Proceedings of the 2022 NHLBI and OASH state of the science in transfusion medicine symposium.

BACKGROUND: State of the Science (SoS) meetings are used to define and highlight important unanswered scientific questions. The National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, and the Office of the Assistant Secretary for Health (OASH), Department of Health and Human Services held a virtual SoS in transfusion medicine (TM) symposium. STUDY DESIGN AND METHODS: In advance of the symposium, six multidisciplinary working groups (WG) convened to define research priorities in the areas of: blood donors and the supply, optimizing transfusion outcomes for recipients, emerging infections, mechanistic aspects of components and transfusion, new computational methods in transfusion science, and impact of health disparities on donors and recipients. The overall objective was to identify key basic, translational, and clinical research questions that will help to increase and diversify the volunteer donor pool, ensure safe and effective transfusion strategies for recipients, and identify which blood products from which donors best meet the clinical needs of specific recipient populations. RESULTS: On August 29-30, 2022, over 400 researchers, clinicians, industry experts, government officials, community members, and patient advocates discussed the research priorities presented by each WG. Dialogue focused on the five highest priority research areas identified by each WG and included the rationale, proposed methodological approaches, feasibility, and barriers for success. DISCUSSION: This report summarizes the key ideas and research priorities identified during the NHLBI/OASH SoS in TM symposium. The report highlights major gaps in our current knowledge and provides a road map for TM research.

Blood transfusion for deep space exploration.

BACKGROUND: Astronauts on exploration missions may be at risk for traumatic injury and medical conditions that lead to life threatening hemorrhage. Resuscitation protocols are limited by the austere conditions of spaceflight. Solutions may be found in low-resource terrestrial settings. The existing literature on alternative blood product administration and walking blood banks was evaluated for applicability to spaceflight. STUDY DESIGN AND METHODS: A literature review was done using PubMed and Google Scholar. References were crosschecked for additional publications not identified using the initial search terms. Twenty-seven articles were identified, including three controlled trials, six retrospective cohort analyses, 15 reviews, one case report, and two experimental studies. RESULTS: Solutions to blood transfusion in austere settings include lyophilized blood products, hemoglobin-based oxygen carriers (HBOCs), and fresh whole blood. Many of these products are investigational. Protocols for walking blood banks include methods for screening and activating donors, transfusion, and monitoring for adverse reactions. Microgravity and mission limitations create additional challenges for transfusion, including baseline physiologic changes, difficulty reconstituting lyophilized products, risk of air emboli during transfusion, equipment constraints, and limited evacuation and surgical options. CONCLUSION: Medical planning for space exploration should consider the possibility of acute blood loss. A model for "floating" blood banks based on terrestrial walking blood bank protocols from austere environments is presented, with suggestions for future development. Constraints on volume, mass, storage, and crew, present challenges to blood transfusion in space and must be weighed against the benefits of expanding medical capabilities.

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

The operational implications of donor behaviors following enrollment in STRIDE (Strategies to Reduce Iron Deficiency in blood donors).

BACKGROUND: Donor behaviors in STRIDE (Strategies to Reduce Iron Deficiency), a trial to reduce iron deficiency, were examined. STUDY DESIGN AND METHODS: Six hundred ninety-two frequent donors were randomized to receive either 19 or 38 mg iron for 60 days or an educational letter based on their predonation ferritin. Compliance with assigned pills, response to written recommendations, change in donation frequency, and future willingness to take iron supplements were examined. RESULTS: Donors who were randomized to receive iron pills had increased red blood cell donations and decreased hemoglobin deferrals compared with controls or with pre-STRIDE donations. Donors who were randomized to receive educational letters had fewer hemoglobin deferrals compared with controls. Of those who received a letter advising of low ferritin levels with recommendations to take iron supplements or delay future donations, 57% reported that they initiated iron supplementation, which was five times as many as those who received letters lacking a specific recommendation. The proportion reporting delayed donation was not statistically different (32% vs. 20%). Of donors who were assigned pills, 58% reported taking them "frequently," and forgetting was the primary reason for non-compliance. Approximately 80% of participants indicated that they would take iron supplements if provided by the center. CONCLUSIONS: Donors who were assigned iron pills had acceptable compliance, producing increased red blood cell donations and decreased low hemoglobin deferrals compared with controls or with pre-STRIDE rates. The majority of donors assigned to an educational letter took action after receiving a low ferritin result, with more donors choosing to take iron than delay donation. Providing donors with information on iron status with personalized recommendations was an effective alternative to directly providing iron supplements.

Qualitative assessment of pica experienced by frequent blood donors.

BACKGROUND: Pica, the compulsive consumption of ice or other nonnutritious substances, is associated with iron deficiency, a common negative consequence of frequent blood donation. Because of this, blood donors, such as those participating in the Strategies to Reduce Iron Deficiency (STRIDE) study, are an ideal population to explore pica and iron deficiency. STUDY DESIGN AND METHODS: STRIDE was a 2-year intervention trial to assess the effectiveness of iron supplementation for mitigating iron deficiency in frequent blood donors. Subjects completed baseline and follow-up questionnaires that included questions about pica symptoms. In-depth telephone interviews were conducted with 14 of these subjects reporting pica symptoms and eight presumed controls (casual ice chewers) to gain a deeper understanding of pica symptoms and their impact on daily life and to make a final determination on the presence of pica. RESULTS: Pica was confirmed in five of the 14 subjects reporting symptoms and in two of eight controls. Outcome misclassification based on the questionnaire was attributed to inadequate assessment of several pica symptoms identified during the interview. Comparison of subjects' repeated quantitative iron measurements taken throughout STRIDE with subjects' final adjudicated pica status revealed a positive relationship between development of pica and worsening iron status; the opposite was found in those whose pica symptoms resolved. CONCLUSION: Continued refinement of pica symptom questions will allow for rapid and accurate detection of pica in frequent blood donors and confirmation of successful treatment with iron supplements.

Estimates of total body iron indicate 19 mg and 38 mg oral iron are equivalent for the mitigation of iron deficiency in individuals experiencing repeated phlebotomy.

Iron deficiency anemia is a common clinical condition often treated with tablets containing 65 mg of elemental iron. Such doses can elicit gastrointestinal side effects lowering patient compliance. Oral iron supplements also increase hepcidin production causing decreased fractional absorption of subsequent doses. Frequent blood donors often become iron deficient. Therefore, they were enrolled in a two-year study involving continued blood donations and randomization to receive no pill, placebo, 19, or 38 mg ferrous gluconate for 60 days. Total body iron (TBI) did not change for the subset of donors in the no pill and placebo groups who completed both enrollment and final visits (P = .21 and P = .28, respectively). However, repeated measures regression analysis on the complete dataset estimated a significant decrease in TBI of 52 mg/year for the placebo and no pill groups (P = .001). The effects of 19 and 38 mg iron supplementation on TBI were indistinguishable (P = .54). TBI increased by 229 mg after the initial 60 days of iron supplementation (P < .0001) and was maintained at this higher level with continued iron supplementation following each subsequent donation. The TBI increase was apportioned 51 mg to red cell iron (P < .0001) and 174 mg to storage iron (P < .0001). Changes in storage iron were negatively impacted by 57 mg due to concurrent antacid use (P = .04). These findings in blood donors suggest that much lower doses of iron than are currently used will be effective for clinical treatment of iron deficiency anemia.

A randomized, blinded, placebo-controlled trial of education and iron supplementation for mitigation of iron deficiency in regular blood donors.

BACKGROUND: The historical approach of offering dietary advice to donors with low hemoglobin (Hb) is ineffective for preventing iron deficiency in frequent donors. Alternative approaches to maintaining donor iron status were explored. STUDY DESIGN AND METHODS: Frequent blood donors were randomly assigned into five arms for 2 years of follow-up. Three double-blinded arms provided 60 once-daily pills after each donation (38, 19, or 0 mg of iron). Two single-blinded arms provided iron status (ferritin) or no information letters after each donation. Ferritin, soluble transferrin receptor, and complete blood count were measured at each donation. RESULTS: There were 692 subjects enrolled and 393 completed the study. Subjects in pill groups deenrolled more than those in letter groups (39% vs. 7%). Adverse events occurred equally in subjects receiving iron or placebo pills. Of those completing the study, the prevalence of ferritin of less than 12 or less than 26 ng/mL declined by more than 50% and was statistically indistinguishable in the three intervention groups (19 or 38 mg of iron; iron status letter). Longitudinal analyses of all subjects showed improved iron status in iron pill groups and worsening iron status in control groups (placebo; no information letter). The iron pill groups experienced a net increase of approximately 0.6 g/dL Hb compared to control groups. The iron status letter group had little change in Hb. CONCLUSION: Providing 19 or 38 mg of daily iron or iron status information were effective and mostly equivalent interventions for mitigating iron deficiency in regular donors when compared at the end of the 2-year longitudinal phase of the study. Donors without intervention had worsened iron deficiency with continued donation.

Development of methyl isoxazoleazepines as inhibitors of BET.

In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.

Comparison of antibody titers in donor specimens and associated AS-1 leukoreduced donor units.

BACKGROUND: Donor units with unexpected antibodies to red blood cell (RBC) antigens have transfusion restrictions and are often discarded by collection facilities. This study examined the antibody titer reduction in AS-1 leukoreduced RBC (LR-RBC) units and potential acceptability of these units for unrestricted transfusions. STUDY DESIGN AND METHODS: Donor specimens and AS-1 LR-RBC segment samples from donors with positive antibody screens and group O donors were analyzed. Antibody identifications were performed, and titer results from the matched serum samples and segment supernatants were compared. RESULTS: During the 5-month study, 39 donor samples with positive antibody screens, five random group O donor samples, and the associated LR-RBC unit segments were assessed. The median donor sample and segment supernatant titers were 4 and 1, respectively. Alloantibodies were undetectable in 28% of the donor segment supernatants. The median anti-A and anti-B titers in the group O donor samples were 128 and were reduced to 32 in the donor segment supernatants. All ABO and other antibodies were diluted by the AS-1 to a titer of not more than 32. CONCLUSION: Antibody titers in AS-1 LR-RBC units were significantly decreased compared to donor specimens and were lower than anti-A and -B titers in group O AS-1 LR-RBC units, which are frequently transfused to non-group O recipients. If a "clinically significant" titer for donor alloantibodies was established, blood centers could determine eligibility of units for unrestricted transfusions. This would decrease unnecessary RBC wastage and increase units available for transfusion.

Targeting MYC dependence in cancer by inhibiting BET bromodomains.

The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G(1) arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.

Ascertainment of iron deficiency and depletion in blood donors through screening questions for pica and restless legs syndrome.

BACKGROUND: Pica and restless legs syndrome (RLS) are associated with iron depletion and deficiency. The presence of pica and RLS was prospectively assessed in blood donors. STUDY DESIGN AND METHODS: During a 39-month period, 1236 donors deferred for fingerstick hemoglobin (Hb) level of less than 12.5 g/dL and 400 nondeferred "control" donors underwent health screening and laboratory testing (complete blood count, ferritin, iron, transferrin). Pica and RLS were assessed by direct questioning. Deferred donors and iron-deficient control donors were given 325 mg of ferrous sulfate daily for 60 days. Reassessments were performed and additional iron tablets dispensed at subsequent visits. RESULTS: Pica was reported in 11% of donors with iron depletion or deficiency, compared with 4% of iron-replete donors (p < 0.0001). Pagophagia (ice pica) was most common and often of extraordinary intensity. Female sex, younger age, and lower mean cell volume and transferrin saturation values were strongly associated with pica. Donors with pica given iron reported a marked reduction in the desire to consume the nonnutritive substance by Days 5 to 8 of therapy, with disappearance of symptoms by Days 10 to 14. RLS was reported in 16% of subjects with iron depletion or deficiency compared with 11% of iron-replete donors (p = 0.012). Iron replacement generally resulted in improvement of RLS symptoms; however, at least 4 to 6 weeks of iron therapy was necessary. CONCLUSION: The presence of pica is associated with a high probability of iron depletion or deficiency in blood donors; however, RLS lacks a strong correlation in this population. Screening questions for pagophagia may be useful in the ascertainment of iron deficiency in donors and may identify those who would benefit from oral iron.

Advancing Understandings of Blood Donation Motivation and Behavior.

In this review, we provide critical analysis of social science research into blood donation motivation and behavior. We first share an understanding of the existing literature and recommendations for future research collectively developed by members of the Working Group on Blood Donors and the Supply: Diversifying while Maintaining the Donor Pool, Donor Selection, and Optimizing Blood Availability and Safety, as part of the National Heart, Lung, and Blood Institute's 2022 State of the Science in Transfusion Medicine symposium. Then, rather than aim for a comprehensive treatment, we review 4 newer manuscripts that exemplify aspects of the group's recommendations and report results from countries where the blood supply is based on voluntary, nonremunerated donations. From the substantial existing literature, we selected: (1) a study that employed motivational interviewing techniques, thematic analysis, and surveys to link donation motivations and barriers reported by diverse young donors in the United States to actual donation behavior over a year of subsequent eligibility; (2) a survey regarding donation motivations and barriers and monetary amounts associated with willingness to participate in whole blood, plasma, or platelet collection; (3) a survey-based assessment of various emotional states reported by donors at 2 time points during donation and the relationship between emotional experience and subsequent vasovagal reactions; and (4) an interpretive discourse analysis of blood collection agency messaging to donors and the public in the beginning of the COVID-19 pandemic. We close by noting several challenges posed by the structure of the United States blood system and the current funding environment to conducting rigorous research and translating findings into practice.

Iron replacement therapy in the routine management of blood donors.

BACKGROUND: Iron depletion or deficiency in blood donors frequently results in deferrals for low hemoglobin (Hb), yet blood centers remain reluctant to dispense iron replacement therapy to donors. STUDY DESIGN AND METHODS: During a 39-month period, 1236 blood donors deferred for a Hb level of less than 12.5 g/dL and 400 nondeferred control donors underwent health history screening and laboratory testing (complete blood counts, iron studies). Iron depletion and deficiency were defined as a ferritin level of 9 to 19 and less than 9 µg/L in females and 18 to 29 and less than 18 µg/L in males. Deferred donors and iron-deficient control donors were given a 60-pack of 325-mg ferrous sulfate tablets and instructed to take one tablet daily. Another 60-pack was dispensed at all subsequent visits. RESULTS: In the low-Hb group, 30 and 23% of females and 8 and 53% of males had iron depletion or deficiency, respectively, compared with 29 and 10% of females and 18 and 21% of males in the control group. Iron-depleted or -deficient donors taking iron showed normalization of iron-related laboratory parameters, even as they continued to donate. Compliance with oral iron was 68%. Adverse gastrointestinal effects occurred in 21% of donors. The study identified 13 donors with serious medical conditions, including eight with gastrointestinal bleeding. No donors had malignancies or hemochromatosis. CONCLUSION: Iron depletion or deficiency was found in 53% of female and 61% of male low-Hb donors and in 39% of female and male control donors. Routine administration of iron replacement therapy is safe and effective and prevents the development of iron depletion and deficiency in blood donors.

Knowledge of food and drug administration reportable deviations.

BACKGROUND: As early as 2001, the Food and Drug Administration (FDA) required blood centers and hospital transfusion services to report events associated with testing, storage, or distribution of blood products that deviated from current good manufacturing practices or affected the safety, purity, or potency of the product. Between 2004 and 2009, an average of only 8.6% of hospitals reported blood product deviations. STUDY DESIGN AND METHODS: Case scenarios designed to evaluate knowledge of FDA reportable deviations were developed and sent for evaluation to the Center for Biologics Evaluation and Research (CBER) and FDA division directors for FDA reportable deviations. A final survey containing eight cases was launched in a web-based online survey tool and sent to blood bank medical technologists. Additional information was queried regarding job title/responsibilities and the size of the blood center and/or transfusion service. RESULTS: There were 176 respondents to the survey. Only 5.7% (10/176) answered all questions correctly. Analysis by job title and place of employment revealed no correlation to the number of correct responses. More importance was attached to deviations involving quality control, blood bank identification, unit specifications, and antibody identification. Less importance was attached to deviations involving phlebotomist's initials, failure to issue units in the computer, and using a recent sample from a previous hospitalization. CONCLUSION: This study revealed that blood bankers did not have clear understanding of what constituted an FDA reportable occurrence. Size or type of blood establishment or individual job title was not associated with more knowledge of FDA reportable deviations.

WBC reduction filtration efficacy performed at varying time intervals post-collection.

BACKGROUND: A multisite blood center experienced unacceptable post-leukoreduction filtration white blood cell (WBC) counts at a few centers. Since prefiltration storage time and temperature were suspect, whole blood (WB) units were stored in transport shippers for at least 2 hours, cooling toward 1-6 ° C, before filtration. This study compared the effect of storage times in transport shippers on the residual WBC counts of leukoreduced units. STUDY DESIGN AND METHODS: Collection and filtration of WB units were accomplished with the use of the Fenwal Express System with Integral Sepacell RZ-2000 WB Leukocyte Reduction Filter. Units were collected and placed in transport shippers containing ice. Leukoreduction filtration was performed at designated intervals post-collection. Acceptable leukoreduction was defined as < 5 × 10(6) residual WBC. RESULTS: Fifty donor units were selected randomly over 3 months. Units were held in transport shippers, and WBC reduction was performed at designated post-collection intervals. Storage times ranged from 28 to 458 minutes. All residual WBC counts were acceptable. CONCLUSION: Storage time of WB units in transport shippers did not play a role in the efficacy of the leukoreduction. This study demonstrated the 2-hour storage time before leukoreduction filtration could be eliminated resulting in time savings and increased efficacy in the component production laboratory.

Therapeutic phlebotomy procedures and their impact on a rural hospital's red blood cell inventory and fiscal stature.

BACKGROUND: Therapeutic phlebotomy (TP) programs offer an important community service and often provide financial and donor unit resources for the hospital. This study assessed the financial impact and red blood cell (RBC) inventory contribution of a small, rural hospital-based TP program. STUDY DESIGN AND METHODS: TP procedures over 13 months were evaluated at a 142-bed rural hospital. The hospital had a Food and Drug Administration variance for a hereditary hemochromatosis (HH) donor program. The revenue for the non-HH therapeutic phlebotomies and the savings attained for units added to RBC inventory from allogeneic eligible HH donors were compiled. RESULTS: During the study, 84 patients were involved in the TP program. Of the 62 HH patients, 43 met eligibility requirements for allogeneic donations resulting in 207 donor units collected for the blood bank inventory and a savings of $21,000 in blood costs. Additionally, 22 non-HH patients underwent 183 TP procedures earning the hospital over $15,000 in net revenue. CONCLUSION: The TP program at this small, rural 142-bed hospital provided a financial gain of $36,000 during the 13-month study period. The HH donor program contributed approximately 4% to the RBC inventory. The TP program at this small, rural 142-bed hospital proved to be financially lucrative and provided a community service to patients.

Gravity sedimentation of granulocytapheresis concentrates with hydroxyethyl starch efficiently removes red blood cells and retains neutrophils.

BACKGROUND: Transfusion of granulocytapheresis concentrates can be limited by the volume of incompatible donor red blood cells (RBCs) in the component. Efficient reduction of RBCs in granulocyte units would result in safe transfusion of RBC-incompatible units. STUDY DESIGN AND METHODS: Granulocyte concentrates were collected by continuous-flow apheresis from granulocyte-colony-stimulating factor (G-CSF) and dexamethasone-stimulated volunteer donors, with 6% hydroxyethyl starch (HES) added continuously during apheresis as a RBC sedimenting agent to enhance granulocyte collection efficiency. After collection, the component was placed in a plasma extractor for 4 hours. A sharp line of demarcation between the starch-sedimented RBCs and the granulocyte-rich supernatant developed, and the supernatant was transferred to a sterilely docked transfer pack. RBC reduction and white blood cell recovery were determined. RESULTS: Gravity sedimentation was performed on 165 granulocyte concentrates. Mean sedimentation time was 267 minutes (range, 150-440 min). RBC depletion was 92% (range, 71%-99%) with mean residual RBC content of 3.2 +/- 1.4 mL. Twelve percent of components contained less than 2 mL of RBCs. Mean granulocyte and platelet (PLT) recoveries were 80 and 81%, respectively. There were no transfusion reactions or signs of hemolysis after transfusion of 66 RBC-incompatible granulocyte concentrates (RBC volume, 1.6-8.2 mL). The remaining concentrates were used for topical or intrapleural applications. CONCLUSIONS: RBCs were significantly reduced and granulocytes and PLTs effectively retained in G-CSF/steroid-mobilized granulocyte components collected with HES and processed by gravity sedimentation. This procedure allows safe transfusion of RBC-incompatible sedimented granulocyte units and may be used to expand the pool of available granulocyte donors for specific recipients.