHL1 autoantibodies among clinical and serologic subgroups did not differ. A higher percentage of Asian patients had anti-FHL1 autoantibodies (11% vs 0.7%; P = 0.002). Myositis-associated autoantibodies (MAAs) [odds ratio (OR) 2.09 (CI 1.03, 4.32)], anti-Ro52 autoantibodies specifically [OR 4.17 (CI 1.83, 9.37)] and V-sign rash [OR 2.59 (CI 1.22, 5.40)] were associated with anti-FHL1 autoantibodies. There were no differences in other features or markers of disease severity. No HLA associations with anti-FHL1 autoantibodies in Caucasian myositis patients were identified. CONCLUSION: Anti-FHL1 autoantibodies are present in ∼11% of juvenile IIM patients and commonly co-occur with MAAs, including anti-Ro52 autoantibodies. In contrast to adult IIM, anti-FHL1 autoantibodies in juvenile myositis are associated with V-sign rash but not with other distinctive clinical features or worse outcomes.