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In the era of blood group genomics, reference collections of complete and fully resolved blood group gene alleles have gained high importance. For most blood groups, however, such collections are currently lacking, as resolving full-length gene sequences as haplotypes (ie, separated maternal/paternal origin) remains exceedingly difficult with both Sanger and short-read next-generation sequencing. Using the latest third-generation long-read sequencing, we generated a collection of fully resolved sequences for all 6 main ABO allele groups: ABO∗A1/A2/B/O.01.01/O.01.02/O.02. We selected 77 samples from an ABO genotype data set (n = 25 200) of serologically typed Swiss blood donors. The entire ABO gene was amplified in 2 overlapping long-range polymerase chain reactions (covering ∼23.6 kb) and sequenced by long-read Oxford Nanopore sequencing. For quality validation, 2 samples per ABO group were resequenced using Illumina and Pacific Biosciences technology. All 154 full-length ABO sequences were resolved as haplotypes. We observed novel, distinct sequence patterns for each ABO group. Most genetic diversity was found between, not within, ABO groups. Phylogenetic tree and haplotype network analyses highlighted distinct clades of each ABO group. Strikingly, our data uncovered 4 genetic variants putatively specific for ABO∗A1, for which direct diagnostic targets are currently lacking. We validated A1-diagnostic potential using whole-genome data (n = 4872) of a multiethnic cohort. Overall, our sequencing strategy proved powerful for producing high-quality ABO haplotypes and holds promise for generating similar collections for other blood groups. The publicly available collection of 154 haplotypes will serve as a valuable resource for molecular analyses of ABO, as well as studies about the function and evolutionary history of ABO.

Background and ObjectivesMixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations like in chimerism, or to rare variants in eitherABOexon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution.Materials and MethodsWe used long-read sequencing by Oxford Nanopore Technologies to sequence the entireABOgene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members.ResultsOur data revealed a novel heterozygous g.10924C>A variant on theABO*A-allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor’s mother, who shared the novel variant and the anti-A specific mixed-field agglutination.ConclusionWe discovered a regulatory variant in the 8-bp RUNX1 motif ofABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3or B3phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as emerging strategy for resolving cases with cryptic ABO phenotypes.

Introduction: Screening of hepatitis B surface antigen (HBsAg) and individual-donation nucleic acid amplification testing (ID-NAT) of blood donors have become standard to detect hepatitis B virus (HBV) infection. However, there is still a residual risk of HBV transmission by blood components of donors suffering from occult HBV infection (OBI). Therefore, many countries implemented universal testing of anti-HBV core antigen (anti-HBc) antibodies in order to increase blood safety. In Switzerland, anti-HBc testing is not part of the routine blood donor-screening repertoire. Therefore, we sought to assess prevalence of donors with OBI in a Swiss blood donor collective. Methods: Blood donations were prospectively investigated for the presence of anti-HBc antibodies during two time periods (I: all donors, March 2017; II: first-time donors only, April 2017 until February 2018). Anti-HBc-positive findings were confirmed by an anti-HBc neutralization test. Discarded plasma samples of anti-HBc-confirmed positive donors were ultracentrifuged and subsequently retested by regular HBV-ID-NAT to search for traces of HBV. Results: During time period I, 78 (1.6%) individuals out of 4,923 donors were confirmed anti-HBc-positive. Sixty-nine (88%) anti-HBc-positive samples were available and processed by ultracentrifugation followed by repeat HBV-ID-NAT. Four samples (5.8%) were found positive for HBV DNA. Sixty-five (94.2%) samples remained HBV NAT-negative upon ultracentrifugation. During time period II, 56 (0.9%) donor samples out of 6,509 exhibited anti-HBc-confirmed positive. Fifty-five (98%) samples could be reassessed by HBV-ID-NAT upon ultracentrifugation. Three (5.5%) samples contained HBV DNA and 52 (94.5%) samples remained HBV NAT-negative. Conclusion: Overall, we detected 7 viremic OBI carriers among 11,432 blood donors, which tested negative for HBV by standard HBV-ID-NAT and HBsAg screening. In contrast, OBI carriers showed positive anti-HBc findings which could be confirmed in 83.8% of the cases. Thus, OBI might be missed by the current HBV screening process of Swiss blood donors. We suggest to review current HBV screening algorithm. Extended donor screening by anti-HBc testing may unmask OBI carriers and contribute to blood safety for the recipient of blood products.

Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against the ectodomain and the receptor-binding domain of the spike protein as well as the nucleocapsid protein of SARS-CoV-2. We used TRABI for continuous seromonitoring of hospital patients and healthy blood donors (n=72’222) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). Seroprevalence peaked in May 2020 and rose again in November 2020 in both cohorts. Validations of results included antibody diffusional sizing and Western Blotting. Using an extended Susceptible-Exposed-Infectious-Removed model, we found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020 in the population of the canton of Zurich. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19 and up to the timepoint of survey participation. Crucially, we found no evidence for a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2- infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Phenotypic plasticity is essential to the immune system, yet the factors that shape it are not fully understood. Here, we comprehensively analyze immune cell phenotypes including morphology across human cohorts by single-round multiplexed immunofluorescence, automated microscopy, and deep learning. Using the uncertainty of convolutional neural networks to cluster the phenotypes of eight distinct immune cell subsets, we find that the resulting maps are influenced by donor age, gender, and blood pressure, revealing distinct polarization and activation-associated phenotypes across immune cell classes. We further associate T cell morphology to transcriptional state based on their joint donor variability and validate an inflammation-associated polarized T cell morphology and an age-associated loss of mitochondria in CD4+ T cells. Together, we show that immune cell phenotypes reflect both molecular and personal health information, opening new perspectives into the deep immune phenotyping of individual people in health and disease., Science Advances, 8 (44), ISSN:2375-2548

The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S–s–U–, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S–s–U–. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.

Neutralizing antibodies are considered a key correlate of protection by current SARS-CoV-2 vaccines. The ability of antibody-based therapies, including convalescent plasma, to affect established disease remains to be elucidated. Only few monoclonal therapies and only when used at a very early stage of infection have shown efficacy. Here, we conducted a proof-of-principle study of convalescent plasma therapy in a phase I trial in 30 COVID-19 patients including immunocompromised individuals hospitalized early after onset of symptoms. A comprehensive longitudinal monitoring of the virologic, serologic, and disease status of recipients in conjunction with detailed post-hoc seroprofiling of transfused convalescent plasma, allowed deciphering of parameters on which plasma therapy efficacy depends. Plasma therapy was safe and had a significant effect on viral clearance depending on neutralizing and spike SARS-CoV-2 antibody levels in the supplied convalescent plasma. Endogenous immunity had strong effects on virus control. Lack of endogenous neutralizing activity at baseline was associated with a higher risk of systemic viremia. The onset of endogenous neutralization had a noticeable effect on viral clearance but, importantly, even after adjusting for their endogenous neutralization status recipients benefitted from therapy with high neutralizing antibody containing plasma.In summary, our data demonstrate a clear impact of exogenous antibody therapy on the rapid clearance of viremia in the early stages of infection and provide directions for improved efficacy evaluation of current and future SARS-CoV-2 therapies beyond antibody-based interventions. Incorporating an assessment of the endogenous immune response and its dynamic interplay with viral production is critical for determining therapeutic effects.One Sentence SummaryThis study demonstrates the impact of exogenous antibody therapy by convalescent plasma containing high neutralizing titers on the rapid clearance of viremia in the early stages of SARS-CoV-2 infection.

Background McLeod syndrome (MLS) is an ultra-rare neurodegenerative X-linked disease caused by mutations in the XK gene, classified as one of the core neuroacanthocytosis syndromes. Together with the clinically very similar chorea-acanthocytosis it belongs to the heterogeneous group of ‘Huntington’s disease (HD) phenocopies’. Aims To characterize a cohort of HD phenocopies with the genetically confirmed diagnosis of McLeod syndrome. Methods This is a retrospective and prospective analysis of genotype and phenotype of sixteen McLeod cases. Results We longitudinally characterized the second largest cohort known to date. We identified novel XK mutations as well as deletions that extend into the PRRG1 gene (novel) and describe two contiguous gene deletion cases of MLS with X-linked chronic granulomatous disease (deletion also effecting the CYBB gene). This study confirms core features of MLS such as late onset hyperkinetic movements in association with neuro/myopathy, neuropsychiatric impairment, cardiac involvement, hyperCKemia. Novel aspects in this MLS series seem obstructive sleep apnea and epileptic seizure onset in childhood. Conclusions Our study expands the limited knowledge on the variable course, the various clinical manifestations and the genetic spectrum of a hereditary HD phenocopy syndrome.

The clinical outcome of SARS-CoV-2 infections can range from asymptomatic to lethal, and is thought to be crucially shaped by the quality of the immune response which includes antibody titres and affinity for their targets. Using Microfluidic Antibody Affinity Profiling (MAAP), we determined the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 23 of whom were confirmed to be SARS-CoV-2-positive by PCR testing. We found that dissociation constants (Kd) of anti-RBD antibodies spanned more than two orders of magnitude from 80 pM to 25 nM, despite having similar antibody concentrations. The tested individuals showed progressively higher antibody concentrations but constant Kd values, suggesting that affinities did not mature over time. 33 sera showed affinities higher than that of the CoV2 spike for its ACE2 receptor. Accordingly, addition of seropositive plasma to pre-formed spike-ACE2 receptor complexes led to their dissociation. Finally, we observed that the RBD of HKU1, OC43, and SARS-CoV coronaviruses, but not unrelated control proteins, were able to compete substantially with the RBD of SARS-CoV-2 in solution. Therefore, the affinity of total plasma immunoglobulins to SARS-CoV-2 is an indicator of the quality of the immune response to SARS-CoV-2, and may help select the most efficacious samples for therapeutic plasmapheresis.

Clonal hematopoiesis (CH) is associated with age and an increased risk of myeloid malignancies, cardiovascular risk, and all-cause mortality. We tested for CH in a setting where hematopoietic stem cells (HSCs) of the same individual are exposed to different degrees of proliferative stress and environments, ie, in long-term survivors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their respective related donors (n = 42 donor-recipient pairs). With a median follow-up time since allo-HSCT of 16 years (range, 10-32 years), we found a total of 35 mutations in 23 out of 84 (27.4%) study participants. Ten out of 42 donors (23.8%) and 13 out of 42 recipients (31%) had CH. CH was associated with older donor and recipient age. We identified 5 cases of donor-engrafted CH, with 1 case progressing into myelodysplastic syndrome in both donor and recipient. Four out of 5 cases showed increased clone size in recipients compared with donors. We further characterized the hematopoietic system in individuals with CH as follows: (1) CH was consistently present in myeloid cells but varied in penetrance in B and T cells; (2) colony-forming units (CFUs) revealed clonal evolution or multiple independent clones in individuals with multiple CH mutations; and (3) telomere shortening determined in granulocytes suggested ∼20 years of added proliferative history of HSCs in recipients compared with their donors, with telomere length in CH vs non-CH CFUs showing varying patterns. This study provides insight into the long-term behavior of the same human HSCs and respective CH development under different proliferative conditions.

Funder: Herchel Smith Fund, Funder: St John’s College Cambridge, Funder: Centre for Misfolding Diseases, Cambridge, Funder: Swiss FCS and the Forschungskredit of the University of Zurich, Funder: Frances and Augustus Newman Foundation, Funder: BBRSC, Funder: NOMIS Foundation, The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Background and objectives A lateral flow assay for simultaneous blood group typing of ABO, RhD, C, E, c, e, Cw and K with stable end-point and without centrifugation is in routine use since several years (MDmulticard® ). The typing of extended phenotype parameters belonging to the Duffy, Kidd, MNSs blood group systems and others, however, has not yet been demonstrated for this technique. Reliable detection of Fyx , a weak Fyb phenotype with a pronounced quantitative reduction of the number of Fyb antigens on the erythrocyte surface, remains a weakness of current serological blood grouping techniques. Material and methods The performance characteristics of the following reagents were evaluated in donor and patient samples in lateral flow technology (MDmulticard® ): Anti-Fya , -Fyb , -Jka , -Jkb , -S, -s, -P1 and -k. The sensitivity to detect Fyx was in addition evaluated with Fyx positive samples, which had been preselected by MALDI-TOF MS-based genotyping. Results All results obtained with the MDmulticard® were in full accordance with those of the CE-certified reference products for all the eight reagent formulations used: Anti-Fya , -Fyb , -Jka , -Jkb , -S, -s, -P1 and -k. Also, all Fyx phenotypes of the selected population of 93 positive samples, originally identified by MALDI-TOF MS-based genotyping, were reliably detected by the lateral flow assay. Conclusion Extended phenotype blood group parameters, including the serologically challenging Fyx phenotype, can be determined simultaneously, rapidly and accurately using the lateral flow (MDmulticard® ) technology, even in cases when IgG class antibodies are the only source of diagnostic antibodies.

BACKGROUND McLeod syndrome (MLS) is hematologically defined by the absence of the red blood cell (RBC) antigen Kx on the transmembrane RBC protein, XK, representing a highly specific diagnostic marker. Direct molecular assessment of XK therefore represents a desirable diagnostic tool. Whereas pathogenic point mutations may be simply identified, partial and complete deletions of XK on Xp21.1, eventually covering adjacent genes and causing multifaceted “continuous gene syndromes,” are difficult to localize. STUDY DESIGN AND METHODS Three different McLeod patient samples were tested using 16 initial positional polymerase chain reaction (PCR) procedures distributed over an approximately 2.8-Mbp Xp-chromosomal region, ranging telomeric from MAGEB16 to OTC, centromeric of XK. The molecular breakpoint of one sample with an apparent large Xp deletion was iteratively narrowed down by stepwise positioning further PCR procedures and sequenced. Two mutant XK genes, one previously published and serving as a positive control, were also sequenced. RESULTS We confirmed the positive control as previously published and listed as XK*N.20 by the International Society of Blood Transfusion (ISBT). The other XK showed a novel four-nucleotide deletion in Exon 1, 195-198delCCGC (newly listed as XK*N.39 by the ISBT). The third sample had an approximately 151-kbp X-chromosomal deletion, reaching from Exon 2 of LANCL3, across XK to Exon 3 of CYBB (newly listed as XK*N.01.016 by the ISBT). Carrier status of the patients' sister was diagnosed using a diagnostic “gap-PCR.” CONCLUSIONS The stepwise partitioning of Xp21.1 is pragmatic and cost-efficient in comparison to other diagnostic techniques such as “massive parallel sequencing” given the rarity of MLS. All males with suspected MLS should be considered for molecular XK profiling.

BACKGROUND Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers. STUDY DESIGN AMD METHODS Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry. RESULTS During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p < 0.001) and PL-EVs increased in 74 PCs (A, C-E; p < 0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage. CONCLUSION PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques.

BACKGROUND Alloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS In a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex-based multiplex assay. RESULTS HPA mismatches were observed in 109 of 241 pregnancies (45%), but child-specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8-193; p = 0.006), whereas the number of pregnancies was not. CONCLUSION Our observational data indicated a low immunogenicity of HPA and suggest that a broader immune response—inferred by HLA sensitization—is probably associated with HPA antibody induction.

BACKGROUND Different types of fresh-frozen plasma (FFP) exist, and the concentrations of plasma proteins vary between individuals and blood groups. Furthermore, processing may also influence the content. Quarantine-stored plasma (qFFP) and plasma that was pathogen-reduced using blood-safety (Intercept) technology (piFFP) were analyzed regarding procoagulant and anticoagulant hemostasis proteins, including endogenous thrombin (thrombin-generation) potential (ETP). MATERIALS AND METHODS Thirty-five samples of each type of FFP were analyzed using only male Blood Group O donors. FFP units were stored frozen for comparable periods of time before plasma protein content was assessed. Once the units were thawed, all tests were completed within 4 hours. The results are presented as means ± standard deviations or as median (minimum; maximum) and were compared using independent-sample t tests (significance, p

Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs.

Background: Roughly one quarter of short-term temporary deferrals (STTD) of blood donors are low-hemoglobin deferrals (LHD), i.e. STTD due to a hemoglobin (Hb) value falling below a cutoff of 125 g/L for female and 135 g/L for male donors. Since voluntarily donating blood is a prosocial activity, donors may perceive deferral as social exclusion, which can cause social pain, decrease self-esteem, and lead to antisocial behavior. However, little is known about the causal impacts of LHD on donor return. Study Design and Methods: We conducted a quasi-experiment with 80,060 donors invited to blood drives in the canton of Zurich, Switzerland, between 2009 and 2014. Within a narrow window of Hb values around the predetermined cutoff, the rate of LHD jumps discontinuously. This discontinuous jump allows us to quantify the causal effects of LHD on donor return, as it is uncorrelated with other unobserved factors that may also affect donor return. Results: We found different behavioral reactions to LHD for female and male donors. Female donors do not react to the first LHD. However, after any repeated LHD, they are 13.53 percentage points (p <0.001) less likely to make at least 1 donation attempt within the next 18 months and make 0.389 fewer donation attempts (p <0.001). Male donors react to the first LHD. They are 5.32 percentage points (p = 0.139) less likely to make at least 1 donation attempt over the next 18 months and make 0.227 (p = 0.018) fewer donation attempts. After any repeated LHD, male donors are 13.30 percentage points (p = 0.004) less likely to make at least 1 donation attempt and make 0.152 (p = 0.308) fewer donation attempts. Conclusion: LHD have detrimental impacts on donor return, especially if they occur repeatedly – suggesting that avoiding false LHD and helping donors to better cope with them helps to maintain the pool of prospective donors.

McLeod syndrome (MLS) is a rare adult-onset, progressive and incurable X-linked multisystemic disorder characterized by chorea, cognitive decline, seizures, polyneuropathy, myopathy and dilated cardiomyopathy with subsequent heart failure and increased risk for arrhythmia [1]. Variable psychiatric symptoms are common; the presence of acanthocytes on blood smears, elevated CK levels and striatal atrophy are other features. MLS is caused by mutation in the XK gene which encodes a membrane transport protein containing the Kx erythrocyte antigen [1]. So far, less than 200 MLS cases have been reported. In general, women harboring XK mutations rarely manifest symptoms. In addition, myoclonus has not been described in association with MLS and functional imaging studies are scarce.

Background: High-frequency blood group antigens (HFA) are present in >90% of the human population, according to some reports even in >99% of individuals. Therefore, patients lacking HFA may become challenging for transfusion support because compatible blood is hardly found, and if the patient carries alloantibodies, the cross-match will be positive with virtual every red cell unit tested. Methods: In this study, we applied high-throughput blood group SNP genotyping on >37,000 Swiss blood donors, intending to identify homozygous carriers of low-frequency blood group antigens (LFA). Results: 326 such individuals were identified and made available to transfusion specialists for future support of patients in need of rare blood products. Conclusion: Thorough comparison of minor allele frequencies using population genetics revealed heterogeneity of allele distributions among Swiss blood donors which may be explained by the topographical and cultural peculiarities of Switzerland. Moreover, geographically localized donor subpopulations are described which contain above-average numbers of individuals carrying rare blood group genotypes.

BACKGROUND Little is known about the long-term effects of interventions aimed at increasing turnout among voluntary blood donors. STUDY DESIGN AND METHODS We use a retrospective natural experiment with all 40,653 donors who were repeatedly invited to blood drives in Zurich, Switzerland, between 2010 and 2013. The intervention is a quasi-randomized phone call informing donors of a current shortage of their blood type. The panel structure of the data allows identification of different types of donors reacting to the phone call. RESULTS Our analysis reveals two types. Type 1 donors make up 27.1% of the population. They are highly motivated and exhibit a baseline donation rate of 59.4% (p

The impact of ABO incompatibility on hematopoietic stem cell transplantation (HSCT) outcome is controversial. As ABH substances are expressed on tissues and secreted in body fluids, they could drive an immune response in minor ABO-incompatible HSCT. The aim of the study was to investigate the prognostic role of the recipients' ABH secretor status.Patients who underwent minor ABO-incompatible HSCT were included. Secretor status was determined either serologically or by molecular genetics.Between March 1996 and June 2012, a total of 176 patients received minor ABO-incompatible HSCT and 150 (85%) were secretors. Incidence and severity of acute graft-versus-host disease (GVHD) and chronic GVHD did not differ between secretors and nonsecretors (cumulative incidences ± standard errors: acute GVHD on Day 100, 41 ± 11 and 46 ± 5%, p = 0.59; chronic GVHD at 2 years, 52 ± 13 and 56 ± 5%, p = 0.62, for secretors and nonsecretors, respectively). Additionally, nonrelapse mortality (NRM) and overall survival (OS) were similar in the two groups (2-year NRM, 27 ± 9 and 23 ± 3%, p = 0.45; 4-year OS, 64 ± 10 and 55 ± 4%, p = 0.28, for secretors and nonsecretors, respectively).The recipients' ABH secretor status in minor ABO-incompatible HSCT has no prognostic impact on major transplant outcomes.

Background: Transfusion-transmitted Chagas disease has been reported from endemic countries in Latin America. Switzerland is a non-endemic country but high prevalence of antibodies against Trypanosoma cruzi was found among immigrants. Immigrants may participate in blood donation; therefore, risk-adapted anti-T. cruzi screening for blood donors was implemented in Switzerland in 2013. Methods: Between January 2013 and July 2015, 1 out of 1,183 at-risk donors, tested at Blood Transfusion Service Zurich, was found anti-T. cruzi IgG-positive. Results and Conclusion: Out of 54 donations given by the index donor (ID), we identified 77 blood products which were delivered to hospitals. Archived serum samples from the donations given during the prior 5 years were available for retrospective testing. All samples from ID revealed positive findings for anti-T.cruzi IgG. Donor-triggered look-back procedure identified a 70-year-old male recipient of a platelet concentrate (PC) donated by ID. The recipient succumbed of acute T. cruzi infection 2 years after transfusion of the PC.

BACKGROUND: Blood group phenotype variation has been attributed to potential resistance to pathogen invasion. Variation was mapped in blood donors from Lampang (northern region) and Saraburi (central region), Thailand, where malaria is endemic. The previously unknown blood group allele profiles were characterized and the data were correlated with phenotypes. The high incidence of the Vel-negative phenotype previously reported in Thais was investigated. STUDY DESIGN AND METHODS: DNA from 396 blood donors was analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Outliers were investigated by serology and DNA sequencing. Allele discrimination assays for SMIM1 rs1175550A/G and ACKR1 rs118062001C/T were performed and correlated with antigen expression. RESULTS: All samples were phenotyped for Rh, MNS, and K. Genotyping/phenotyping for RhD, K, and S/s showed 100% concordance. Investigation of three RHCE outliers revealed an e-variant antigen encoded by RHCE*02.22. Screening for rs147357308 (RHCE c.667T) revealed a frequency of 3.3%. MN typing discrepancies in 41 samples revealed glycophorin variants, of which 40 of 41 were due to Mia. Nine samples (2.3%) were heterozygous for FY*01W.01 (c.265C>T), and six samples (1.5%) were heterozygous for JK*02N.01. All samples were wildtype SMIM1 homozygotes with 97% homozygosity for rs1175550A. CONCLUSIONS: Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is an efficient method for rapid routine genotyping and investigation of outliers identified novel variation among our samples. The expected high prevalence of the Mi(a+) phenotype was observed from both regions. Of potential clinical relevance in a region where transfusion-dependent thalassemia is common, we identified two RHCE*02 alleles known to encode an e-variant antigen. (Less)

Background The impact of ABO incompatibility on hematopoietic stem cell transplantation (HSCT) outcome is controversial. As ABH substances are expressed on tissues and secreted in body fluids, they could drive an immune response in minor ABO-incompatible HSCT. The aim of the study was to investigate the prognostic role of the recipients' ABH secretor status. Study Design and Methods Patients who underwent minor ABO-incompatible HSCT were included. Secretor status was determined either serologically or by molecular genetics. Results Between March 1996 and June 2012, a total of 176 patients received minor ABO-incompatible HSCT and 150 (85%) were secretors. Incidence and severity of acute graft-versus-host disease (GVHD) and chronic GVHD did not differ between secretors and nonsecretors (cumulative incidences ± standard errors: acute GVHD on Day 100, 41 ± 11 and 46 ± 5%, p = 0.59; chronic GVHD at 2 years, 52 ± 13 and 56 ± 5%, p = 0.62, for secretors and nonsecretors, respectively). Additionally, nonrelapse mortality (NRM) and overall survival (OS) were similar in the two groups (2-year NRM, 27 ± 9 and 23 ± 3%, p = 0.45; 4-year OS, 64 ± 10 and 55 ± 4%, p = 0.28, for secretors and nonsecretors, respectively). Conclusion The recipients' ABH secretor status in minor ABO-incompatible HSCT has no prognostic impact on major transplant outcomes.

Starting in 2013, blood donors must be tested at least using: (1) one monoclonal anti-D and one anti-CDE (alternatively full RhCcEe phenotyping), and (2) all RhD negative donors must be tested for RHD exons 5 and 10 plus one further exonic, or intronic RHD specificity, according to the guidelines of the Blood Transfusion Service of the Swiss Red Cross (BTS SRC). In 2012 an adequate stock of RHD screened donors was built. Of all 25,370 RhD negative Swiss donors tested in 2012, 20,015 tested at BTS Berne and 5355 at BTS Zürich, showed 120 (0.47%) RHD positivity. Thirty-seven (0.15%) had to be redefined as RhD positive. Routine molecular RHD screening is reliable, rapid and cost-effective and provides safer RBC units in Switzerland.

A revolution occurred during the last decade in the comprehension of the physiology as well as in the physiopathology of iron metabolism. The purpose of this review is to summarize the recent knowledge that has accumulated, allowing a better comprehension of the mechanisms implicated in iron homeostasis. Iron metabolism is very fine tuned. The free molecule is very toxic; therefore, complex regulatory mechanisms have been developed in mammalian to insure adequate intestinal absorption, transportation, utilization, and elimination. ‘Ironomics' certainly will be the future of the understanding of genes as well as of the protein-protein interactions involved in iron metabolism.

Blood bankers have focused their energy to secure blood transfusion, and only recently have studies been published on the effect of blood donation on iron metabolism. In many facilities, hemoglobin measurement is only performed just before or even during blood donation, but the determination of iron stores is largely ignored. The 2013 paradox of transfusion medicine is due to the fact that blood donation may be harmful and leads to iron deficiency with or without anemia, but for other individuals, it may be a healthy measure preventing type 2 diabetes. The purpose of this review is to discuss iron metabolism in the perspective of blood donation, notably regarding their possible genetic profiles that eventually will discriminate “good” iron absorbers from “bad” iron responders.

Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has previously been reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This review describes the development of a large cooperative Swiss-German project, aiming to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. Recent reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost-efficiency when used in genetic analysis, including forensics, pharmacogenetics, oncology and hematology. Furthermore, Kell and RhD genotyping have been performed on fetal DNA from maternal plasma with excellent results. In summary, this article introduces a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS. Although all data presented are preliminary, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method.

Background: Data on blood donor status obtained from general surveys and health interview surveys have been widely used. However, the integrity of data on self-reported blood donor status from surveys may be threatened by sampling and non-sampling error. Our study aimed to compare self-reported blood donors (including one-time as well as regular donors) from the Swiss Health Survey 2012 (SHS) with register-based blood donors recorded by blood establishments and evaluate the direction and magnitude of bias in the SHS. Methods: We compared population-weighted SHS point estimates of the number of blood donors with their corresponding 95% confidence intervals to the respective figures from blood donor registries (birth cohorts 1978-1993) and estimates of donors based on period donor tables derived from blood donor registries (birth cohorts 1920-1993). Results: In the birth cohorts 1978-1993, the SHS-predicted number of donors was 1.8 times higher than the respective number of donors based on registry data. Adjusting for foreign and naturalized Swiss nationals that immigrated after their 18th birthday, the SHS overall predicted number of donors was 1.6 times higher. Similarly, SHS estimates for the 1920-1993 birth cohorts were 2.4 and 2.1 times higher as compared to register-based estimates. Generally, the differences between SHS and register-based donors were more pronounced in men than in women. Conclusion: Self-reported blood donor status in the SHS is biased. Estimates of blood donors are substantially higher than respective estimates based on blood donor registries.

The GYPC gene encodes the glycophorins C and D. The two moieties express 12 known antigens of the Gerbich blood group system and functionally stabilize red blood cell membranes through their intracellular interaction with protein 4.1 and p55. Three GYPC exon deletions are responsible for the lack of the high-frequency antigens Ge2 (Yus type, exon 2 deletion), Ge2 and Ge3 (Gerbich type, exon 3 deletion), and Ge2 to 4 (Leach type, exons 3 and 4 deletion), but lack exact molecular description. A total of 29 rare blood samples with Yus (GE:-2,3,4) and Gerbich (GE:-2,-3,4) phenotypes, including individuals of Middle-Eastern, North-African or Balkan ancestry were examined genetically. All phenotypes could be explained by 4 different Yus alleles, characterized by deletions of exon 2 and adjacent introns, and 3 different Gerbich alleles, with deletions of exon 3 and adjacent introns. A 3600 base pair GYPC region, encompassing exon 2 and flanking region, shares a high degree of sequence homology with a region flanking exon 3, probably representing an evolutionary duplication event. Defining the expression of Gerbich variants presently relies on rare serological reagents. Our approach substitutes the serological characterization with a precise genotype approach to identify the rare Yus and Gerbich alleles.

McLeod syndrome (MLS) is hematologically defined by the absence of the red blood cell (RBC) antigen Kx on the transmembrane RBC protein, XK, representing a highly specific diagnostic marker. Direct molecular assessment of XK therefore represents a desirable diagnostic tool. Whereas pathogenic point mutations may be simply identified, partial and complete deletions of XK on Xp21.1, eventually covering adjacent genes and causing multifaceted "continuous gene syndromes," are difficult to localize.Three different McLeod patient samples were tested using 16 initial positional polymerase chain reaction (PCR) procedures distributed over an approximately 2.8-Mbp Xp-chromosomal region, ranging telomeric from MAGEB16 to OTC, centromeric of XK. The molecular breakpoint of one sample with an apparent large Xp deletion was iteratively narrowed down by stepwise positioning further PCR procedures and sequenced. Two mutant XK genes, one previously published and serving as a positive control, were also sequenced.We confirmed the positive control as previously published and listed as XK*N.20 by the International Society of Blood Transfusion (ISBT). The other XK showed a novel four-nucleotide deletion in Exon 1, 195-198delCCGC (newly listed as XK*N.39 by the ISBT). The third sample had an approximately 151-kbp X-chromosomal deletion, reaching from Exon 2 of LANCL3, across XK to Exon 3 of CYBB (newly listed as XK*N.01.016 by the ISBT). Carrier status of the patients' sister was diagnosed using a diagnostic "gap-PCR."The stepwise partitioning of Xp21.1 is pragmatic and cost-efficient in comparison to other diagnostic techniques such as "massive parallel sequencing" given the rarity of MLS. All males with suspected MLS should be considered for molecular XK profiling.

Tissue engineering (TE) combines the three components - cells, scaffolds and growth factors - to generate tissues for functional replacement of damaged or diseased organs upon transplantation. Further, regenerative medicine (RM) combines TE with other strategies such as cell-based therapies, gene therapy, and immunomodulation, using stem and progenitor cells from various sources in order to induce in vivo organ regeneration [1,2,3]. TERM, the combination of TE and RM, combines basic sciences such as nanotechnology, biomechanics, bioinformatics, material science, and polymer chemistry with cell biology and medical sciences to promote functional organo(re)genesis. In the light of a growing and aging population with ever increasing demand of organ replacements, TERM might become the appropriate strategy to meet future needs of the patients [4].

Alloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia.In a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex-based multiplex assay.HPA mismatches were observed in 109 of 241 pregnancies (45%), but child-specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8-193; p = 0.006), whereas the number of pregnancies was not.Our observational data indicated a low immunogenicity of HPA and suggest that a broader immune response-inferred by HLA sensitization-is probably associated with HPA antibody induction.

or organs to restore impaired function resulting from any cause, including congenital defects, disease, trauma and aging. It uses a combination of several technological approaches that moves it beyond traditional transplantation and replacement therapies. These approaches may include, but are not limited to, the use of soluble molecules, gene therapy, stem cell transplantation, tissue engineering and reprogramming of cell and tissue types’. The interdisciplinarity of TERM goes far beyond medical sciences such as transplantation, immunology, and stem cell biology. It rather integrates disciplines such as system biology, nanotechnology, material science, genetics, and bioengineering in an effort to restore or replace organs and their function. However, spectacular successes in experimental settings, such as cloning of mammalians, the discovery of human embryonic stem cells and finally the reprogramming of terminally differentiated cells into various body cells via induced pluripotent stem cells [11–15], ignited hope and overreaching expectations of scientific and lay auditorium leading Time magazine to claim tissue engineer as being ‘The Hottest Job’ for the future [16]. By 2000, there were 66 companies in the USA working in the field and having a capital value of USD 2.5 billion [16]. In the meantime, failures in early applications and mature understanding of (patho)biology of diseased or deteriorated tissue have fostered new and holistic concepts of TERM: Regenerative technologies and services strive to boost innate repair processes and provide scalable production and standardized application of clinical grade biotherapeutics. Critical areas include regulatory pathways for RM therapies, strategic partnerships, coordination of resources, developing of standards, priorities for industry, biobanking, and integration of new technologies [17, 18]. According to the US Department of Health and Human Services, RM offers the potential to cure countless fatal and debilitating conditions through therapies that spur in vivo regeneration and in vitro creation of healthy tissue for implantation [19]. In the prospect, 7 of 10 deaths in the world will be caused by non-communicable diseases, Uncountable dreams, myths and excitements on artificial generation of tissues, organs, and even whole organisms accompany mankind’s history to master health and disease. The creation of Eve out of Adam’s rip, the generation of Prometheus without sexual reproduction in the Greek mythology, and the failing trial by Doctor Faustus in Goethe’s poetry to build a living Homunculus illustrate the desire of mankind to overcome sickness and perishability [1]. With the emergence of empirical sciences during and after the Enlightenment era, the physicochemical understanding of man and his failures led to the discoveries of modern developmental biology [2], which were culminating in seismic achievements such as cloning of the sheep Dolly, growing of a human ear on a mouse – the so-called auriculosaurus – or the isolation of omnipotent human embryonic stem cells [2, 3]. Although tissue engineering (TE) and regenerative medicine (RM) – often reflected by the acronym ‘TERM’ – are used synonymously and interchangeably in literature, the two areas of medical science pursue different focus and at the same time are complementary to each other [4]. TE goes back to early work by Vacanti, Langer, Green, Matapurkar and many others, combining in vitro isolated and expanded organ-specific cells in co-existence with artificial biodegradable matrix scaffolds to replace diseased, defect or missing organ tissue such as bone, cartilage, liver, ureter and others in attempt to generate in vivo engineered (induced) organ tissue [5–8]. Publications on TE started to appear as early as 1990 while the more comprehensive entity of RM appeared in the literature only by 2000 and thereafter [9]. There are many, ever changing definitions of RM in the literature reflecting evolving science and technologies involved but also taking care of society’s needs driven by population growth and demographic changes. One of the most comprehensive outline of today’s RM is given by Greenwood et al. [10] in 2006 which covers also the progresses made since then: ‘Regenerative Medicine is an emerging interdisciplinary field of research and clinical application focused on the repair, replacement or regeneration of cells, tissue Received: July 15, 2016 Accepted: July 18, 2016 Published online: July 26, 2016

Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers.Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry.During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p0.001) and PL-EVs increased in 74 PCs (A, C-E; p0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage.PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques.

Different types of fresh-frozen plasma (FFP) exist, and the concentrations of plasma proteins vary between individuals and blood groups. Furthermore, processing may also influence the content. Quarantine-stored plasma (qFFP) and plasma that was pathogen-reduced using blood-safety (Intercept) technology (piFFP) were analyzed regarding procoagulant and anticoagulant hemostasis proteins, including endogenous thrombin (thrombin-generation) potential (ETP).Thirty-five samples of each type of FFP were analyzed using only male Blood Group O donors. FFP units were stored frozen for comparable periods of time before plasma protein content was assessed. Once the units were thawed, all tests were completed within 4 hours. The results are presented as means ± standard deviations or as median (minimum; maximum) and were compared using independent-sample t tests (significance, p 0.01).Significantly higher concentrations of adintegrin-like and metalloprotease with thrombospondin type-13 motifs (ADAMTS13), fibrinogen, Factor (F)V, FVIII, FXIII, protein S, protein S activity, antithrombin, microvesicle (900 nm), and α2 antiplasmin were observed in qFFP. The variability of factors was significantly lower in piFFP. Tissue factor (TF) at 1 picomolar (pM) exhibited significantly longer lag time, a lower peak, lower ETP, and a lower velocity index in qFFP compared with piFFP. In TF at 5 pM, significant differences in lag time (longer in qFFP), velocity index (lower in qFFP), and peak (lower in qFFP) were observed. Rotational thromboelastometry revealed a significantly longer (p = 0.002) clot-formation time with intrinsic thromboelastometry for piFFP and a significantly shorter clotting time (p = 0.004) with thromboelastometry fibrinogen testing for piFFP.Pathogen reduction reduces procoagulant and anticoagulant coagulation factors as well as variability. A thrombin-generation assay showed no reduced ETP and no supraphysiological thrombin generation. None of the FFP preparations is likely to be effective for treating fibrinogen deficiency.