Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.

Tissue factor pathway inhibitor is required for cerebrovascular development in mice.

Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.

Associations between ABO non-identical platelet transfusions and patient outcomes-A multicenter retrospective analysis.

BACKGROUND: Due to platelet availability limitations, platelet units ABO mismatched to recipients are often transfused. However, since platelets express ABO antigens and are collected in plasma which may contain ABO isohemagglutinins, it remains controversial as to whether ABO non-identical platelet transfusions could potentially pose harm and/or have reduced efficacy. STUDY DESIGN AND METHODS: The large 4-year publicly available Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) database was used to investigate patient outcomes associated with ABO non-identical platelet transfusions. Outcomes included mortality, sepsis, and subsequent platelet transfusion requirements. RESULTS: Following adjustment for possible confounding factors, no statistically significant association between ABO non-identical platelet transfusion and increased risk of mortality was observed in the overall cohort of 21,176 recipients. However, when analyzed by diagnostic category and recipient ABO group, associations with increased mortality for major mismatched transfusions were noted in two of eight subpopulations. Hematology/Oncology blood group A and B recipients (but not group O) showed a Hazard Ratio (HR) of 1.29 (95%CI: 1.03-1.62) and intracerebral hemorrhage group O recipients (but not groups A and B) showed a HR of 1.75 (95%CI: 1.10-2.80). Major mismatched transfusions were associated with increased odds of receiving additional platelet transfusion each post-transfusion day (through day 5) regardless of the recipient blood group. DISCUSSION: We suggest that prospective studies are needed to determine if specific patient populations would benefit from receiving ABO identical platelet units. Our findings indicate that ABO-identical platelet products minimize patient exposure to additional platelet doses.

The relationship between heavy menstrual bleeding, iron deficiency, and iron deficiency anemia.

For reproductive-aged women, the symptom of heavy menstrual bleeding is highly prevalent and a major contributor to iron deficiency and its most severe manifestation, iron deficiency anemia. It is recognized that these 2 clinical entities are not only highly prevalent, but their interrelationship is poorly appreciated and frequently normalized by society, healthcare providers, and affected girls and women themselves. Both heavy menstrual bleeding and iron deficiency, with or without anemia, adversely impact quality of life-heavy menstrual bleeding during the episodes of bleeding and iron deficiency on a daily basis. These combined issues adversely affect the lives of reproductive-aged girls and women of all ages, from menarche to menopause, and their often-insidious nature frequently leads to normalization. The effects on cognitive function and the related work and school absenteeism and presenteeism can undermine the efforts and function of women in all walks of life, be they students, educators, employers, or employees. There is also an increasing body of evidence that suggests that iron deficiency, even in early pregnancy, may adversely impact fetal neurodevelopment with enduring effects on a spectrum of cognitive and psychological disorders, critically important evidence that begs the normalization of iron stores in reproductive-aged women. The authors seek to raise individual, societal, and professional awareness of this underappreciated situation in a fashion that leads to meaningful and evidence-based changes in clinical guidance and healthcare policy directed at preventing, screening, diagnosing, and appropriately managing both disorders. This manuscript provides evidence supporting the need for action and describes the elements necessary to address this pervasive set of conditions that not only affect reproductive-aged girls and women but also the lives of children everywhere.

Iron status of blood donors.

PURPOSE OF REVIEW: This review examines recent research on the prevalence and importance of iron deficiency in blood donors, and on efforts to mitigate it. RECENT FINDINGS: Premenopausal females, teenagers, and high-frequency donors are at the highest risk for donation-induced iron deficiency, in both high-resource and low-resource settings. The physiology relating iron stores to hemoglobin levels and low hemoglobin deferral is well elucidated in blood donor populations, yet the clinical effects attributable to iron loss in the absence of anemia are challenging to identify. Expanded adoption of ferritin testing is improving donor management but may cause decreases in the blood supply from temporary donor loss. The potential for personalized donor management is emerging with development of computational models that predict individual interdonation intervals that aim to optimize blood collected from each donor while minimizing low hemoglobin deferrals. SUMMARY: Measures to reduce iron deficiency are available that can be deployed on a standardized or, increasingly, personalized basis. Blood centers, regulators, and donors should continue to evaluate different tactics for addressing this problem, to obtain a balanced approach that is optimal for maintaining adequate collections while safeguarding donor health.

Sex-specific genetic modifiers identified susceptibility of cold stored red blood cells to osmotic hemolysis.

BACKGROUND: Genetic variants have been found to influence red blood cell (RBC) susceptibility to hemolytic stress and affect transfusion outcomes and the severity of blood diseases. Males have a higher susceptibility to hemolysis than females, but little is known about the genetic mechanism contributing to the difference. RESULTS: To investigate the sex differences in RBC susceptibility to hemolysis, we conducted a sex-stratified genome-wide association study and a genome-wide gene-by-sex interaction scan in a multi-ethnic dataset with 12,231 blood donors who have in vitro osmotic hemolysis measurements during routine blood storage. The estimated SNP-based heritability for osmotic hemolysis was found to be significantly higher in males than in females (0.46 vs. 0.41). We identified SNPs associated with sex-specific susceptibility to osmotic hemolysis in five loci (SPTA1, KCNA6, SLC4A1, SUMO1P1, and PAX8) that impact RBC function and hemolysis. CONCLUSION: Our study established a best practice to identify sex-specific genetic modifiers for sexually dimorphic traits in datasets with mixed ancestries, providing evidence of different genetic regulations of RBC susceptibility to hemolysis between sexes. These and other variants may help explain observed sex differences in the severity of hemolytic diseases, such as sickle cell and malaria, as well as the viability of red cell storage and recovery.

FcRn augments induction of tissue factor activity by IgG-containing immune complexes.

Thromboembolism complicates disorders caused by immunoglobulin G (IgG)-containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), ß-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix.

[Figure: see text].

Demographic, clinical, and biochemical predictors of pica in high-intensity blood donors.

BACKGROUND: Frequent blood donors who contribute multiple times annually are important for maintaining an adequate blood supply. However, repeated donations exacerbate iron deficiency, which can lead to pica, a condition characterised as repeated eating or chewing of a non-nutritious substance such as ice, clay and dirt. Understanding characteristics of frequent donors that are associated with increased risk for developing pica will help to identify them and prevent this adverse consequence of blood donation. METHODS: Demographic, clinical, haematological, and biochemical factors associated with pica were investigated using univariable and multivariable logistic regression analysis in a cohort of 1693 high-intensity donors who gave nine or more units of whole blood in the preceding 2 years. Pica was classified by questionnaire responses as consuming at least 8 oz of ice daily and/or consumption of non-ice substances regardless of the amount and frequency. RESULTS: Pica was present in 1.5% of the high-intensity donors, and only occurred in those with ferritin <50 ng/ml. Of 16 candidate variables, only haematocrit (OR = 0.835, p = 0.020) was independently associated with pica. Although severe iron deficiency was more prevalent in high-intensity donors, pica behaviours were less prevalent than in less frequent donors (2.2%). CONCLUSION: We have uncovered predictors of pica in high-intensity donors, which further emphasises the need to continue to implement iron replacement programs to reduce the prevalence of pica and maintain a robust pool of frequent donors.

Demographic, clinical, and biochemical predictors of pica in a large cohort of blood donors.

BACKGROUND: Pica is characterized as repeatedly eating or chewing a non-nutritious substance including, but not limited to ice, clay and dirt, starch, raw pasta, chalk, coal, paint, or paper. Pica symptoms can be intense and addiction-like and disrupt quality of life. It is strongly linked to iron deficiency. Since substantial iron loss occurs during blood donation, blood donors may be susceptible to development of pica behaviors. METHODS: We investigated demographic, clinical, hematological, and biochemical factors associated with pica using univariable and multivariable logistic regression analysis in a cohort of 11,418 racially diverse blood donors. Pica was defined by questionnaire responses as consuming at least 8 oz of ice daily and/or consumption of non-ice substances regardless of the amount and frequency. RESULTS: Pica was present in 2.2% of the donors. The sensitivity and specificity of pica in iron-deficient donors were 36% and 82%, respectively. Lower ferritin (p = .001), non-Asian race (p < .001), higher red cell distribution width (p < .001), younger age, and restless legs syndrome (p = .008) were independently associated with pica. Female sex is associated with iron deficiency but was not an independent predictor of pica suggesting that iron deficient males and females were equally susceptible to the development of pica behaviors. Donors with normal ferritin levels also reported pica, reinforcing the role of non-iron related factors in its presentation. CONCLUSIONS: We have identified demographic, clinical, and biochemical predictors of pica that help identify those most at risk for developing pica behaviors, and thereby assist in its clinical diagnosis and treatment.

Iron status and self-reported fatigue in blood donors.

Fatigue is a reported symptom of iron depletion, but studies in blood donors show no conclusive link. We conducted an observational analysis of data from the STRIDE randomized trial to evaluate association of iron status with self-reported fatigue. STUDY DESIGN AND METHODS: Three blood centers randomly assigned 692 frequent donors to education or iron supplementation treatments. Biomarkers for iron status were measured during 20 to 24 months of follow-up. A fatigue score was derived from an 11-item questionnaire at baseline and final visits, and associations between iron status and fatigue were assessed. RESULTS: Final lab and questionnaire data were evaluable from 337 subjects. At baseline, female sex, older age, and anemia were associated with fatigue, but iron status was not. Mean (±SD) fatigue score change was 0.0 (±0.5). Mean (±SD) increase in iron stores was 1.0 (±3.5) mg/kg, but changes in body iron stores were not associated with fatigue score changes (0.01 per mg/kg; 95% CI, -0.01 to 0.02) or with fatigue (RR, 1.01; 95% CI, 0.99 to 1.04). The only factor associated with fatigue score changes was baseline fatigue (0.36; 95% CI, 0.25 to 0.48). CONCLUSION: Among high-frequency donors, neither iron status at baseline nor changes in iron status predicted fatigue during follow-up, with improvements limited to those with higher levels of baseline fatigue. Assessment of the association between iron and fatigue in blood donors benefits from careful consideration of study design and the study population.

Red blood cell transfusion does not increase risk of venous or arterial thrombosis during hospitalization.

Previous observational studies suggest associations between red blood cell (RBC) transfusion and risk for arterial or venous thrombosis. We determined the association between thrombosis and RBC transfusion in hospitalized patients using the Recipient Database from the National Heart Lung and Blood Institute (NHLBI) Recipient Epidemiology and Donor Evaluation Study-III. A thrombotic event was a hospitalization with an arterial or venous thrombosis ICD-9 code and administration of a therapeutic anticoagulant or antiplatelet agent. Patients with history of thrombosis or a thrombosis within 24 hours of admission were excluded. A proportional hazards regression model with time-dependent covariates was calculated. Estimates were adjusted for age, sex, hospital, smoking, medical comorbidities, and surgical procedures. Of 657 412 inpatient admissions, 67 176 (10.2%) received at least one RBC transfusion. Two percent (12927) of patients experienced a thrombosis. Of these, 2587 developed thrombosis after RBC transfusion. In unadjusted analyses, RBC transfusion was associated with an increased thrombosis risk [HR = 1.3 (95% CI 1.23-1.36)]. After adjustment for surgical procedures, age, sex, hospital, and comorbidities, no association between RBC transfusion on risk of venous and arterial thrombosis was found [HR 1.0 (95% CI: 0.96-1.05)]. Thus, RBC transfusion does not appear to be an important risk factor for thrombosis in most hospitalized patients.

Genetic and behavioral modification of hemoglobin and iron status among first-time and high-intensity blood donors.

BACKGROUND: Some people rapidly develop iron deficiency anemia following blood donation, while others can repeatedly donate without becoming anemic. METHODS: Two cohorts of blood donors were studied. Participants (775) selected from a 2-year longitudinal study were classified into six analysis groups based on sex, donation intensity, and low hemoglobin deferral. Associations with iron supplement use, cigarette smoking, and four genetic variants of iron metabolism were examined at enrollment and with longitudinal regression models. An unbiased assessment of genetic variability and ability to repeatedly donate blood without experiencing low hemoglobin deferral was conducted on participants (13,403) in a cross-sectional study who were examined by genome wide association (GWA). RESULTS: Behaviors and genetic variants were associated with differences in hemoglobin and ferritin change following repeated donation. At least weekly iron supplement use was associated with improved status in first-time donors, while daily use was associated with improved status in high-intensity donors. Cigarette smoking was associated with 0.5 g/dL increased hemoglobin in high-intensity donors. A736V in TMPRSS6 was associated with a rapid drop in hemoglobin and ferritin in first-time females following repeated donation. Conversely, the protective TMPRSS6 genotype was not enriched among high-intensity donors. H63D in HFE was associated with increased hemoglobin in female high-intensity donors. However, no differences in genotype between first-time and high-intensity donors were found in GWA analyses. CONCLUSION: Behavioral and genetic modifiers contributed to first-time donor hemoglobin and iron status, while iron supplement use was more important than underlying genetics in high-intensity donors.

The benefits of iron supplementation following blood donation vary with baseline iron status.

Whole blood donation rapidly removes approximately 10% of a donor's blood volume and stimulates substantial changes in iron metabolism and erythropoiesis. We sought to identify donors who benefit from iron supplementation, describe the nature of the benefit, and define the time course for recovery from donation. Blood samples were collected over 24 weeks following whole blood donation from 193 participants, with 96 participants randomized to 37.5 mg daily oral iron. Changes in total body, red blood cell (RBC), and storage iron, hepcidin, erythropoietin, and reticulocyte count were modeled using semiparametric curves in a mixed model. and the changes were compared among six groups defined by baseline ferritin (<12; 12-50; ≥50 ng/mL) and iron supplementation. The effect of oral iron on storage and RBC iron recovery was minimal in donors with baseline ferritin ≥50 ng/mL, but sizeable when ferritin was <50 ng/mL. Iron initially absorbed went to RBC and storage iron pools when ferritin was <12 ng/mL but went mostly to RBCs when ferritin was ≥12 ng/mL. Donors with ferritin ≥12 ng/mL had a "ripple" increase in reticulocytes ~100 days after donation indicating physiological responses occur months following donation. Thus, iron supplements markedly enhance recovery from whole blood donation in donors with ferritin <50 ng/mL. However, full recovery from donation requires over 100 days when taking iron. The findings also highlight the value of the study of blood donors for understanding human hemoglobin and iron metabolism and their usefulness for future studies as additional biomarkers are discovered.

Iron status and risk factors for iron depletion in a racially/ethnically diverse blood donor population.

BACKGROUND: The optimal approach for reducing iron depletion (ID) in blood donors may vary depending on biologic or behavioral differences across donors. STUDY DESIGN AND METHODS: More than 12,600 successful whole blood donors were enrolled from four US blood centers for ferritin testing. The study population was enriched for racial/ethnic minorities (1605 African American, 1616 Asian, 1023 Hispanic). Subjects completed questionnaires on ID risk factors. Logistic regression identified predictors of absent iron stores (AIS; ferritin <12 ng/mL) and low ferritin (LF; ferritin <26 ng/mL). RESULTS: Across all subjects, 19% had AIS and 42% had LF, with a sharp increase in risk observed with increasing donation intensity and among women a large decrease in risk in those more than 50 years old. When other factors were controlled for, African American and Asian donors showed 20% to 25% decreased risk for AIS compared to non-Hispanic Caucasian donors, while Hispanic donors had 25% higher risk. Daily iron supplementation reduced risk for LF and AIS by 30% to 40%, respectively, while the benefit from less frequent use was lower (7%-19% protection). Regular antacid use was associated with at least 20% increment to risk. Use of oral contraceptives or estrogen in females reduced risk by 16% to 22%, while males who reported supplemental testosterone use had a 50% to 125% greater risk for LF and AIS. CONCLUSIONS: This study confirms high prevalence of LF and AIS in US donors and the principal risk factors of age, sex, and donation frequency. Additional demographic and behavioral risk factors of secondary importance might allow for refinement of ID mitigation strategies.

Elevated risk for iron depletion in high-school age blood donors.

BACKGROUND: High school students 16 to 18 years-old contribute 10% of the US blood supply. Mitigating iron depletion in these donors is important because they continue to undergo physical and neurocognitive development. STUDY DESIGN AND METHODS: Study objectives were to determine the prevalence of iron depletion in 16- to 18-year-old donors and whether their risk for iron depletion was greater than adult donors. Successful, age-eligible donors were enrolled from high school blood drives at two large US blood centers. Plasma ferritin testing was performed with ferritin less than 12 ng/mL as our primary measure of iron depletion and ferritin less than 26 ng/mL a secondary measure. Multivariable repeated-measures logistic regression models evaluated the role of age and other demographic/donation factors. RESULTS: Ferritin was measured from 4265 enrollment donations September to November 2015 and 1954 follow-up donations through May 2016. At enrollment, prevalence of ferritin less than 12 ng/mL in teenagers was 1% in males and 18% in females making their first blood donation, and 8% in males and 33% in females with prior donations. Adjusted odds for ferritin less than 12 ng/mL were 2.1 to 2.8 times greater in 16- to 18-year-olds than in 19- to 49-year-olds, and for ferritin less than 26 ng/mL were 3.3- to 4.7-fold higher in 16- to 18-year-olds. Progression to hemoglobin deferral was twice as likely in 16- to 18-year-old versus 19- to 49-year-old females. CONCLUSION: Age 16 to 18 years-old is an independent risk factor for iron deficiency in blood donors at any donation frequency. Blood centers should implement alternate eligibility criteria or additional safety measures to protect teenage donors from iron depletion.

Blood, sweat, and tears: Red Blood Cell-Omics study objectives, design, and recruitment activities.

BACKGROUND: The Red Blood Cell (RBC)-Omics study was initiated to build a large data set containing behavioral, genetic, and biochemical characteristics of blood donors with linkage to outcomes of the patients transfused with their donated RBCs. STUDY DESIGN AND METHODS: The cohort was recruited from four US blood centers. Demographic and donation data were obtained from center records. A questionnaire to assess pica, restless leg syndrome, iron supplementation, hormone use, and menstrual and pregnancy history was completed at enrollment. Blood was obtained for a complete blood count, DNA, and ferritin testing. A leukocyte-reduced RBC sample was transferred to a custom storage bag for hemolysis testing at Storage Days 39 to 42. A subset was recalled to evaluate the kinetics and stability of hemolysis measures. RESULTS: A total of 13,403 racially/ethnically diverse (12% African American, 12% Asian, 8% Hispanic, 64% white, and 5% multiracial/other) donors of both sexes were enrolled and ranged from 18 to 90 years of age; 15% were high-intensity donors (nine or more donations in the prior 24 mo without low hemoglobin deferral). Data elements are available for 97% to 99% of the cohort. CONCLUSIONS: The cohort provides demographic, behavioral, biochemical, and genetic data for a broad range of blood donor studies related to iron metabolism, adverse consequences of iron deficiency, and differential hemolysis (including oxidative and osmotic stress perturbations) during RBC storage. Linkage to recipient outcomes may permit analysis of how donor characteristics affect transfusion efficacy. Repository DNA, plasma, and RBC samples should expand the usefulness of the current data set.

Frequent blood donations alter susceptibility of red blood cells to storage- and stress-induced hemolysis.

BACKGROUND: Frequent whole blood donations increase the prevalence of iron depletion in blood donors, which may subsequently interfere with normal erythropoiesis. The purpose of this study was to evaluate the associations between donation frequency and red blood cell (RBC) storage stability in a racially/ethnically diverse population of blood donors. STUDY DESIGN: Leukoreduced RBC concentrate-derived samples from 13,403 donors were stored for 39 to 42 days (1-6°C) and then evaluated for storage, osmotic, and oxidative hemolysis. Iron status was evaluated by plasma ferritin measurement and self-reported intake of iron supplements. Donation history in the prior 2 years was obtained for each subject. RESULTS: Frequent blood donors enrolled in this study were likely to be white, male, and of older age (56.1 ± 5.0 years). Prior donation intensity was negatively associated with oxidative hemolysis (p < 0.0001) in multivariate analyses correcting for age, sex, and race/ethnicity. Increased plasma ferritin concentration was associated with increased RBC susceptibility to each of the three measures of hemolysis (p < 0.0001 for all), whereas self-reported iron intake was associated with reduced susceptibility to osmotic and oxidative hemolysis (p < 0.0001 for both). CONCLUSIONS: Frequent blood donations may alter the quality of blood components by modulating RBC predisposition to hemolysis. RBCs collected from frequent donors with low ferritin have altered susceptibility to hemolysis. Thus, frequent donation and associated iron loss may alter the quality of stored RBC components collected from iron-deficient donors. Further investigation is necessary to assess posttransfusion safety and efficacy in patients receiving these RBC products.

Development and evaluation of a transfusion medicine genome wide genotyping array.

BACKGROUND: Many aspects of transfusion medicine are affected by genetics. Current single-nucleotide polymorphism (SNP) arrays are limited in the number of targets that can be interrogated and cannot detect all variation of interest. We designed a transfusion medicine array (TM-Array) for study of both common and rare transfusion-relevant variations in genetically diverse donor and recipient populations. STUDY DESIGN AND METHODS: The array was designed by conducting extensive bioinformatics mining and consulting experts to identify genes and genetic variation related to a wide range of transfusion medicine clinical relevant and research-related topics. Copy number polymorphisms were added in the alpha globin, beta globin, and Rh gene clusters. RESULTS: The final array contains approximately 879,000 SNP and copy number polymorphism markers. Over 99% of SNPs were called reliably. Technical replication showed the array to be robust and reproducible, with an error rate less than 0.03%. The array also had a very low Mendelian error rate (average parent-child trio accuracy of 0.9997). Blood group results were in concordance with serology testing results, and the array accurately identifies rare variants (minor allele frequency of 0.5%). The array achieved high genome-wide imputation coverage for African-American (97.5%), Hispanic (96.1%), East Asian (94.6%), and white (96.1%) genomes at a minor allele frequency of 5%. CONCLUSIONS: A custom array for transfusion medicine research has been designed and evaluated. It gives wide coverage and accurate identification of rare SNPs in diverse populations. The TM-Array will be useful for future genetic studies in the diverse fields of transfusion medicine research.

Intradonor reproducibility and changes in hemolytic variables during red blood cell storage: results of recall phase of the REDS-III RBC-Omics study.

BACKGROUND: Genetic determinants may underlie the susceptibility of red blood cells (RBCs) to hemolyze in vivo and during routine storage. This study characterized the reproducibility and dynamics of in vitro hemolysis variables from a subset of the 13,403 blood donors enrolled in the RBC-Omics study. STUDY DESIGN AND METHODS: RBC-Omics donors with either low or high hemolysis results on 4°C-stored leukoreduced (LR)-RBC samples from enrollment donations stored for 39 to 42 days were recalled 2 to 12 months later to donate LR-RBCs. Samples of stored LR-RBCs from the unit and from transfer bags were evaluated for spontaneous and stress-induced hemolysis at selected storage time points. Intradonor reproducibility of hemolysis variables was evaluated in transfer bags over two donations. Hemolysis data at serial storage time points were generated on LR-RBCs from parent bags and analyzed by site, sex, race/ethnicity, and donation frequency. RESULTS: A total of 664 donors were successfully recalled. Analysis of intradonor reproducibility revealed that osmotic and oxidative hemolysis demonstrated good and moderate reproducibility (Pearson's r = 0.85 and r = 0.53, respectively), while spontaneous hemolysis reproducibility was poor (r = 0.40). Longitudinal hemolysis in parent bags showed large increases over time in spontaneous (508.6%) and oxidative hemolysis (399.8%) and smaller increases in osmotic (9.4%) and mechanical fragility (3.4%; all p < 0.0001). CONCLUSION: Spontaneous hemolysis is poorly reproducible in donors over time and may depend on site processing methods, while oxidative and osmotic hemolysis were reproducible in donors and hence could reflect consistent heritable phenotypes attributable to genetic traits. Spontaneous and oxidative hemolysis increased over time of storage, whereas osmotic and mechanical hemolysis remained relatively stable.