ALBERT

Description: Introduction Daratumumab (DARA), an IgG1k human monoclonal antibody (Ab) against CD38, is a promising novel therapy for multiple myeloma. However, direct binding of DARA to endogenous CD38 on reagent red blood cells (RBCs) interferes with routine blood bank serologic testing. We recently showed that treating reagent RBCs with DTT eliminates the DARA interference by denaturing cell surface CD38, allowing the safe transfusion of patients on DARA.1 This multicenter international study was aimed at validating the DTT method for use by blood banks worldwide. Methods Participating blood banks received two plasma sample unknowns. Sample 1 was spiked with DARA alone (5 mcg/mL). Sample 2 was spiked with DARA plus a clinically significant RBC Ab (anti-D (Rh immune globulin) or monoclonal anti-Fya or anti-s). Sites were instructed to first perform an Ab screen using their usual method (tube, gel, or solid phase), then to repeat the Ab screen using DTT-treated RBCs (gel or tube). If the Ab screen remained positive with DTT-treated RBCs (Sample 2), sites were to identify the unknown Ab using a DTT-treated RBC panel (gel or tube.) The primary outcome measure was the proportion of sites able to successfully identify the unknown Ab in the presence of DARA. Qualitative data were collected by online survey. Results Paired plasma sample unknowns were shipped to 25 study sites in North America, South America, Europe, Asia, and Australia/New Zealand. Data were received from 23 sites to date (Table). For the initial Ab screen, 10 sites used tube testing, 7 sites used gel, and 6 sites used solid phase. All sites observed DARA interference with the Ab screen (false positive agglutination reactions). All sites reported no DARA interference using DTT-treated RBCs. For Ab identification (Sample 2), 13 sites used tube testing and 10 sites used gel. 23/23 sites (100%) were able to correctly identify the unknown Ab using the DTT method. The Abs identified were: anti-Fya (9/9), anti-s (8/8), and anti-D (6/6). Feedback on the DTT method was mainly positive, with 86% of sites that responded to the survey indicating that they planned to use the DTT method to manage clinical samples from DARA-treated patients. Conclusion DARA consistently interferes with all three Ab screening methods currently used by blood banks (tube, gel, and solid phase.) Using DTT-treated RBCs, 23/23 (100%) of blood bank laboratories from around the world were able to identify a clinically significant Ab initially masked by the presence of DARA. The DTT method is robust, reproducible, and can be implemented by blood banks globally to help provide safe blood products to patients on DARA. As DTT denatures Kell antigens, K- RBC units should be provided when using the DTT method. 1. Chapuy CI, Nicholson RT, Aguad MD, et al. Resolving the daratumumab interference with blood compatibility testing. Transfusion. 2015;55(6pt2):1545-1554. Disclosures Unger: Janssen: Employment. Doshi:Janssen: Employment. Kaufman:Janssen: Consultancy, Research Funding.

Description: BACKGROUND: Apheresis platelet collection from healthy normal blood donors can reduce the donor peripheral blood platelet concentration by 50% or more. The kinetics of peripheral blood platelet count (PLT) recovery in the apheresis donors over the first 24 hours has not been described. The objective of this study was to determine the recovery kinetics of the donor peripheral blood platelet count following apheresis platelet donation. METHODS: Healthy apheresis platelet donors were enrolled following informed consent. The apheresis platelet collection was performed using the Gambro Trima system (Gambro BCT, Lakewood, CO) following local SOP and manufacturer’s directions for use. The minimum predicted post-count was configured in the Trima to no less than 78K plt/μL. PLT was determined pre-procedure (Pre), immediately post procedure (Post), 4–11 h (FU1) and 11–41 h (FU2) post-donation using standard methods. The PLT recovery was evaluated as the increase in PLT following the donation (Delta). The effects of study site, time of sample, and the fraction of platelets collected (Fpc) at donation on Delta were evaluated using a random effects generalized linear regression model. A full regression model of Delta as a function of study site, follow-up period and Fpc with all main and interaction effects was used to test hypotheses. RESULTS: 548 subjects were entered into the study at 3 study sites; Pre-PLT 276±59 × 103 plt/μL, Post-PLT 205±47 × 103 plt/μL, Fpc 25±10%. No adverse events were reported by any subjects. Recovery of platelet count following apheresis platelet donation is variable between subjects; and the independent variables of study site, follow-period and Fpc accounted for 25% of the total variation in Delta. PLT increased 12.4±0.9 × 103 plt/μL by the time of follow-up sampling (p=0.01), although there was no difference between PLT at FU1 (214±49 × 103 plt/μL) and FU2 (212±47 × 103 plt/μL; p=0.15). None of the donors reached their pre-donation platelet count during the follow-up period. There was no difference in Delta between centers (p=0.23). Fpc had a significant affect on Delta (p

Description: Abstract 3149 Poster Board III-86 Background The plasticizer di-2-ethylhexyl phthalate (DEHP) is a common component in medical plastics. Unique to the storage of RBC, DEHP is a necessary component of these storage bags, and is required to prevent excessive hemolysis over storage. Due to concerns related to the long term effects of DEHP exposure and issues related to disposal of these materials, there is a drive to replace this component. Our objective is to evaluate a candidate replacement plasticizer (Hexamoll TM DINCH, Cyclohexane-1,2-dicarboxylic acid, dinonylester, branched and linear) compared to control DEHP in an in vitro feasibility study. We hypothesize that the candidate will provide at least equivalent protection against hemolysis for RBC stored in additive solution for 42 days, and periodic mixing of RBC stored in additive solution for 42 days will add additional protection against hemolysis. Methods Whole blood was collected into CPD in standard collection sets (Terumo), combined into pools of 2 ABO identical RBC, divided, leukocyte-reduced, centrifuged and separated into plasma and packed RBC, then additive solution (AS-5) was added to the RBC, and the RBC/AS-5 were transferred into 2 study bags (matched pair) for storage up to 42 days under standard blood bank conditions of 4°C. In the first part, both study bags were Hexamoll TM DINCH, and one bag of each pair was mixed once per week, the other was held undisturbed. In the second part, DEHP bags were used as control held 42 days undisturbed, and the DINCH test bag was mixed once per week during storage. Standard in vitro RBC characteristics were determined at days 0 and 42. Results DINCH bags mixed weekly retained RBC morphology better, had lower extracellular potassium and had lower hemolysis compared to static storage. There were no detectable differences between DEHP static stored bags and DINCH bags mixed weekly when comparing in vitro parameters. Conclusion Periodic mixing of RBC stored in DINCH bags provides additional protection against hemolysis over 42 days of storage. DINCH plasticizer provided at least equivalent protection against hemolysis and potassium leakage for RBC stored in additive solution for 42 days. These initial observations should be extended to larger sample sizes and to in vivo autologous RBC recovery studies. The need for periodic mixing will need to be evaluated further. The plasticizer DINCH may be a viable candidate for replacement DEHP in RBC storage bags. Disclosures Dumont: BASF: Research Funding. Baker:BASF: Research Funding. Waters:BASF: Research Funding. Herschel:BASF: Research Funding. Dumont:BASF: Research Funding. David:BASF: Employment. Otter:BASF: Employment.

Description: Availability of platelets (plts) is severely limited by shelf life in some military as well as civilian settings. Additionally, some bleeding, thrombocytopenic patients do not have a therapeutic response to a standard plt transfusion. Methods for cryopreservation of apheresis plts for up to two years in 6% DMSO at

Description: Blood transfusion is a life-saving intervention for millions of recipients worldwide every year. However, refrigerated storage of red blood cells (RBCs) for up to 42 days promotes impairments in energy and redox homeostasis, which impact RBC hemolytic propensity and post-transfusion performance of the storage-damaged RBC. Since mature RBCs are devoid of de novo protein synthesis - owing to the lack of organelles and nuclei - they evolved metabolic mechanisms to cope with oxidative stress. Two of these involve (i) activation of the pentose phosphate pathway (PPP), which generates reducing equivalents (NADPH) to preserve glutathione homeostasis and recharge NADPH-dependent antioxidant enzymes; and (ii) recycling of oxidatively damaged proteins via methylation of dehydrated and deamidated aspartate and asparagine residues, a process that consumes methionine as the main methyl group donor. Thus, we hypothesize that the latter mechanism is relevant to routine blood bank storage, especially in glucose-6-phosphate dehydrogenase (G6PD)-deficient donors. In this routinely accepted donor population (~10% of donors of African descent), mutations of G6PD, the rate-limiting enzyme of the PPP, result in instability and decreased enzymatic activity (e.g.,

Description: Introduction Crohn's disease and Ataxia Telangiectasia have been managed by extended release of dexamethasone from autologous red blood cells (RBC) with encapsulated dexamethasone sodium phosphate DSP. The EryDexSystem (EDS) is an automated system that loads RBC ex vivo using hypotonic opening of RBC followed by hypertonic resealing of the RBC and washing to prepare the DSP-RBC for infusion. In vivo, DSP is dephosphorylated within the RBC to dexamethasone which passively diffuses into the plasma. The objective of this two-phase study was to elucidate pharmacokinetics (PK) and in vivo 24-hour recovery of RBCs as well as RBC survival (T50) properties of RBC encapsulated DSP. Materials and Methods The study was conduct in two separate Phases, A and B. Phase A, the 24-hour RBC recovery and T50 survival phase, was designed as a randomized, concurrently controlled, single-blind, single-center study to determine the in vivo kinetics of EDS-processed autologous RBC. Healthy volunteer consenting subjects were randomized to receive autologous RBCs prepared using EDS and loaded with either 15-20mg DSP (Group 1A) or sham hypotonic saline (Group 2A). EDS prepared RBC were radiolabeled with 51-Cr following standard methods, and the in vivo labeled RBC followed over 49 days post infusion. The Phase B PK study was designed as an open-label, single-center Phase I study that evaluated two dose levels of DSP encapsulated in RBCs using the EDS. Healthy volunteer consenting subjects were randomized to receive autologous RBCs loaded with either 2.5-5 mg DSP (Group 1B) or 15-20 mg (Group 2B). Post-infusion plasma levels of dexamethasone were followed (over 42 days). Both studies conformed to the Declaration of Helsinki. Results Phase A: Ten subjects (3male; 7 female) were randomized to Groups 1A or 2A. The mean 24-hour RBC recovery ± SD [95% CL] was 77.9 ± 3.3% [73.8-81.9%] and 72.7 ± 10.5% [57.8-85.7%] for Groups 1A and 2A, respectively. The mean ± SD RBC life span in Group 1A was 84.3 ± 8.3 days with a mean T50 of 42.1 ± 4.1 [95% CL: 37.0, 47.3] days, whereas these values were 88.9 ± 6.2 days and 44.4 ± 3.1 [95% CL: 40.6, 48.3] days, respectively, in Group 2A. Sixteen (16) treatment-emergent adverse events (TEAEs) were recorded in Group 1A and 23 in Group 2A. All TEAEs were judged to be unlikely related to the treatment. Phase B: Eighteen subjects (12 male; 6 female) were randomized to Groups 2A and 2B. The actual DSP loading doses (mean ± SEM) were 4.2±0.27 mg and 16.9±0.90 mg. Release of dexamethasone from RBCs in vivo peaked at 1 hour after the end of IV infusion independent of the dose. A detailed summary of the PK parameters for dexamethasone for each treatment group is shown in Table 1. A sustained release of dexamethasone could be detected until 14 and 35 days after the single IV infusion ofEryDex in Group 1B and 2B, respectively. Six (6) TEAEs were reported in each group and were judged to be unlikely related to the study drug or procedure. Conclusion The results for the mean RBC in vivo recovery for DSP-loaded EDS-processed cells meet the FDA criteria for 24-hour RBC recovery of ≥ 75%, without adverse impact on the survival of EDS-processed RBCs. Most of the dexamethasone was rapidly released from the RBCs in vivo with a maximum peak occurring 1 hour after the end of the intravenous infusion, independent of the dose administered, but sustained release of dexamethasone could be detected until 14 and 35 days post infusion for the low and high doses, respectively. DSP-loaded autologous RBCs prepared using the EDS delivered a sustained dose of dexamethasone in vivo. Additional efficacy studies in targeted patient populations are indicated. Disclosures Szczepiorkowski: EryDel S.P.A.: Research Funding. Ferrari:EryDel S.P.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Benatti:EryDel S.P.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Mambrini:EryDel S.P.A.: Employment, Equity Ownership. Hartman:EryDel S.P.A.: Consultancy. Dumont:EryDel S.P.A.: Research Funding.

Description: Abstract 844 Background: RBC transfusion has clear efficacy in treating various types of anemia. However, in recent decades, there is a renewed focus on the potential for negative clinical sequelae from transfusing stored RBCs. Whether or not older, stored RBC units are associated with adverse outcomes remains controversial. However, there are clear cellular and biochemical data that RBC units accumulate particles and molecules over time with known toxicity when administered to animals and/or humans. Among the most potent of these are prostaglandins and leukotrienes (jointly known as eicosanoids), which are potent mediators of inflammation and vascular pathology. Indeed, arachidonic acid (AA), and 5-, 12-, and 15-hydroxyeicsotetranoic acid (HETE) accumulate during storage of human RBCs and are biologically active in priming neutrophils (Silliman et al., Transfusion 2011 51(12):2549-54). It is well known that there is substantial donor-to-donor variation in how well RBCs store from the standpoint of post-transfusion RBC recovery; however, it is unclear whether the accumulation of eicosanoids varies substantially among donors. If differences exist, then it may be useful to screen RBC units prior to transfusion into patients with illnesses likely to be affected by eicosanoid exposure. Using a well characterized mouse model of RBC storage, and different strains of donor mice, we tested the hypothesis that there are genetic determinants affecting eicosanoid levels in stored RBCs. Methods: RBCs from C57BL/6 (B6) and FVB mice were collected in CPDA-1, filter leukoreduced, and stored under conditions previously shown to model human RBC storage. Samples collected on days 0, 5, 9, and 14 were analyzed by small molecule mass spectrometry. The study was repeated 3 times and combined data were analyzed. Results: AA accumulated over storage time in both B6 and FVB RBC units to a similar level. In contrast, although essentially no accumulation of eicosanoids was observed in B6 RBC units, substantial time-dependent increases (compared to day of collection) were observed in FVB RBC units for 5-HETE (4-fold) and 15-HETE (12-fold). In addition, a greater than 10-fold increase was observed for prostaglandin E2 in FVB RBC units with no detectable prostaglandin E2 in B6 RBC units [ findings were consistent in all 3 experiments and all differences had p values of

Description: Mixing of allogeneic serum with RBC that are ABO-incompatible is well known to cause lysis of the RBC. However, the severity and extent of such hemolysis may not be the same for every mismatched pairing and is not documented in the literature. We were interested in quantitating the lytic potential of a large panel of normal sera when exposed to ABO-incompatible RBC. We collected 80mL of whole blood in red-top tubes from 110 random healthy subjects (84 female 26 male) with blood Groups A (n=6), AB (n=5), B (n=10) and O (n=89). Sera were prepared by clotting at room temp, frozen in aliquots, stored at −80°C. CPD whole blood (N=14) from healthy subjects were obtained commercially, shipped and stored per standard methods (A1: 8, A2: 2, O: 4). Forward ABO typing on all serum subjects and whole blood was with Immucor-Gamma murine monoclonal antibody reagents. Within 48 h of donation, packed RBC were prepared by centrifugation of an aliquot at 900xg for 5 min. RBC were resuspended in AS-3 to a final hematocrit of 10%. Total Hb was measured on a BayerAdvia120 analyzer, and hematocrit was determined by capillary tube centrifugation. Aliquots of 125μL of this RBC suspension were incubated with 250μL aliquots of the serum panel for 30 min at 37°C followed by double centrifugation at 900xg for 5 min. Supernatant (sup) was assayed for Hb by the cyanmethemoglobin method reading on a spectrophotometer at 540nm with a turbidity correction for 680nm absorbance. Results were corrected for test serum and RBC plasma absorbances. Percent hemolysis was calculated as 100xHb(sup, mg/dL)x(1-hct)/Hb(total, mg/dL). A repeated measures analysis of variance mixed-effects model was used to test the effect of serum-donor sex, serum-donor ABO group and RBC ABO group on hemolysis. Sup Hb was categorized into visually detectable hemolysis (〉56 mg/dL; Elliot et al, Transfusion2003;43:297) or not. A total of 1462 plasma-RBC incubations were carried out, with each of the 110 test sera tested versus the test RBC. As expected, no significant hemolysis was detected for ABO-compatible combinations (p〉0.8). Complete hemolysis was not observed in any combination. Of the 89 O-sera, median hemolysis with A1-RBC was 3.9% (max 33.4%), but only a median of 0.9% (max of 17.5%) with A2-RBC. There was no significant effect of serum-donor gender on hemolysis (p=0.8). Group O-sera caused higher average hemolysis than B sera for both A1 and A2-RBC. A1-RBC had a larger hemolytic response to both sera types. Notably, not all sera of the same ABO-group caused equivalent hemolysis. We observed a larger variation in hemolysis between serum donors, with an intraclass correlation coefficient ρ=0.55, than between RBC units (ρ=0.028). Therefore, in vitro testing of hemolysis potential should be against a large panel of sera to capture the inherent person-to-person variability. Hemolysis of ABO Incompatible Mixtures of RBC and Sera Phenotype Hemolysis (%) Hb (mg/dL) Visual Lysis RBC Serum N Mean±SE (min-max) p Mean±SE (min-max) 〉 56 mg/dL (p