Longitudinal Comparison of Automated SARS-CoV-2 Serology Assays in Assessing Virus Neutralization Capacity in COVID-19 Convalescent Sera.

CONTEXT.­: Serologic tests on automated immunology analyzers are increasingly used to monitor acquired immunity against SARS-CoV-2. The heterogeneity of assays raises concerns about their diagnostic performance and comparability. OBJECTIVE.­: To test sera from formerly infected individuals for SARS-CoV-2 antibodies by using 6 automated serology assays and a pseudoneutralization test (PNT). DESIGN.­: Six SARS-CoV-2 serology assays were used to assess 954 samples collected during a 12-month period from 315 COVID-19 convalescents. The tests determined either antibodies against the viral nucleocapsid (anti-NC) or spike protein (anti-S). Two assays did not distinguish between antibody classes, whereas the others selectively measured immunoglobulin G (IgG) antibodies. PNT was used to detect the presence of neutralizing antibodies. RESULTS.­: Comparison of qualitative results showed only slight to moderate concordance between the assays (Cohen κ < 0.57). Significant correlations (P < .001) were observed between the antibody titers from all quantitative assays. However, titer changes were not detected equally. A total anti-S assay measured an increase in 128 of 172 cases (74%) of a suitable subset, whereas all IgG anti-S tests reported decreases in at least 118 (69%). Regarding the PNT results, diagnostic sensitivities of 89% or greater were achieved with positive predictive values of at least 93%. In contrast, specificity changed substantially over time, varying from 20% to 100%. CONCLUSIONS.­: Comparability of serologic SARS-CoV-2 antibody tests is rather poor. Owing to different diagnostic specificities, the tested assays were not equally capable of capturing changes in antibody titers. However, with thoroughly validated cutoffs, IgG-selective anti-S assays are a reliable surrogate test for SARS-CoV-2 neutralizing antibodies in former COVID-19 patients.

Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays.

Since the emergence of SARS-CoV-2 numerous antibody assays have become available, demonstrating different performance characteristics. This study focused on a quantitative correlation between different commercial assays and a neutralization test (NT). Comparative data is needed as a basis for the production of convalescent plasma and potential interpretations COVID-19 immunity. Sera of 100 SARS-CoV-2 convalescent plasma donors were collected and SARS-CoV-2 antibodies were characterized using three different IgG-ELISAs (EUROIMMUN IgG and NCP-IgG ELISA, Wantai ELISA), two CLIA (Elecsys, LIAISON) and two lateral flow tests (MEDsan IgM/IgG-Rapid-Test, Wantai Rapid Test) and subsequently correlated to neutralization titers. The Wantai ELISA and the Elecsys provide the highest sensitivities in this sample (98 and 95 percent respectively). Titers with the best overall quantitative correlation to the NT titer were obtained with the Euroimmun IgG ELISA assay (Rho=0.759) and the Wantai ELISA assay (Rho=0.729). An infection without fever and negative or weakly positive reactions in the Wantai Rapid test were negative predictive factors for NT titers >1:200 (negative predictive value of 92 % and 92 % respectively, combination of both 100 %). The Wantai ELISA titer could be a suitable substitute for NT. An adequate pooling strategy of plasma units additionally could compensate deviations of individual antibody titers.

A 24-base pair deletion in the ABO gene causes a hereditary splice site defect: a novel mechanism underlying ABO blood group O.

BACKGROUND: Blood group A and B antigens are synthesized by glycosyltransferases regulated by a complex molecular genetic background. A multibase deletion in the ABO gene was identified in two related blood donors. To define its hereditary character and to evaluate genotype-phenotype associations, a detailed study including 30 family members was conducted. METHODS AND MATERIALS: ABO phenotyping was performed with agglutination techniques and adsorption-elution tests. The secretor status was determined. Allele-specific sequencing of ABO and genotyping of family members by a mutation-specific polymerase chain reaction were carried out. Functional analysis included cloning of complementary DNA and transfection experiments in HeLa cells. The antigen expression was investigated by flow cytometry and adsorption-elution method. RESULTS: Sequencing analysis revealed a 24-bp deletion in Exon 5 and the adjacent intronic region of ABO. The alteration was inherited by 16 family members. Nine of them being heterozygous for the mutated allele failed to express A antigen on their erythrocytes as found by routine typing. In particular samples, however, adsorption-elution studies indicated inconclusive results. HeLa cells transfected with aberrant gene transcripts did not express blood group antigen A. CONCLUSION: The variation causes defects in messenger RNA splicing, most likely inactivating the transferase as observed by serological typing and in vitro expression analysis. These data suggest a novel mechanism associated with blood group O and extend the knowledge of exceptionally rare ABO splice site mutations and deletions. With increased understanding of the molecular bases of ABO, the diagnostics may be further enhanced to ensure the safest possible use of the blood supply.

The Effect of Parenteral or Oral Iron Supplementation on Fatigue, Sleep, Quality of Life and Restless Legs Syndrome in Iron-Deficient Blood Donors: A Secondary Analysis of the IronWoMan RCT.

Background: Besides anemia, iron deficiency may cause more subtle symptoms, including the restless legs syndrome (RLS), the chronic fatigue syndrome (CFS) or sleeping disorders. Objective: The aim of this pre-planned secondary analysis of the IronWoMan randomized controlled trial (RCT) was to compare the frequency and severity of symptoms associated with iron deficiency before and after (intravenous or oral) iron supplementation in iron deficient blood donors. METHODS/DESIGN: Prospective, randomized, controlled, single-centre trial. (ClinicalTrials.gov: NCT01787526). SETTING: Tertiary care center in Graz, Austria. PARTICIPANTS: 176 (138 female and 38 male) whole-blood and platelet apheresis donors aged ≥ 18 and ≤ 65 years with iron deficiency (ferritin ≤ 30ng/mL at the time of blood donation). INTERVENTIONS: Intravenous iron (1 g ferric carboxymaltose, n = 86) or oral iron supplementation (10 g iron fumarate, 100 capsules, n = 90). MEASUREMENTS: Clinical symptoms were evaluated by a survey before iron therapy (visit 0, V0) and after 8-12 weeks (visit 1, V1), including questions about symptoms of restless legs syndrome (RLS), chronic fatigue syndrome (CFS), sleeping disorders, quality of life and symptoms like headaches, dyspnoea, dizziness, palpitations, pica and trophic changes in fingernails or hair. RESULTS: We found a significant improvement in the severity of symptoms for RLS, fatigue and sleep quality (p < 0.001). Furthermore, a significant decrease in headaches, dyspnoea, dizziness and palpitations was reported (p < 0.05). There was no difference between the type of iron supplementation (intravenous versus oral) and clinical outcome data. CONCLUSION: Iron supplementation in iron-deficient blood donors may be an effective strategy to improve symptoms related to iron deficiency and the wellbeing of blood donors.

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

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

High-dose intravenous versus oral iron in blood donors with iron deficiency: The IronWoMan randomized, controlled clinical trial.

INTRODUCTION: Frequent blood donation often leads to iron deficiency and even anemia but appropriate strategies for detection and prevention are currently not mandatory. At the Medical University of Graz, we conducted a single-center prospective clinical trial to compare oral and IV iron supplementation in iron deficient blood donors including Austrian regular whole blood and platelet apheresis donors. We aimed to determine the difference of transferrin saturation between the treatment groups 8-12 weeks iron administration besides other parameters of iron status and blood count. METHODS: 176 healthy male and female blood donors with iron deficiency (ferritin ≤30 ng/mL) were randomized to either a single dose of IV ferric carboxymaltose (1000 mg, n = 86) or oral iron (II)fumarate (100 tablets of 100 mg [10 per week], n = 90). RESULTS: Between 2014 and 2016, 172 donors (137 women) completed the study; 4 in the oral group were lost to follow-up. At follow-up, median (IQR) transferrin saturation and ferritin were significantly higher in the intravenous group (27 [23-35]%, vs 21.0 [16-32]%; p < 0.001 and 105 [75-145] ng/mL vs 25 [17-34] ng/mL; p < 0.001, respectively) while median (IQR) hemoglobin levels were comparable (IV, 13.6 [13.0-14.4] g/dL vs oral, 13.6 [13.0-14.2] g/dL). The frequency of adverse effects was comparable (38% in both groups) and no serious adverse events occurred. CONCLUSIONS: A single dose of 1000 mg of intravenous iron is highly effective to counteract iatrogenic iron deficiency in blood donors. Oral iron appears to be an acceptable alternative. The assessment of body iron stores should play a key role in maintaining blood donors' health. This trial was registered at www.clinicaltrials.gov as NCT01787526 on February 8, 2013 and at www.clinicaltrialsregister.eu (EudraCT identifier: 2013-000327-14) on September 24, 2013.

High-dose intravenously administered iron versus orally administered iron in blood donors with iron deficiency: study protocol for a randomised, controlled trial.

BACKGROUND: About 2-3 % of the population participates in blood donation programmes. Each whole blood donation or ten apheresis donations cause a loss of 200-250 mg of iron. As a result, one of the most common risks of regular blood donors is iron deficiency. Although this has been known for decades, in most countries, iron status is currently not assessed or treated in this population. Premenopausal women are particularly affected, as they have lower iron reserves and higher daily requirements. Besides anaemia, iron deficiency may lead to fatigue and impaired cognitive and physical performance. Current iron preparations for intravenous administration are well tolerated and allow for application of large doses up to 1 g in one visit. Our hypothesis is that in blood donors with iron deficiency, intravenously administered iron is more efficient and as safe as oral iron supplementation. Since anaemia is one of the most frequent reasons for permanent or intermittent donor deferral, maintaining an iron-replete donor pool may help to prevent shortages in blood supply and to avoid iron deficiency-related comorbidities. METHODS/DESIGN: In this randomised clinical trial we include male and female blood donors aged ≥18 and ≤65 years with a ferritin value of ≤30 ng/ml. Stratified by gender, participants are randomized with a web-based randomisation tool in a 1:1 ratio to either 1 g of intravenously administered ferric carboxymaltose or 10 g of iron fumarate supplements at one to two daily doses of 100 mg each. Eight to 12 weeks after the first visit, iron status, blood count and symptoms are assessed in both groups. The primary endpoint is the difference in transferrin saturation (%) following the intervention between both groups. Secondary endpoints include other parameters of iron metabolism and red blood cell count, the number of patients with drug-related adverse events, and subjective symptoms including those of the restless legs syndrome, quality of life, and fatigue. DISCUSSION: Iron supplementation administered intravenously in non-anaemic but iron-deficient blood donors could represent an effective strategy to protect blood donors from comorbidities related with iron deficiency and therefore improve blood donor wellbeing. Furthermore, iron supplementation will help to maintain an iron-replete blood donor pool. TRIAL REGISTRATION: EudraCT: 2013-000327-14, Clinical Trials Identifier: NCT01787526 . Registered on 6 February 2013.

A novel ABO O allele caused by a large deletion covering two exons of the ABO gene identified in a Caucasian family showing discrepant ABO blood typing results.

BACKGROUND: The presence of ABO subgroup alleles and unusual O alleles often is associated with discrepant serologic findings in ABO blood group typing. In the ABO gene of a Caucasian female and her daughters who had aberrant ABO phenotypes, a novel ABO O allele characterized by a large deletion that included two exons was identified. METHODS: ABO phenotypes were determined by standard agglutination tests and adsorption-elution studies. Allele-specific sequencing analyses of the ABO gene as well as messenger RNA transcripts were carried out. RESULTS: All three samples showed the same discrepant ABO blood typing results lacking A and B antigens, indicating Blood Group O, whereas anti-A1 and anti-A2 isoagglutinins were not detectable in reverse typing. Analyses of the ABO gene revealed a novel allele characterized by a deletion of 2169 base pairs, including sequences of Intron 1, Exon 2, Intron 2, Exon 3, and Intron 3. Exon 1 was directly joined to Exon 4 in the ABO transcript. CONCLUSION: Because the novel allele was associated with a well-described O allele, the absence of A-antigens in the inherited ABO subtype phenotype may be due to the identified mutation affecting the transmembrane-spanning domain of the encoded protein and impairing the transferase activity.

Platelet antibody analysis by three different tests.

BACKGROUND: Platelet-reactive antibodies lead to thrombocytopenia and bleeding disorders, and diverse assays are used for their detection. In this retrospective analysis, the applicability of three different test systems was compared and antibody specificities were assessed. METHODS: Sera of 1,234 patients were tested with an enzyme-linked immunosorbent assay (ELISA; Lifecodes PAKPLUS(®) or PAK 12(®), Gen-Probe) and a solid-phase assay (Capture-P Ready Screen(®), Immucor Inc.). In cases of suspected anti-HLA class I antibodies, a specific lymphocytotoxicity test (LCT, Bio-Rad(®)) was performed. RESULTS: Platelet antibodies were detected in 366 of 1,234 samples (29.7%). In 70.3% concordant negative but only in 8.4% concordant positive results were obtained with both the methods; 185 of 1,053 in the solid-phase assay negative samples were positive in the ELISA (15.0%). In samples positive in both methods, most antibodies reacted against HLA class I antigens. Glycoprotein (GP) specific platelet antibodies, mainly against GPIIb/IIIa and GPIa/IIa, were more frequently detectable in the ELISA than in the solid-phase assay, whereas weakly positive results have to be interpreted cautiously. CONCLUSION: ELISA, solid-phase assay, and LCT showed highly divergent results. Due to several limitations, the additional analysis by the "monoclonal antibody-specific immobilization of platelet antigen" (MAIPA)-assay is highly recommended.

Sclerostin and its association with physical activity, age, gender, body composition, and bone mineral content in healthy adults.

CONTEXT: Sclerostin is produced by osteocytes and inhibits bone formation through the Wnt/ß-catenin-signaling pathway. Only limited data are available on circulating sclerostin levels in healthy subjects. OBJECTIVE: We aimed to evaluate the correlation between sclerostin and physical activity, anthropometric, and biochemical variables. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional observational study in 161 healthy adult men and premenopausal women aged 19 to 64 yr (mean age, 44 ± 10). INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURE(S): Serum sclerostin levels were associated with body composition, bone mineral density, physical activity, and various biochemical parameters. RESULTS: A positive correlation between age and sclerostin in both men (r = 0.37; P < 0.001) and premenopausal women (r = 0.66; P < 0.001) was found. Men had significantly higher sclerostin levels than women (49.8 ± 17.6 vs. 37.2 ± 15.2 pmol/liter; P < 0.001). However, after adjustment for age, bone mineral content (BMC), physical activity, body mass index (BMI), and renal function, sclerostin levels did not differ (P = 0.543). Partial correlation analysis adjusted for age, gender, and kidney function revealed a significant positive correlation between sclerostin levels and BMC, bone mineral density, BMI, and android/gynoid fat and a significant negative correlation with serum osteocalcin and calcium. The most physically active quartile had significantly lower sclerostin levels compared to the least active quartile in a univariate analysis. CONCLUSIONS: In healthy adults, sclerostin serum levels correlate positively with age, BMI, and BMC and negatively with osteocalcin and calcium. Further studies in larger populations are needed to confirm our findings and to better understand their clinical implications.

Adverse events and safety issues in blood donation--a comprehensive review.

Although blood donation is generally safe, a variety of risks and complications exist, the most common being iron deficiency, vasovagal reactions and citrate-related events. In the last decades, extensive efforts have significantly improved recipient and product safety, but there is still great potential to optimise donor care. Many therapies in modern medicine depend on the prompt availability of blood products, therefore it is crucial to maintain a motivated and healthy donor pool in view of a limited number of healthy volunteers willing and able to give blood or blood components. We present a comprehensive review on adverse events addressing all types of blood donation including whole blood, plasma, platelet, peripheral blood stem cell, leucocyte and bone marrow donation. In addition, we outline strategies for the prevention and treatment of these events and give a blueprint for future research in this field.

Identification of 14 new alleles at the fucosyltransferase 1, 2, and 3 loci in Styrian blood donors, Austria.

BACKGROUND: Genes for fucosyltransferases 1 (FUT1:H), 2 (FUT2:Secretor), and 3 (FUT3:Lewis) encode enzymes crucial for ABH and Lewis blood group antigen synthesis. They are highly polymorphic and ethnically and geographically specific. STUDY DESIGN AND METHODS: Genetic variations and allele frequencies of FUT1, FUT2, and FUT3 encoding regions and flanking sequences were analyzed in 100 Styrian blood donors by systematic sequencing. Haplotypes were verified with sequence-specific primers. To identify discrepancies, serologically determined ABO and Lewis blood groups were correlated to respective genotypes. RESULTS: Two novel FUT1 alleles were defined by 9C>T (silent) and 991C>A (P331T) mutations, the latter located in the catalytic domain of the enzyme. Five new alleles of FUT2 were found: three were characterized by new variants and two resulted from new combinations of known polymorphisms. The new 412G>A (G138S) mutation also is located in the catalytic domain. A new nonsecretor allele, based on the presence of 428G>A (nonsense), was found. Another FUT2 allele may have resulted from an intragenic crossover event. FUT3 analysis revealed seven novel alleles, partly based on the new mutations 41G>A (R14H), 1060C>G (R354G), 735G>C (silent), and 882C>T (silent). While 41G>A is placed in the cytoplasmic domain and functional, 1060C>G is placed in the catalytic domain. CONCLUSION: Multiple common and sporadic sequence variations including 14 new alleles at FUT1, FUT2, and FUT3 loci were identified. Four novel mutations result in amino acid substitution in the protein. Three of them are predicted to have adverse effects on the enzyme activity. A novel nonsecretor allele was found.

Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.

Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of > 2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.

Blood donor screening for parvovirus B19 in Germany and Austria.

BACKGROUND: Although the main transmission pathway of parvovirus B19 (B19) is typically via the respiratory route, several transfusion-transmitted infections have been reported. To increase blood safety, all blood donations to our blood donor service have been screened by a B19 minipool real-time nucleic acid testing (NAT) since April 2000. Additional customers have been screened since the summer of 2003. STUDY DESIGN AND METHODS: In total, 2.8 million donations from Germany and Austria were screened for B19 by real-time minipool NAT. A subgroup of 50 B19 DNA-positive donors was screened for B19 immunoglobulin G (IgG) and IgM antibodies and B19 DNA over a 6-month period. Results were compared to those of 100 B19 DNA-negative donors. RESULTS: Data accumulated over the past 6 years indicate a high incidence period from May 2004 to January 2006. In total, the incidence was 12.7 and 261.5 per 100,000 donations with high virus loads equal to or above 10(5) and below 10(5) IU per mL, respectively. Median virus concentration in the case group was 4.85 x 10(7) IU per mL at Time Point T0 and was reduced to 4 x 10(2) IU per mL at the time of the next donation (3 months later). Neutralizing antibodies (VP2) were detected in all donations if virus load was reduced to less than 10(5) IU per mL. CONCLUSION: The release of B19 DNA-positive blood products with a concentration of less than 105 IU per mL is thought to be safe due to the high level of neutralizing VP2 antibodies and is currently examined in a donor recipient infectivity study. In contrast, blood products with a high B19 DNA concentration (> or =10(5) IU/mL), some of which did not contain neutralizing antibodies, were discarded to protect at risk individuals.

Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.

BACKGROUND: Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodulatory cellular therapies. Translation of auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS). STUDY DESIGN AND METHODS: The capacity of human platelet lysate (HPL) to replace FBS for clinical-scale MSC propagation was analyzed. RESULTS: HPL could be efficiently produced from buffy coats. Multiplex analyses allowed a distinct HPL growth factor profile to be delineated. With a previously established two-step clinical-scale procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. With only 3 x 10(5) primary culture-derived MSCs, a mean of 4.36 x 10(8) HPL-MSCs (range, 3.01 x 10(8)-5.40 x 10(8)) was obtained within a single secondary 11- to 13-day culture step. Although morphologically distinct, HPL-MSCs and FBS-MSCs did not differ significantly in terms of immunophenotype, differentiation potential in vitro, and lack of tumorigenicity in nude mice in vivo. CONCLUSIONS: Replacing FBS with HPL prevents bovine prion, viral, and zoonose contamination of the stem cell product. This new efficient FBS-free two-step procedure for clinical-scale MSC propagation may represent a major step toward challenging new stem cell therapies.

Blood group chimerism.

PURPOSE OF REVIEW: With improved methods for detecting chimeras and growing numbers of stem cell transplantations, blood group and other forms of chimerism are observed with increasing frequency. This review will focus on the state of science and new insights into the multifaceted subject of blood group chimerism. RECENT FINDINGS: Recognition that the immune system tolerates chimeric cells under certain conditions has led to efforts to elucidate related immune-modulating processes. The chimeric state following transplantation of hematopoietic stem cells, as well as after solid organ transplantation, is of special interest. Also, chimerism is considered to be a potential trigger for certain autoimmune diseases. Natural chimerism is more frequent than previously recognized. Using improved laboratory techniques, investigators can often trace chimeric tissues to their origin. New therapeutic strategies have been applied after stem cell transplantation depending on the chimeric state. Also, recent research has resulted in methods for determining chimerism in maternal peripheral blood that reflect fetal blood type and certain congenital diseases. SUMMARY: The subject of human chimerism has evolved from a curiosity of nature to an important field of research and, potentially, holds the key to advancing our understanding of the fundamental mechanisms of immune tolerance.