How do we achieve blinding in modern electronic and paper medical records during the conduct of transfusion trials?

BACKGROUND: Regulatory aspects of transfusion medicine add complexity in blinded transfusion trials when considering various electronic record keeping software and blood administration processes. The aim of this study is to explore strategies when blinding transfusion components and products in paper and electronic medical records. METHODS: Surveys were collected and interviews were conducted for 18 sites across various jurisdictions in North America to determine solutions applied in previous transfusion randomized control trials. RESULTS: Sixteen responses were collected of which 11 had previously participated in a transfusion randomized control trial. Various solutions were reported which were specific to the laboratory information system (LIS) and electronic medical record (EMR) combinations although solutions could be grouped into four categories which included the creation of a study product code in the LIS, preventing the transmission of data from the LIS to the EMR, utilizing specialized stickers and labels to conceal product containers and documents in the paper records, and modified bedside procedures and documentation. DISCUSSION: LIS and EMR combinations varied across sites, so it was not possible to determine combination-specific solutions. The study was able to highlight solutions that may be emphasized in future iterations of LIS and EMR software as well as procedural changes that may minimize the risk of unblinding.

How do I provide rare red cells to patients?

BACKGROUND: The demand for rare blood is expected to increase in Canada as its population continues to expand through immigration from diverse regions of the world. MATERIAL AND METHODS: This paper outlines a national approach to providing rare red cells for patients through the Rare Blood Program of Canadian Blood Services (CBS). Data detailing the rare red cell requests and inventory managed by CBS' Rare Blood Program is provided. RESULTS: The provision of rare red cells involves multiple considerations such as multidisciplinary communication, serologic/molecular confirmation, donor recruitment, inventory optimization and logistical factors. CONCLUSION: The description of CBS' Rare Blood Program will inform others that seek to create, optimize, or expand programs that facilitate the provision of rare blood. New technologies such as next-generation sequencing may also affect how rare donors are identified and recruited in the future.

Severe fetal anemia caused by anti-Jra : Burst forming unit-erythroid colony formation inhibition assay suggesting possible erythroid suppression mechanism.

BACKGROUND: The Jr blood group system includes a single, high-prevalence antigen, Jra , encoded by the ABCG2 gene. The impact of anti-Jra in pregnancy is variable, ranging from no clinical effect to severe anemia including some fetal deaths. Case reports have postulated that anti-Jra mediated fetal anemia is poorly hemolytic, suggesting other mechanisms of anemia may be involved. STUDY DESIGN AND METHODS: We describe the case of severe anti-Jra mediated fetal anemia. At Canadian Blood Services laboratories, maternal anti-Jra was tested for phagocytic activity via a monocyte monolayer assay (MMA) and erythroid suppression via inhibition of burst forming unit-erythroid (BFU-E) colony formation assays. The New York Blood Center sequenced exons 4 and 7 of the ABCG2 gene. RESULTS AND DISCUSSION: Sequencing of exons 4 and 7 of the ABCG2 gene revealed maternal compound heterozygosity for two nonsense mutations at exon 7 (c.706 C > T and c.784G > T). Fetal sequencing revealed the c.706C > T polymorphism. The MMA showed a borderline phagocytic index (around the cutoff of five for both donor segments tested [5 ± 1 and 7 ± 3]). The BFU-E colony formation inhibition assay suggested a dose-dependent inhibition of BFU-E colony formation with inhibition percentages of 4%, 11%, and 43% at maternal serum concentrations of 2%, 5%, and 10%, respectively. Our findings support the hypothesis that anti-Jra may impair erythropoiesis leading to clinically significant fetal/neonatal anemia. A referral to maternal fetal medicine is recommended if anti-Jra is detected in pregnancy, regardless of the titer.

Transfusion of ABO-group identical red blood cells following uncrossmatched transfusion does not lead to higher mortality in civilian trauma patients.

BACKGROUND: Questions persist about the safety of switching non-group O recipients of group O uncrossmatched red blood cells (RBC) or low titer group O whole blood (LTOWB) to ABO-identical RBCs during their resuscitation. METHODS: The database of an earlier nine-center study of transfusing incompatible plasma to trauma patients was reanalyzed. The patients were divided into three groups based on 24-h RBC transfusion: (1) group O patients who received group O RBC/LTOWB units (control group, n = 1203), (2) non-group O recipients who received only group O units (n = 646), (3) non-group O recipients who received at least one unit of group O and non-group O units (n = 562). Fixed marginal effect of receipt of non-O RBC units on 6- and 24-h and 30-day mortality was calculated. RESULTS: The non-O patients who received only group O RBCs received fewer RBC/LTOWB units and had slightly but significantly lower injury severity score compared to control group; non-group O patients who received both group O and non-O units received significantly more RBC/LTOWB units and had a slightly but significantly higher injury severity score compared to control group. In the multivariate analysis, the non-O patients who received only group O RBCs had significantly higher mortality at 6-h compared to the controls; the non-group O recipients of O and non-O RBCs did not demonstrate higher mortality. At 24-h and 30-days, there were no differences in survival between the groups. CONCLUSION: Providing non-group O RBCs to non-group O trauma patients who also received group O RBC units is not associated with higher mortality.

Serologic reactivity of unidentified specificity in antenatal testing and hemolytic disease of the fetus and newborn: The BEST collaborative study.

BACKGROUND: The clinical significance of serologic reactivity of unidentified specificity (SRUS) in pregnancy is not clear based on available literature. The aim of this study is to determine if SRUS is associated with hemolytic disease of the fetus and newborn (HDFN). STUDY DESIGN AND METHODS: Retrospective data were collected from eight institutions over an 11-year study period (2010-2020), when available (5/8 sites). The outcome of the pregnancies with SRUS-no, mild, moderate, or severe HDFN-was determined. RESULTS: SRUS was demonstrated in 589 pregnancies. After excluding those with incomplete data, a total of 284 pregnancies were included in the primary HDFN outcome analysis. SRUS was detected in 124 (44%) pregnancies in isolation, and none were affected by HDFN. Of 41 pregnancies with SRUS and ABO incompatibility, 37 (90%) were unaffected, and 4 (10%) were associated with mild HDFN. Of 98 pregnancies with SRUS and concurrent identifiable antibody reactivity(s), 80 (81%) were unaffected, and 19 (19%) were associated with mild to severe HDFN. There was 1 case of mild HDFN and 1 case of severe HDFN in the 21 pregnancies with SRUS, ABO incompatibility, and concurrent identifiable antibody reactivity(s), and 19 (90%) were unaffected by HDFN. Among all patients with repeat testing, newly identified alloantibodies or other antibodies were identified in 63 of 212 (30%) patients. Although most were not clinically significant, on occasion SRUS preceded clinically significant antibody(s) associated with HDFN (3%, 5/188). CONCLUSION: The antenatal serologic finding of SRUS in isolation is not associated with HDFN but may precede clinically significant antibodies.

An online immunohematology educational resource for post-graduate hematology trainees: LearnSerology.ca.

BACKGROUND: Canadian hematology residents are required to demonstrate competencies in transfusion medicine by the end of their 2-year training. Prior evaluation of final year trainees revealed significant variation in knowledge. To address the lack of standardization in serology teaching, an online educational immunohematology resource was created and evaluated. STUDY DESIGN AND METHODS: All Canadian post-graduate trainees completing a residency program in adult hematology during the 2018/2019 academic year were invited to participate. Only trainees from one university were exposed to the program curriculum. A validated exam was administered to trainees at both exposed and unexposed sites at the start of the academic year as a pre-test and in the following year as a post-test. The effectiveness of the program was assessed by both comparing the degree of improvement from pre- to post-test, and by comparing performance on the post-test. RESULTS: 57 trainees from 13 universities completed the pre-test, and 45 trainees from 14 universities completed the post-test. A strong trend towards better performance in the exposed vs non-exposed trainees on the post-test was observed, and the difference was more pronounced, and statistically significant, when analysis was limited to two questions relating to interpretation of an antibody investigation panel. DISCUSSION: LearnSerology.ca is effective and may be potentially superior to traditional immunohematology teaching. The interactive capability of the platform can improve skills related to the resolution of red cell antibody panels.

Impacts of COVID-19 and elective surgery cancellations on platelet supply and utilization in the Canadian Province of British Columbia.

BACKGROUND AND OBJECTIVES: The coronavirus disease 2019 (COVID-19) pandemic raised concerns about the vulnerability of platelet supply and the uncertain impact of the resumption of elective surgery on utilization. We report the impact of COVID-19 on platelet supply and utilization across a large, integrated healthcare system in the Canadian province of British Columbia (BC). MATERIALS AND METHODS: Historical platelet use in BC by indication was compiled for fiscal year 2010/2011-2019/2020. Platelet collections, initial daily inventory and disposition data were assessed pre-COVID-19 (1 April 2018-15 March 2020) and for two COVID-19 time periods in BC: a shutdown phase with elective surgeries halted (16 March-17 May, 2020) and a renewal phase when elective surgeries resumed (18 May-27 September 2020); comparisons were made provincially and for individual health authorities. RESULTS: Historically, elective surgeries accounted for 10% of platelets transfused in BC. Initial daily supplier inventory increased from baseline during both COVID-19 periods (93/90 units vs. 75 units pre-COVID-19). During the shutdown phase, platelet utilization decreased 10.4% (41 units/week; p < 0.0001), and remained significantly decreased during the ensuing renewal period. Decreased platelet utilization was attributed to fewer transfusions during the shutdown phase followed by a decreased discard/expiry rate during the renewal phase compared to pre-COVID-19 (15.2% vs. 18.9% pre-COVID-19; p < 0.0001). Differences in COVID-19 platelet utilization patterns were noted between health authorities. CONCLUSION: Decreased platelet utilization was observed in BC compared to pre-COVID-19, likely due to a transient reduction in elective surgery as well as practice and policy changes triggered by pandemic concerns.

Epigenetic Heterogeneity and Mitotic Heritability Prime Endothelial Cell Gene Induction.

Homogeneous populations of mature differentiated primary cell types can display variable responsiveness to extracellular stimuli, although little is known about the underlying mechanisms that govern such heterogeneity at the level of gene expression. In this article, we show that morphologically homogenous human endothelial cells exhibit heterogeneous expression of VCAM1 after TNF-α stimulation. Variability in VCAM1 expression was not due to stochasticity of intracellular signal transduction but rather to preexisting established heterogeneous states of promoter DNA methylation that were generationally conserved through mitosis. Variability in DNA methylation of the VCAM1 promoter resulted in graded RelA/p65 and RNA polymerase II binding that gave rise to a distribution of VCAM1 transcription in the population after TNF-α stimulation. Microarray analysis and single-cell RNA sequencing revealed that a number of cytokine-inducible genes shared this heterogeneous response pattern. These results show that heritable epigenetic heterogeneity is fundamental in inflammatory signaling and highlight VCAM1 as a metastable epiallele.

Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA.

Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell type-specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for spliced-transcript endothelial-enriched lncRNA (STEEL) in angiogenic potential, macrovascular/microvascular identity, and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear enriched, and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse ECs. Of interest, STEEL up-regulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL up-regulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity.

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests.

BACKGROUND: West Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)-reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination. CASE SERIES: Cases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine. STUDY DESIGN AND METHODS: WNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV-reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed. RESULTS: Supplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT-positive, WNV NAT-negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT. CONCLUSIONS: We recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

Transfusion of blood components containing ABO-incompatible plasma does not lead to higher mortality in civilian trauma patients.

BACKGROUND: This study investigated the effect on mortality of transfusing ABO-incompatible plasma from all sources during trauma resuscitation. METHODS: Demographic, transfusion, and survival data were retrospectively extracted on civilian trauma patients. Patients were divided by receipt of any quantity of ABO-incompatible plasma from any blood product (incompatible group) or receipt of solely ABO-compatible plasma (compatible group). The primary outcome was 30-day mortality, while other outcomes included 6- and 24-hour mortality. Mixed-effects logistic regression was used to model the effect of various predictor variables, including receipt of incompatible plasma, on mortality outcomes. RESULTS: Nine hospitals contributed data on a total of 2618 trauma patients. There were 1282 patients in the incompatible group and 1336 patients in the compatible group. In both the unadjusted and adjusted models, the 6-hour, 24-hour, and 30-day mortality rates were not significantly different between these groups. The patients in the incompatible group were then divided into high volume (>342 mL) and low volume (≤342 mL) incompatible plasma recipients. In the adjusted model, the high-volume group had higher 24-hour mortality when the Trauma Injury Severity Score survival prediction was >50%. Mortality at 6 hours and 30 days was not higher in this model. The low-volume group did not have increased mortality at any of the time points in this adjusted model. CONCLUSION: The transfusion of incompatible plasma in civilian trauma resuscitation does not lead to higher 30-day mortality. The finding of higher mortality in a select group of recipients in the secondary analysis warrants further study.

Utilising red cell antigen genotyping and serological phenotyping in sickle cell disease patients to risk-stratify patients for alloimmunisation risk.

BACKGROUND: Alloimmunisation and haemolytic transfusion reactions (HTRs) can occur in patients with sickle cell disease (SCD) despite providing phenotype-matched red blood cell (RBC) transfusions. Variant RBC antigen gene alleles/polymorphisms can lead to discrepancies in serological phenotyping. We evaluated differences between RBC antigen genotyping and phenotyping methods and retrospectively assessed if partial antigen expression may lead to increased risk of alloimmunisation and HTRs in SCD patients at a tertiary centre in Canada. METHODS: RBC antigen phenotyping and genotyping were performed by a reference laboratory on consenting SCD patients. Patient demographic, clinical and transfusion-related data were obtained from a local transfusion registry and chart review after research ethics board approval. RESULTS: A total of 106 SCD patients were enrolled, and 91% (n = 96) showed additional clinically relevant genotyping information when compared to serological phenotyping alone. FY*02N.01 (FY*B GATA-1) (n = 95; 90%) and RH variant alleles (n = 52, 49%; majority accompanied by FY*02N.01) were common, the latter with putative partial antigen expression in 25 patients. Variability in genotype-phenotype antigen prediction occurred mostly in the Rh system, notably with the e antigen (kappa: 0.17). Fifteen (14.2%) patients had a history of alloimmunisation, with five having HTR documented; no differences in clinical outcomes were found in patients with partial antigen expression. Genotype/extended-phenotype matching strategies may have prevented alloimmunisation events. CONCLUSION: We show a high frequency of variant alleles/polymorphisms in the SCD population, where genotyping may complement serological phenotyping. Genotyping SCD patients before transfusion may prevent alloimmunisation and HTRs, and knowledge of the FY*02N.01 variant allele increases feasibility of finding compatible blood.

The learning curves of a validated virtual reality hip arthroscopy simulator.

INTRODUCTION: Decreases in trainees' working hours, coupled with evidence of worse outcomes when hip arthroscopies are performed by inexperienced surgeons, mandate an additional means of training. Though virtual reality simulation has been adopted by other surgical specialities, its slow uptake in arthroscopic training is due to a lack of evidence as to its benefits. These benefits can be demonstrated through learning curves associated with simulator training-with practice reflecting increases in validated performance metrics. METHODS: Twenty-five medical students with no previous experience of hip arthroscopy completed seven weekly simulated arthroscopies of a healthy virtual hip joint using a 70° arthroscope in the supine position. Twelve targets were visualised within the central compartment, six via the anterior portal, three via the anterolateral portal and three via the posterolateral portal. Task duration, number of collisions (bone and soft-tissue), and distance travelled by arthroscope were measured by the simulator for every session of each student. RESULTS: Learning curves were demonstrated by the students, with improvements in time taken, number of collisions (bone and soft-tissue), collision length and efficiency of movement (all p < 0.01). Improvements in time taken, efficiency of movement and number of collisions with soft-tissue were first seen in session 3 and improvements in all other parameters were seen in session 4. No differences were found after session 5 for time taken and length of soft-tissue collision. No differences in number of collisions (bone and soft-tissue), length of collisions with bone, and efficiency of movement were found after session 6. CONCLUSIONS: The results of this study demonstrate learning curves for a hip arthroscopy simulator, with significant improvements seen after three sessions. All performance metrics were found to improved, demonstrating sufficient visuo-haptic consistency within the virtual environment, enabling individuals to develop basic arthroscopic skills.

Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell gene regulation.

Although the functional role of chromatin marks at promoters in mediating cell-restricted gene expression has been well characterized, the role of intragenic chromatin marks is not well understood, especially in endothelial cell (EC) gene expression. Here, we characterized the histone H3 and H4 acetylation profiles of 19 genes with EC-enriched expression via locus-wide chromatin immunoprecipitation followed by ultra-high-resolution (5 bp) tiling array analysis in ECs versus non-ECs throughout their genomic loci. Importantly, these genes exhibit differential EC enrichment of H3 and H4 acetylation in their promoter in ECs versus non-ECs. Interestingly, VEGFR-2 and VEGFR-1 show EC-enriched acetylation across broad intragenic regions and are up-regulated in non-ECs by histone deacetylase inhibition. It is unclear which histone acetyltransferases (KATs) are key to EC physiology. Depletion of KAT7 reduced VEGFR-2 expression and disrupted angiogenic potential. Microarray analysis of KAT7-depleted ECs identified 263 differentially regulated genes, many of which are key for growth and angiogenic potential. KAT7 inhibition in zebrafish embryos disrupted vessel formation and caused loss of circulatory integrity, especially hemorrhage, all of which were rescued with human KAT7. Notably, perturbed EC-enriched gene expression, especially the VEGFR-2 homologs, contributed to these vascular defects. Mechanistically, KAT7 participates in VEGFR-2 transcription by mediating RNA polymerase II binding, H3 lysine 14, and H4 acetylation in its intragenic region. Collectively, our findings support the importance of differential histone acetylation at both promoter and intragenic regions of EC genes and reveal a previously underappreciated role of KAT7 and intragenic histone acetylation in regulating VEGFR-2 and endothelial function.

Evaluation of immunohematology knowledge in hematology trainees.

BACKGROUND: Canadian hematology trainees are expected to attain clinical knowledge in the subject of red blood cell and platelet antigen systems and the principles of transfusion medicine. However, the relative degree of expertise required in blood bank serology is not well defined. STUDY DESIGN AND METHODS: A modified Delphi approach involving 10 Canadian hematology program directors was utilized to identify 12 relevant topics in immunohematology. A multiple-choice exam was developed and validated among hematology trainees from 13 hematology training programs across Canada. A Rasch analysis was used to determine fit of the examination before deploying the exam the following year to ascertain the level of knowledge in hematology trainees. RESULTS: The exam was piloted with 62 hematology trainees. The reliability of the exam was 0.93 with a mean item fit score of 1.01. The exam was able to discriminate between training years and self-rated expertise with better performance attained by more advanced trainees (p < 0.01). No differences were seen between geographic regions. A modified version of the exam was deployed the following year to 85 trainees, with a mean score of 58.9% ± 15.3%. Trainees scored poorest on topics concerning antibody investigations and D variants. CONCLUSION: A standardized exam for assessing hematology trainees on their expected expertise in transfusion immunohematology has been developed and can be used to assess the efficacy of educational resources provided in the subject. Trainees had a low overall mean score indicating additional educational initiatives are warranted.