BACKGROUND: In the past 10 years, PCR‐based methods have been described to allow the detection of gene polymorphisms responsible for many blood group antigens. These methods are routinely used to test samples of fetal origin and to resolve serologic discrepancies. Another interesting application of blood group genotyping could be the extended typing of blood donors for minor antigens to facilitate the procurement of compatible blood for alloimmunized patients. STUDY DESIGN AND METHODS: PCR‐based tests have been modified to allow multiplex amplification of specific fragments of blood group genes and the convenient detection of hybridized amplicons by ELISA in a microplate format. RESULTS: The results obtained show that fragments of the Rh (D, c, C, e, E), Kell (K, k), Duffy (Fy a , Fy b ), and Kidd (Jk a , Jk b ) genes could be amplified along with controls in multiplex PCR reactions. Labeling of amplicons with digoxigenin allowed their solid‐phase detection in microplate wells previously coated with individual blood group‐specific oligonucleotides. A comparative study performed with 100 individuals showed a 99.7 percent concordance between genotypes and phenotypes for the 11 antigens assayed, with only three discrepant Fy b genotypes. CONCLUSION: Extended genotyping could be performed once on regular donors and confirmed when needed by standard serologic RBC assays. The format of these tests will allow easy automation of the procedure including the interpretation and downloading of the results with existing ELISA software.
ABSTRACT Background Emerging evidence suggests that COVID-19 vaccination decreases the sensitivity of anti-nucleocapsid (N) serologies, making them less reliable to assess recently-acquired infections. We therefore developed and tested a new approach based on the ratio of the anti-N absorbance of longitudinal samples to overcome this limitation. Methods Previously vaccinated repeat plasma donors provided at least one pre-infection (reference) and one post-infection (test) sample. All samples were tested using an in-house anti-N ELISA. Seropositivity was determined based on the ratio between the anti-N absorbance of the test and reference samples. The ratio approach was tested in a real-world setting during three cross-sectional serosurveys carried out among plasma donors in Québec, Canada. Results Using a cut-off ratio of 1.5, the approach had a sensitivity of 95.2% among the 248 previously vaccinated and infected donors compared with 63.3% for the conventional approach. When tested in a real-world setting, the ratio-based approach yielded an adjusted seroprevalence of 27.4% (95% confidence interval [CI]=23.8%-30.9%) at the latest time point considered, compared to 15.1% (95% CI=12.2%-18.0%) for the conventional approach. Conclusions This article describes a new and highly-sensitive approach that captures a significantly greater proportion of vaccinated individuals with a recent history of SARS-CoV-2 infection.
A substantial proportion of individuals infected with SARS-CoV-2 do not experience noticeable symptoms typical of COVID-19. Our objectives were to evaluate the impact of the first wave of the pandemic in Québec by measuring SARS-CoV-2 antibody seroprevalence in a convenience sample of healthy blood donors and to study the association between seropositivity and the occurrence of COVID-19 symptoms.The study design was a cross-sectional serological survey with a nested case-control study. Residual blood samples from donations collected between May 25 and July 9, 2020 (well before vaccination rollout) in the province of Québec were tested for anti-Spike RBD antibodies by ELISA. Seropositive donors and a control group of seronegative donors were questioned about prior COVID-19 symptoms. All qualified blood donors were eligible for participation.A total of 7691 blood donors were included in the study. After adjustments, the seroprevalence rate was 2.2% (95% CI 1.9-2.6). Seropositive donors reported one or more symptoms in a proportion of 52.2% (95% CI 44.2-60.1); this proportion was 19.1% (95% CI 13.4-26.1) among seronegative donors, suggesting that approximately 50-66% of all infections were asymptomatic. Univariate analysis of associations between symptoms and seropositivity revealed that except for rhinorrhea, all symptoms were significantly associated with seropositivity.Assuming that blood donors are fairly representative of the general adult population, this study shows that less than 3% of 18-69-year-olds have been infected during the first wave of the pandemic in the province of Québec. Our data also confirm that many infections escaped detection, including a substantial proportion that were asymptomatic.RéSUMé: OBJECTIFS: Une proportion substantielle de personnes infectées par le SRAS-CoV-2 ne présentent pas de symptômes visibles typiques de la COVID-19. Nos objectifs étaient d’évaluer l’impact de la première vague de la pandémie au Québec en mesurant la séroprévalence des anticorps anti-SRAS-CoV-2 chez les donneurs de sang en bonne santé, et d’étudier l’association entre la séropositivité et la survenue des symptômes de la COVID-19. MéTHODES: Le design de l’étude était une enquête de sérologie transversale avec une étude cas-témoins nichée dans la cohorte. Des échantillons de sang provenant de dons recueillis entre le 25 mai et le 9 juillet 2020 (bien avant le déploiement de la vaccination) dans la province de Québec ont été testés pour les anticorps anti-spicule RBD (Receptor Binding Domain) par ELISA. Les donneurs séropositifs et un groupe témoin de donneurs séronégatifs ont été interrogés sur les symptômes spécifiques à la COVID-19. Tous les donneurs qualifiés pour le don de sang étaient éligibles à participer à l’étude. RéSULTATS: Au total, 7 691 donneurs de sang ont été inclus dans l’étude. Après ajustements, le taux de séroprévalence était de 2,2 % (IC à 95% 1,9–2,6). Les donneurs séropositifs ont signalé un ou plusieurs symptômes dans une proportion de 52,2 % (IC à 95% 44,2–60,1); cette proportion était de 19,1 % (IC à 95% 13,4–26,1) parmi les donneurs séronégatifs, ce qui suggère qu’entre 50 % et 66 % de toutes les infections étaient asymptomatiques. Une analyse univariée des associations entre les symptômes et la séropositivité a révélé qu’à l’exception de la rhinorrhée, tous les symptômes étaient significativement associés à la séropositivité. CONCLUSION: En supposant que les donneurs de sang sont assez représentatifs de la population adulte générale, cette étude montre que moins de 3 % des 18–69 ans ont été infectés lors de la première vague de la pandémie dans la province du Québec. Nos données confirment également que de nombreuses infections n’ont pas fait l’objet d’un test moléculaire de dépistage, y compris une proportion importante qui était asymptomatique.
Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost.A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further.Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins.With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.
SUMMARY Objectives The goal of this study was to establish a red blood cell antigen portrait of self‐identified Black donors for the province of Quebec, Canada. Background The demand for extensively phenotyped red blood cells is on the rise. A good example is the sickle cell patient cohort. To better answer their transfusion needs, Héma‐Québec put forward great efforts to increase the recruitment of donors among cultural communities. Materials and Methods In October 2009, an optional question was added on the record of donation to indicate the donor's ethnicity. Self‐identified Black donors were extensively phenotyped by the Immunohematology Laboratory, whereas the Research and Development team genotyped red blood cell antigens to complete the picture. Results Approximately 1500 self‐identified Black donors have donated blood at least once since the beginning of the programme. Genotyping results predicted rare phenotypes: 18 S−s− (3 U−, 15 U+ w ), 15 Js(a+b−), 5 Hy−, 3 Jo(a−), 34 hr B + w /− and 15 hr B −. Conclusion These Black donors, with or without a rare phenotype, are precious to the patient cohort depending on blood transfusions and to our organisation as the blood provider for the whole province of Quebec.
Abstract Background The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus is the cause of the ongoing coronavirus disease 2019 (COVID‐19) pandemic, infecting millions of people and causing more than two million deaths. The SARS‐CoV‐2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID‐19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike declines rapidly after the resolution of the infection. Study Design and Methods To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full‐length SARS‐CoV‐2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS‐CoV‐2 Spike and used it to develop a high‐throughput flow cytometry‐based assay to detect SARS‐CoV‐2 Spike‐specific antibodies in the plasma of convalescent donors. Results and Conclusion We found that the level of antibodies targeting the full‐length SARS‐CoV‐2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD‐specific antibodies and the number of days post‐symptom onset. These findings help to better understand the decline of humoral responses against the SARS‐CoV‐2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.
John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status.Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies.Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7AArg347 and variant Sema7ALeu347 proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7AArg347 protein and not the variant one.A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein.
Abstract Background A global downtrend in blood usage has been observed by many countries, while the demand for antigen‐negative red blood cell (RBC) units used in antigen‐matched transfusions keeps increasing. The declining number of units collected exposes blood providers to a rapidly evolving supply challenge. Methods This study was conducted retrospectively with use of internal data analysis to weigh Québec's situation regarding global and antigen‐negative RBC demand, to measure the effects of community‐directed recruitment and blood drives, and to evaluate the benefits of mass‐scale RBC genotyping. Results Our findings confirm a global RBC usage downtrend of over 20% total in the past 10 years with a steady antigen‐negative usage and highlight the most requested negative antigen combinations. Our data also show our +39.5% progress regarding the number of Black donors recruited for antigen matching of patients with sickle cell disease in the past 3 years, as well as a constantly growing number of just‐in‐time blood collection for complex orders. Finally, our data summarize the efficiency of our mass‐scale RBC genotyping efforts. Conclusion Altogether, this study confirms the demand trends for regular and antigen‐negative RBC units in Québec and the efficient effects of our recruitment and typing strategies.
Abstract The Omicron BQ.1.1 variant is now the major SARS-CoV-2 circulating strain in many countries. Because of the many mutations present in its Spike glycoprotein, this variant is resistant to humoral responses elicited by monovalent mRNA vaccines. With the goal to improve immune responses against Omicron subvariants, bivalent mRNA vaccines have recently been approved in several countries. In this study, we measure the capacity of plasma from vaccinated individuals, before and after a fourth dose of mono-or bivalent mRNA vaccine, to recognize and neutralize the ancestral (D614G) and the BQ.1.1 Spikes. Before and after the fourth dose, we observe a significantly better recognition and neutralization of the ancestral Spike. We also observe that fourth-dose vaccinated individuals who have been recently infected recognize and neutralize better the BQ.1.1 Spike, independently of the mRNA vaccine used, than donors who have never been infected or have an older infection. Our study supports that hybrid immunity, generated by vaccination and a recent infection, induces higher humoral responses than vaccination alone, independently of the mRNA vaccine used.
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