Abstract Presentations from the AABB Annual Meeting Philadelphia, PA, October 25-28, 2014

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Plenary P1-030A The Genetic Basis of A3, an Enigmatic ABO Subgroup Phenotype with Dual Cell Populations A K Hult1,2, B Thuresson1, Hellberg1,2, R Ajore1, M L Olsson1,2. 1 Department of Laboratory Medicine, Lund University, Division of Hematology and Transfusion Medicine, Lund, Sweden; 2Division of Laboratory Medicine, Office for Medical Services, Department of Clinical Immunology and Transfusion Medicine, Lund, Sweden Background/Case Studies: A3 has been called the most common weak ABO subgroup. It represents a longstanding and principally interesting conundrum, since no explanation has been presented to elucidate its underlying mechanism. First described in 1936 by Friedenreich, A3 red cells display an inherited mixed-field reaction with a few large agglutinates among large amounts of free cells when typing with anti-A. In 1942, Gammelgaard reported the A3 prevalence to be ∼0.1% in Denmark. The A3 samples encountered in our reference laboratory all display heterozygosity for a consensus A1 allele in the coding region of the ABO gene. In the absence of a genetic marker, we recently identified a diagnostic flow cytometry pattern. In 2010, a regulatory element was reported in ABO intron 1 with a functional GATA-1 erythroid transcription factor binding site. Mutations in this motif cause subgroups Am/Bm and prompted us to investigate this region further in a cohort of A3 samples. Study Design/Methods: DNA sequencing and allele-specific primer (ASP) polymerase chain reaction (PCR) was used to examine parts of ABO intron 1 (+5,784 to +6,267) in blood samples (n = 51) defined as A3 by serology and/or flow cytometry. Screening by PCR-ASP was performed on donor samples with common ABO phenotypes (n = 321) and on other Aw/Bw samples (n = 28) with defined genetic backgrounds. The upstream CBF/NF-Y enhancer region was examined in all A3 samples. Luciferase assay was performed using two different cell lines, K562 and HEL. Results/Findings: A previously unreported single nucleotide substitution (G > A) close to the GATA site mentioned above was found in 42 of the A3 samples. Constructs with the new change showed lower relative Luciferase activity in both cell lines compared to consensus. Re-analysis of flow cytometry patterns of samples with or without the mutation revealed a previously overlooked difference between them regarding distribution of the two main populations. The A3 mutation was not found in common phenotypes or other rare subgroup samples. Of the 9 remaining A3-like samples, 2 had other mutations close to the GATA site, whilst 7 samples displayed consensus sequences. Analysis of the enhancer region showed expected results in all A3 samples. Conclusion: An apparently A3-specific genetic basis underlying the first reported weak ABO subgroup was found outside the ABO coding region. The majority (82%) of the A3 samples had the same alteration in a regulatory portion of intron 1, whilst another two (4%) had other mutations but still close to the GATA site, resulting in similar phenotypes. The genetic background in the remaining seven samples still needs to be resolved as does the mechanism by which the characteristic A3 pattern with dual populations arises. Our work yielded new genetic markers useful in blood banks for correct definition of ABO group in blood donors and patients. Disclosure of Commercial Conflict of Interest R. Ajore: No Answer; Hellberg: Nothing to disclose; A. K. Hult: No Answer; M. L. Olsson: No Answer; B. Thuresson: Nothing to disclose

P2-030A Randomized Trial Results: Red Cell Storage Age is Not Associated with a Significant Difference in Multiple-Organ Dysfunction Score or Mortality in Transfused Cardiac Surgery Patients M E Steiner1, D J Triulzi2, S F Assmann3, S R Sloan4, M Delaney5, M A Blajchman6, S Granger3, P A D’Andrea2, S Pulkrabek7, C P Stowell8, F the Transfusion Medicine/Hemostasis Network9. 1Pediatrics, University of Minnesota, Minneapolis, MN, United States; 2University of Pittsburgh, Pittsburgh, PA, United States; 3NERI, Watertown, MA, United States; 4 Boston Children’s Hospital, Boston, MA, United States; 5Puget Sound Blood Center, Seattle, WA, United States; 6McMaster University, Hamilton, ON, Canada; 7University of Minnesota, Minneapolis, MN, United States; 8 MGH, Boston, MA, United States; 9NHLBI, Bethesda, MD, United States Background/Case Studies: Equipoise exists over whether red blood cell (RBC) storage age affects recipient health outcomes. Many studies of RBC storage age are observational and have conflicting conclusions about the impact of RBC storage time. Cardiac surgery patients were chosen for the study as they often require multiple RBC transfusions, and because observational studies suggest they may be at risk from longer-stored RBCs. Study Design/Methods: A randomized trial was conducted from 2010-2014 at 33 centers. Participants ≥12 years old and >40 kg undergoing complex/re-do cardiac surgery and likely to receive RBCs were randomized preoperatively to receive leukocyte-reduced AS RBC stored either ≤10 d or ≥21 d for all intra- and post-op transfusions through hospital discharge, death, or Day 28 (end of study). Participants were randomized only if the crossmatch request could be filled both with RBC units stored ≤10 d and stored ≥21 d. The primary endpoint was change in Multiple-Organ Dysfunction Score (Δ MODS) from pre-op to the highest composite MODS through Day 7 (or death/discharge, if earlier), adjusted for baseline MODS. Secondary endpoints included adverse events and Day 28 mortality. The study was powered to have precision for a 2-sided 95% CI for the treatment group difference in Δ MODS of ≤ ± 0.85 points. Results/Findings: Of 1613 eligible consenting patients, 1481 were randomized, and 1098 were evaluable (transfused by 96 hr post-op); 132 patients were not randomized because blood banks were unable to provide enough RBC units of both storage times. Results are summarized in the Table. Conclusion: The trial had good compliance with the randomized storage age assignment with a minimal amount of overlap. RBC storage duration was not significantly associated with 7-day Δ MODS, serious adverse events, or 28-day mortality. Disclosure of Commercial Conflict of Interest S. F. Assmann: Nothing to disclose; M. A. Blajchman: No Answer; P. A. D’Andrea: Nothing to disclose; M. Delaney: Nothing to disclose; S. Granger: Nothing to disclose; S. Pulkrabek: Nothing to disclose; S. R. Sloan: Nothing to disclose; M. E. Steiner: Nothing to disclose; C. P. Stowell: Nothing to disclose; F. the Transfusion Medicine/Hemostasis Network: Nothing to disclose; D. J. Triulzi: Nothing to disclose Disclosure of Grants Conflict of Interest S. F. Assmann: Nothing to disclose; M. A. Blajchman: No Answer; P. A. D’Andrea: Nothing to disclose; M. Delaney: Nothing to disclose; S. Granger: Nothing to disclose; S. Pulkrabek: Sekisui Diagnostics,LLC,Grants or Research Support; S. R. Sloan: Nothing to disclose; M. E. Steiner: Nothing to disclose; C. P. Stowell: Nothing to disclose; F. the Transfusion Medicine/ Hemostasis Network: Nothing to disclose; D. J. Triulzi: Nothing to disclose

Disclosure of Grants Conflict of Interest R. Ajore: No Answer; Hellberg: Nothing to disclose; A. K. Hult: No Answer; M. L. Olsson: No Answer; B. Thuresson: Nothing to disclose TABLE. Trial Results Arm Outcome RBC Transfusions Median units/patient (Q1,Q3) N (%) subjects with all units of assigned storage duration Median storage days (Q1, Q3) Primary outcome Mean Composite Δ MODS Day 7 (+ SD) Adj. Treatment group difference (95% CI) Secondary outcomes N (%) subjects with > 1 SAE Median SAEs/subject (Q1,Q3) N (%) All-Cause Mortality Day 28

≥21 d N = 560

≤10 d N = 538

p

3.0 (2.0, 6.0) 488 (87%) 28 (24, 33)

4.0 (2.0, 6.0) 478 (89%) 7 (5, 9)

0.80 0.35 90%) of recipients failed to make detectable alloantibody responses to 50 microliters of KEL2 high RBCs, even with repeat transfusions. However, in 2/2 experiments (n = 10 recipients total), 100% of recipients who were transfused with smaller volumes (5 microliters) of the same KEL2 high RBCs made detectable, albeit low-level (adjusted mean MFI 20) anti-KEL IgG alloantibody responses. If mice were pretreated with an inducer of systemic inflammation, then significant alloimmunization was seen to both doses of KEL2 high RBCs. Conclusion: These studies demonstrate that both RBC copy number and dose of transfused RBCs are important variables in determining recipient immune responses. “High dose tolerance” has been described for non-RBC antigens. It was recently reported that transfusing large amounts of RBCs expressing another highdensity antigen (human glycophorin A, hGPA), resulted in antigen-specific immune tolerance, and studies are underway to assess the possibility in the KEL2 system. A better understanding of donor factors that influence recipient responsiveness may allow for strategies to better predict and prevent RBC alloimmunization. Disclosure of Commercial Conflict of Interest C. M. Arthur: No Answer; J. E. Hendrickson: Nothing to disclose; N. H. Smith: Nothing to disclose; S. R. Stowell: Nothing to disclose; J. C. Zimring: Nothing to disclose Disclosure of Grants Conflict of Interest C. M. Arthur: No Answer; J. E. Hendrickson: Terumo, Grants or Research Support; N. H. Smith: Nothing to disclose; S. R. Stowell: Nothing to disclose; J. C. Zimring: Immucor, Grants or Research Support P5-030A Innate Immune Factors Specifically Target Blood Group-positive Microbes C M Arthur1, H C Sullivan1, L C Rodriguez1, X Zhao1, S Patel1, R Cummings1, S R Stowell1. 1Emory University, Decatur, GA, United States Background/Case Studies: Despite Landsteiner’s discovery of carbohydrate ABO blood group antigens over a century ago, many questions remain regarding the development and regulation of naturally occurring anticarbohydrate blood group antibodies. Unlike the development of red blood cell-induced alloantibodies, early studies suggested that carbohydratebased blood group-positive (cBG+) microbes might stimulate naturally occurring anti-carbohydrate blood group antibodies in carbohydrate blood group-negative (cBG−) individuals within the first few months of life. However, as immunological tolerance limits adaptive immunity toward cBG+ microbes in cBG+ individuals, the specific mechanism(s) responsible for protecting cBG+ individuals from cBG+ microbes remains unknown. We hypothesized that innate immune factors may exist that specifically target cBG+ microbes, thereby protecting cBG+ individuals from cBG+ microbes. Study Design/Methods: Using a recently developed microbial glycan microarray that includes hundreds of isolated microbial carbohydrates, including carbohydrate blood group antigens, we prepared and screened more than 20 recombinant human innate immune factors with putative carbohydrate-binding activity. Positive interactions with microbial glycans were further characterized by examining potential engagement with intact microbes by flow cytometric examination. The potential impact of innate immune factors on microbial viability was assessed by incubating each innate immune factor with target microbes followed by colony forming unit enumeration. Results/Findings: Among innate immune factors examined, several members of the galectin family, including galectin-4 (Gal-4), galectin-7 (Gal-7), and galectin-8 (Gal-8), possessed remarkable specificity for microbial carbohydrate blood group antigens. Gal-4, Gal-7, and Gal-8 recognized ABO(H), I, and i blood group antigens from diverse microbial genera, including E. coli, P. alcalifaciens, K. pneumoniae, S. marcescens,

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and S. agalactiae, while failing to bind glycans isolated from cBG− microbes. Consistent with microarray results, each galectin displayed a similar preference for intact cBG+ microbes. Incubation of each galectin with cBG+ microbes resulted in rapid microbial death, while galectins failed to recognize or kill cBG− microbes. Conclusion: These results suggest that innate immune factors exist that specifically target cBG+ microbes, thus filling an important gap in adaptive immunity against cBG+ microbes in cBG+ individuals. The ability of these innate immune factors to specifically target BG+ microbes further suggests that galectin-mediated immunity may also target cBG+ microbes in cBG− individuals and thus regulate the development of naturally occurring anti-carbohydrate blood group antibodies.

service. These findings document the poor sensitivity of sepsis criteria, the value of active surveillance, and the need for improved recognition and reporting of septic platelet transfusion reactions. Disclosure of Commercial Conflict of Interest H. Hong: Nothing to disclose; M. R. Jacobs: No Answer; R. W. Maitta: Nothing to disclose; W. Xiao: Nothing to disclose Disclosure of Grants Conflict of Interest H. Hong: Nothing to disclose; M. R. Jacobs: No Answer; R. W. Maitta: Nothing to disclose; W. Xiao: Nothing to disclose

Disclosure of Commercial Conflict of Interest C. M. Arthur: No Answer; R. Cummings: No Answer; S. Patel: No Answer; L. C. Rodriguez: No Answer; S. R. Stowell: Nothing to disclose; H. C. Sullivan: Nothing to disclose; X. Zhao: No Answer Disclosure of Grants Conflict of Interest C. M. Arthur: No Answer; R. Cummings: No Answer; S. Patel: No Answer; L. C. Rodriguez: No Answer; S. R. Stowell: Nothing to disclose; H. C. Sullivan: Nothing to disclose; X. Zhao: No Answer

P6-030A Sensitivity and Specificity of Septic Transfusion Reaction Criteria in Identifying Septic Reactions to Platelet Transfusions H Hong1,2, W Xiao1,2, M R Jacobs1,2, R W Maitta1,2. 1Pathology, University Hospitals Case Medical Center, Cleveland, OH, United States; 2School of Medicine, Case Western Reserve University, Cleveland, OH, United States Background/Case Studies: Criteria for recognition of transfusiontransmitted bacterial contamination have been set up to trigger recognition, management, and further investigation. However, these criteria broadly overlap with the diagnostic criteria for febrile nonhemolytic transfusion reactions (FNHTRs). We retrospectively evaluated the sensitivity and specificity of current septic transfusion reaction criteria in identifying patients with true transfusion-transmitted bacterial contamination of platelets. Study Design/ Methods: Patients receiving platelets from January 1, 2007, through December 31, 2013, at a large medical center with transfusion reactions reported to the transfusion service were analyzed (passive surveillance). Bacterial culture was performed on every platelet unit at time of issue (active surveillance). Patients’ transfusion reactions were correlated with platelet and posttransfusion blood cultures. Transfusion reactions were categorized based on the CDC Biovigilance Guidelines. Data were analyzed using the two-tailed Fisher’s exact test. Significance was set at a p of 1.5 mg/mL, but DIIHA has been described only once. Anti-gentamicin was reported once as an in vitro problem only. We report a 61-year-old female with an infected knee joint who underwent surgical irrigation, debridement, and placement of antibiotic impregnated polymethylmethacrylate articulating spacers containing vancomycin and gentamicin. Postoperative medications included cefazolin on days 0 (surgery) and 1, ceftriaxone on days 2 and 6-15, cefotaxime on day 8, and vancomycin on days 1-20. On day 1, her antibody screen was negative. She was transfused RBCs due to blood loss, but continued to require RBC transfusions (10 units on days 1-16) without signs of bleeding, and she had laboratory evidence of hemolysis (day 15: hemoglobin 5.2 g/dL, retics 253 x 109/L, LDH 1083 IU/L, haptoglobin < 6 mg/dL; total bilirubin 1.2 mg/mL). On day 7, autoanti-N and an unidentified antibody were detected, the direct antiglobulin test (DAT) was positive (IgG neg, C3 1+), and the eluate was nonreactive. A cold antibody was detected on day 15. Study Design/Methods: Drug antibody testing was performed by two methods: 1) with drug-treated N- RBCs, and/or 2) with untreated (UT) & enzyme-treated (ET) N- RBCs in the presence of a solution of drug (1 mg/ mL). Testing was initially performed with the three cephalosporins and later with vancomycin and gentamicin. Results/Findings: Antibodies to ceftriaxone, cefotaxime, and cefazolin were not detected. On day 18, the DAT was positive (IgG 2+, C3 2+); by day 51, the DAT was reactive only with anti-C3 (1+w). An agglutinating anti-vancomycin (3+) was detected in sera from days 18 and 51 (agglutinin titers = 2) and in an eluate from the day 18 RBCs. The AGTs were reactive only if saline containing vancomycin was used for the washing step. The vancomycin antibody reacted only with UT RBCs in the presence of vancomycin; it was not detected when testing either vancomycin-treated RBCs or ET RBCs in the presence of vancomycin. The serum and eluate anti-vancomycin agglutinins were not destroyed by dithiothreitol and thus were IgG. An antibody to gentamicin was detected in the patient’s day 18 and 51 sera but not in the day 18 eluate. After discontinuation of intravenous vancomycin on day 20, the patient’s hemoglobin remained stable. Conclusion: The patient’s clinical course and laboratory testing confirm a second report of vancomycin-induced immune hemolytic anemia and a second example of anti-gentamicin. This case highlights the importance of considering all medications with good temporal relationships to apparent immune hemolytic anemias as possible causes of DIIHA.

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RBCs. Study Design/Methods: This was a retrospective study from January 1994 to June 30, 2013. The alloimmunization rate and specificity of additional alloantibodies following IUTs with the use of allogeneic RBCs were determined and compared to those following IUTs using maternal RBCs. Results/Findings: During the study period, 167 patients with alloantibodies implicated in HDFN received 650 IUTs during 196 pregnancies. Thirty patients underwent 96 IUTs with the use of maternal RBCs. Following IUT, 18/167 patients (10.8%) developed 23 new RBC alloantibodies. Of these 18 patients, 6/30 (20%) produced 6 additional antibodies after IUTs with maternal RBCs, and 12/137(8.8%) patients developed alloantibodies following IUTs with allogeneic RBCs. Details of alloantibody specificity and frequency in these 18 patients before and after IUTs are summarized in the Table. Despite using C-, E-, K-negative allogeneic RBCs or maternal RBCs for IUTs in this study, 18 of 23 (78.3%) additional alloantibodies could not be induced by either allogeneic RBCs or maternal RBCs. Conclusion: Conclusion: Rh(D)-sensitized women had a high incidence of producing additional alloantibodies within the Rh system following IUTs due to fetal RBCexposure from FMH. The use of more extended antigen-matched allogeneic RBCs may reduce some but not the preponderance of new alloantibodies that develop after IUT. Disclosure of Commercial Conflict of Interest M. J. Biscocho: Nothing to disclose; K. Lau: No Answer; T. Mnich: No Answer; X. Qi: Nothing to disclose; N. Shehata: No Answer; A. Wagner: No Answer Disclosure of Grants Conflict of Interest M. J. Biscocho: Nothing to disclose; K. Lau: No Answer; T. Mnich: No Answer; X. Qi: Nothing to disclose; N. Shehata: No Answer; A. Wagner: No Answer TABLE. Alloantibody Specificity and Frequency before and after IUT Antibodies before 1st IUT

Additional antibodies after IUTs Frequency

Specificity -D

-D&-C

-K

Frequency 11

5

2

Specificity -C -E -K -Fya -Lea -E -Jkb -Fyb -S -s -Jkb -e

After 1st IUT

After 2nd IUT

After 3rd IUT

After 4th IUT

1* 1*

4 4 1

1

2 (1*)

1* 1 1 1 1 1 1 1* 1*

* The additional antibody was formed after the IUT with maternal RBCs.

Disclosure of Commercial Conflict of Interest P. A. Arndt: Nothing to disclose; E. Y. Cheng: Nothing to disclose; R. M. Leger: Nothing to disclose; N. D. Zantek: Nothing to disclose; D. Zydowicz: No Answer Disclosure of Grants Conflict of Interest P. A. Arndt: Nothing to disclose; E. Y. Cheng: Nothing to disclose; R. M. Leger: Nothing to disclose; N. D. Zantek: Sekisui Diagnostics,Grants or Research SupportAstraZeneca,Grants or Research Support; D. Zydowicz: No Answer S3-010A High Incidence of Formation of Additional Rh Antibodies in Rh(D)sensitized Patients after Intrauterine Transfusion X Qi1, A Wagner1, T Mnich1, M J Biscocho1, K Lau1, N Shehata1. 1 Transfusion Medicine Service, Mount Sinai Hospital, Toronto, ON, Canada Background/Case Studies: Anti-D is the most common cause of severe hemolytic disease of the fetus and newborn (HDFN). Intrauterine transfusion (IUT) has improved the survival of fetuses affected by HDFN. However, IUT is often associated with fetomaternal hemorrhage (FMH), which may result in formation of additional red blood cell (RBC) alloantibodies. The objective of this study was to determine the frequency and specificity of additional RBC alloantibodies after IUT in patients receiving allogeneic or maternal

S4-010A Resource Utilization after First Trimester Noninvasive Fetal RHD Screening for Targeted Antenatal Anti-D Prophylaxis in RhD-Negative Swedish Women M Neovius2, E Tiblad1,3, M Westgren3, K Neovius2, A T Wikman1,4. 1 Karolinska University Hospital, Stockholm, Sweden; 2Clinical Epidemiology Unit, Karolinska Institutet, Stockholm, Sweden; 3Clintec, Karolinska Institutet, Stockholm, Sweden; 4Labmedicine, Karolinska Institutet, Stockholm, Sweden Background/Case Studies: With routine antenatal anti-D prophylaxis (RAADP), the incidence of anti-D immunization in RhD-negative women has decreased to approximately 0.2%. Arguments against RAADP for all RhDnegative women are that almost 40% carry an RHD-negative fetus, and the fetus does not benefit of a preparation that can be potentially harmful and has limited availability. Noninvasive fetal RHD screening has made it possible to administer RAADP selectively to the women carrying an RHDpositive fetus. The aim of the present study was to investigate resource utilization of targeted RAADP using noninvasive antenatal RHD screening in early pregnancy, compared to Swedish standard care, which is administration of postnatal anti-D prophylaxis but not RAADP, among Swedish RhDnegative pregnant women. Study Design/Methods: A prospective, nonrandomized intervention study with historical controls was conducted

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based on population-based data from the Swedish National Health Service in Stockholm County. The study included RhD-negative pregnant women who received first-trimester fetal RHD screening and targeted anti-D prophylaxis, with deliveries between January 1, 2010, and December 31, 2011 (n = 6723). Data on obstetric and neonatal outcome were retrieved from medical records. Historical controls who received only postnatal anti-D prophylaxis with deliveries between January 1, 2008, and December 31, 2009, were identified via the same data source (n = 7099). The proportions of sensitizations, amniocenteses, length of stay, and anti-D titers were compared between the groups, adjusted for maternal age, country of birth, and multiple birth. Results/Findings: In the screening cohort, 13 (0.19%) sensitizations were registered compared with 33 (0.46%) in the control cohort (adjusted odds ratio 0.41; 95%CI 0.22-0.78; P = 0.006). A difference was also detected between the screening cohort and the control cohort regarding the proportion of women with amniocentesis (6.1% vs. 6.8%; adjusted odds ratio 0.81; 95%CI 0.70-0.93; P = 0.004) and anti-D titers registered (0.95% vs. 1.38%; adjusted odds ratio 0.67; 95%CI 0.49-0.93; P = 0.01). However, no difference could be detected in length of hospital stay for the infants after delivery, between the two groups (3.3 vs. 3.4 days; P = 0.08). Conclusion: Noninvasive RHD screening was found to be associated with a lower incidence of sensitization, as well as less use of amniocentesis and anti-D titrations. How much of the increased costs associated with the screening test that are offset by these and potentially other resource utilization reductions needs to be quantified. These data need to be complemented by costs for fetal and neonatal blood transfusions and photo therapy, as well as screening test and administration costs, in order to perform a formal costeffectiveness analysis. Disclosure of Commercial Conflict of Interest K. Neovius: No Answer; M. Neovius: Nothing to disclose; E. Tiblad: No Answer; M. Westgren: Nothing to disclose; A. T. Wikman: Nothing to disclose Disclosure of Grants Conflict of Interest K. Neovius: No Answer; M. Neovius: Nothing to disclose; E. Tiblad: No Answer; M. Westgren: Nothing to disclose; A. T. Wikman: Nothing to disclose S5-010A Comparison of Alloimmunization Rates in Sickle Cell Disease Patients in the United States vs. Other Countries Y Zheng2,1, R W Maitta2,1. 1Case Western Reserve University School of Medicine, Cleveland, OH, United States; 2University Hospitals Case Medical Center, Cleveland, OH, United States Background/Case Studies: Sickle cell disease (SCD) patients treated with chronic transfusion therapy are at a high risk of red blood cell (RBC) alloimmunization. In general, alloimmunization rate is determined by factors such as the frequency of transfusions (exposure) and the genetic makeup characterizing the recipient vs. donor pool. It is not clear whether there is a lower threshold for RBC transfusion in the United States compared to other countries, thus influencing alloimmunization. We evaluated the alloimmunization rates of SCD patients in the United States vs. the cumulative alloimmunization rates in other countries. Study Design/Methods: We searched MEDLINE for studies on alloimmunization of SCD patients (case reports were excluded). Studies were divided into two groups: US-based and non-US-based studies. The following parameters were recorded from each study: 1) number of transfused patients, 2) number of non-ABO/ non-naturally occurring alloantibodies, and 3) number of alloimmunized patients to non-ABO antigens. Alloimmunization rate (number of alloimmunized patients/ number of transfused patients) and the number of alloantibodies per transfused patient of each study were calculated first. Due to the variable patient numbers among studies, a weighted average was used to calculate the average alloimmunization rates and number of alloantibodies per transfused patient. Results/Findings: Twenty-four studies published between 1971 to 2012 were used to derive the alloimmunization rate of SCD patients; 16 of these studies, with a total of 3,609 patients, were from the United States, and 8 of these studies, with a total of 2,088 patients, were from other regions, including South America, Middle East, Africa, and Europe. The alloimmunization rate in the United States was higher than that in other countries (22.8 ± 0.1% vs.17.0 ± 0.3%, p < 0.0001). Similarly, US patients had a higher number of alloantibodies per person compared to patients from other countries (0.3 vs. 0.2, p < 0.0001). To determine if this higher alloimmunization rate in the United States is influenced by a Caucasian-dominant US donor pool, the alloimmunization rate in Brazil, whose donor pool consists of a mixed ethnic background, was used for comparison. Brazil had a lower alloimmunization rate (14.59% ± 0.40, p < 0.0001) and less alloantibodies per person (0.20 ± 0.01, p < 0.0001) than did the United States. Conclusion: SCD patients in the United States

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had an apparent higher alloimmunization rate, which could be reduced by a more diverse donor pool and a restricted transfusion strategy. Though the differences between the donor pool and SCD patients who are negative for many antigens may in part explain the high US alloimmunization rate, this alone does not explain the observed differences. Disclosure of Commercial Conflict of Interest R. W. Maitta: Nothing to disclose; Y. Zheng: Nothing to disclose Disclosure of Grants Conflict of Interest R. W. Maitta: Nothing to disclose; Y. Zheng: Nothing to disclose

S6-010A Inflammation Significantly Alters Splenic Composition and Erythrophagocytosis K E Hudson1, J E Hendrickson2, J C Zimring1. 1Research Institute, Puget Sound Blood Center, Seattle, WA, United States; 2Yale University, New Haven, CT, United States Background/Case Studies: Antigen-presenting cells (APCs) directly influence the specificity and magnitude of an immune response through antigen presentation to T cells. Dendritic cells, macrophages, and B cells are capable of processing and presenting antigen to CD4+ T cells, which can elicit antibodies from B cells. Generation of RBC-specific antibodies has been correlated with inflammation (both in the context of autoantibodies in Autoimmune Hemolytic Anemia and alloantibodies upon transfusion). Herein, we test the hypothesis that different APCs participate in red blood cell (RBC) consumption between baseline and inflamed states. Study Design/Methods: C57BL/6 (B6) RBCs were labeled with fluorescent lipophilic dye, DiO. Labeled RBCs were analyzed by flow cytometry to confirm fluorescence prior to transfusion. B6 mice were treated with 100 ug of Poly(I:C) intraperitoneally to induce inflammation or control PBS. One hour after treatment, mice were transfused with 100 uL of packed DiO+ B6 RBCs. Mice were sacrificed 18 hours post- transfusion, and spleens were harvested, collagenase-digested, and stained with antibodies to delineate subsets of APCs. Stained cells were analyzed on an LSRII. For all analysis, cells were gated on live events that were TER119-Thy1.2- to exclude RBCs stuck to the surface of cells and T cells. Results/Findings: Compared to control mice, Poly(IC)-treated mice had >15% increase in DiO+ cells (4 independent experiments, n = 12 mice). RBC consumption, as a measure of DiO+ fluorescence, was detectable in conventional dendritic cells (DCs) (CD11chiCD11b+ myeloid and CD11chiCD8+ lymphoid), CD11cintCD11b+F4/ 80+MHCII+ red pulp macrophages, and IgMhiCD21/35hi marginal zone B cells with similar MFI between control and Poly(IC)-treated groups. However, absolute numbers of conventional DCs and red pulp macrophages were significantly decreased upon Poly(IC) administration. Preliminary data demonstrated that Poly(IC) treatment resulted in increased numbers and MFI of DiO+CD115+Ly6Clo inflammatory monocytes, DiO+Ly6G+ granulocytes, and plasmacytoid-like DCs that are DiO+ and express CD11cintCD11b+PDCA1+CD19+. Conclusion: Dendritic cell and macrophage populations participate in erythrophagocytosis. At baseline, DiO+ RBCs are predominately detected in red pulp macrophages by flow cytometry. Upon administration of inflammatory agent Poly(IC), splenic APC populations are significantly altered, with cDCs and red pulp macrophage numbers significantly reduced. Additionally, Poly(IC) induces the appearance of new populations of DiO+ APCs that are associated with inflammation, antigen presentation, and T cell activation. Ongoing studies will assess which APC subsets can induce proliferation of CD4+ T cells and lead to anti-RBC antibody production and will evaluate whether these parameters are applicable to RBC alloantigens. Disclosure of Commercial Conflict of Interest J. E. Hendrickson: Nothing to disclose; K. E. Hudson: Nothing to disclose; J. C. Zimring: Nothing to disclose Disclosure of Grants Conflict of Interest J. E. Hendrickson: Terumo,Grants or Research Support; K. E. Hudson: Nothing to disclose; J. C. Zimring: Immucor,Grants or Research Support

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Platelet and Leukocyte Biology

S7-010B Kinesin-dependent Transport of Stress Granule into Microparticles during Platelet Apoptosis P Schubert1,2, B Culibrk1,2, S Karwal1,2, D Devine1,2. 1Centre for Innovation, Canadian Blood Services, Vancouver, BC, Canada; 2Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada Background/Case Studies: Platelets possess the capacity for protein translation, and some mRNAs exhibit a surprising stability throughout platelet storage. In nucleated cells, protection of mRNA from degradation involves the incorporation of mRNA into stress granules (SG) complexed with mRNAbinding proteins. We have previously reported the relocalization of stress granule proteins from the cytoplasm to the membrane upon stress stimulation and have now investigated whether platelets have a transport process for this movement. Kinesins are motor proteins responsible for organelle transport along microtubules in mammalian cells, and they might be involved. Furthermore, a potential delivery of SG into platelet microparticles was analyzed. Study Design/Methods: Leukoreduced apheresis platelet concentrates were fractionated using differential centrifugation, resulting in a membrane- and actin-cytoskeleton fraction and the cytoplasm. Platelet microparticles (MPs) were obtained at 100,000 rpm after removing the platelets at 3,300 rpm. MP lysates prepared in 1% Triton were analyzed by immunoblot. As stress granules are quite labile, crosslinking using dithiobis(succinimidyl)propionate (DSP, Pierce) according to the manufacturer’s protocol was carried out to stabilize SG complexes. To test whether SG bind to transport proteins, immunoprecipitation (IP) of G3BP-1 (SigmaAldrich) with kinesin light chain (KLC, Abcam) was used to examine the interaction. Inhibition of kinesin activity was achieved with Monastrol (Santa Cruz Biotechnologies) at a final concentration of 10 mM at room temperature for 10 minutes before calcium-ionophore (CI, 0.5 mM) stimulation. Results/ Findings: Immunoblot analyses demonstrated that stress granules not only assembled in the actin cytoskeleton, but also appeared in the membrane fraction of platelets upon induction of apoptosis with CI. Proteomic analyses identified stress granule proteins such as G3BP-1 in the platelet microparticles upon treatment with CI, but not without the stimulation. KLC is the kinesin subunit for cargo binding to the motor component, and immunoblot analyses demonstrated the existence of KLC in platelets. IP revealed interaction between the stress granule component G3BP-1 and KLC. Kinesins mediate anterograde transport from the cytoplasm to the membrane, which is in agreement with the results obtained from the fractionation analyses. Inhibition of kinesin activity by monastrol prior to the CI treatment reduced the protein level of G3BP-1 in micoparticles by 2.5-3.5fold. This potential transport process could be verified in five independent experiments. Conclusion: The observation that stress granules move to the platelet membrane in a kinesin-dependent mechanism and accumulate in microparticles suggests a defined pathway for the transport of mRNA loading into microparticles and subsequent delivery into other cells. Disclosure of Commercial Conflict of Interest B. Culibrk: Terumo BCT, Grants or Research Support; D. Devine: Nothing to disclose; S. Karwal: Nothing to disclose; P. Schubert: Terumo BCT, Grants or Research Support Disclosure of Grants Conflict of Interest B. Culibrk: Terumo BCT, Grants or Research Support; D. Devine: Terumo BCT, Grants or Research Support; MacoPharma, Grants or Research Support; Fresenius, Consulting or Board of Director Fees; S. Karwal: Nothing to disclose; P. Schubert: Terumo BCT, Grants or Research Support S8-010B Fas Receptor Signaling in Platelets is Involved in Platelet Clot Fibrinolysis P Schubert1,2, B Culibrk1,2, A F Arbaeen1,2, D Devine1,2. 1Centre for Innovation, Canadian Blood Services, Vancouver, BC, Canada; 2Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada Background/Case Studies: It has become increasingly apparent that the production of platelets and their subsequent life span in the circulation are regulated by apoptotic mechanisms that seem to be involved in the regulation of platelet functional responses. Activated platelets express the Fas ligand (FasL) on its surface, and we have identified its partner, the Fas receptor (FasR), and several signaling components. In nucleated cells, engagement of the receptor by the ligand results in cell death mediated by

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caspase activation. Whether this FasR-FasL interaction is involved in platelet clot formation or destruction is still unknown. Study Design/Methods: Leukoreduced apheresis platelet concentrates were used in both protein expression studies and thromboelastography (TEG) experiments. FasR (CD95) expression and fibrinogen binding were measured on a flow cytometer (FACS Canto, Beckman-Coulter) using CD95 antibodies (Abcam) and an anti-fibrinogen antibody (DAKO), respectively. Fibrinolysis was initiated by 10-nM tissue plasminogen activator prior to calcification of platelets using 17-mM CaCl2. Anti-Fas blocking antibody, ZB4 (Millipore), was used at 10 mM final concentration, and its effectiveness was determined by caspase 8 activation (Biovision). Results/Findings: Platelet activation with 10 mM ADP led to 20 ± 3% (n = 4) surface expression of FasR, while no signal was detected on resting platelets. Quantification of FasR protein levels in membrane fractions comparing untreated and activated platelets revealed a 2.7-fold increase, indicating relocalization to the membrane, while activation did not alter the protein level. This observation suggests that both FasR and FasL are synergistically expressed with platelet activation and may have a role in platelet clot formation. TEG technology was used to monitor clot formation and destruction by analyzing the role of this interaction. Clot formation was initiated by platelet calcification, and the TEG profile revealed a clot lysis of 3% at 30 min (CL30). The addition of ZB4 before calcification, but not an isotype control antibody, increased the CL30 to 12%, while the parameters such as MA, R- and k-time were similar to the untreated samples. This accelerated clot destruction was correlated with reduced caspase 8 activation from 18 ± 3% to 8 ± 2% in the presence of ZB4, indicating that the antibody successfully blocked FasR. Interestingly, fibrinogen binding to platelets treated with ZB4 prior to calcification was reduced by 20 ± 4% (n = 4). Conclusion: Membrane-bound FasL can activate FasR in a manner that seems to downregulate apoptosis signaling in platelets. At the same time, FasR activation contributes to efficient activation of GPIIbIIIa signaling. The physiological significance of these interactions requires further investigation, but FasL-FasR interactions appear to delay fibrinolysis. Disclosure of Commercial Conflict of Interest A. F. Arbaeen: Nothing to disclose; B. Culibrk: Terumo BCT, Grants or Research Support; D. Devine: Nothing to disclose; P. Schubert: Terumo BCT, Grants or Research Support Disclosure of Grants Conflict of Interest A. F. Arbaeen: Nothing to disclose; B. Culibrk: Terumo BCT, Grants or Research Support; D. Devine: Terumo BCT, Grants or Research Support; MacoPharma, Grants or Research Support; Fresenius, Consulting or Board of Director Fees; P. Schubert: Terumo BCT, Grants or Research Support S9-010B Polytrauma and Hemorrhage Affects Platelet Function in Rats D Darlington1, X Wu1, A P Cap1. 1US Army Institute of Surgical Research, JBSA-FT Sam Houston, TX, United States Background/Case Studies: Acute coagulopathy of trauma (aCOT) can occur after polytrauma and hemorrhage in both the civilian and military patients and is associated with an increased mortality rate. The purpose of this study was to determine whether platelet function is altered after polytrauma and hemorrhage. Study Design/Methods: Sprague-Dawley rats (n = 9) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, and the right leg skeletal muscle and by fracturing the right femur. The rats were bled to a mean arterial pressure of 40 mmHg, and held there until 40% of the blood volume was removed. No fluid resuscitation was given. Blood samples were taken before the procedure (time 0) and at 30, 60, 120 and 240 min. Platelet aggregation was measured on a Multiplate (Diapharma) after stimulation with ADP, collagen, thrombin (PAR4), or arachidonic acid and expressed as area under the curve per 1000 platelets. Platelet-rich plasma from samples at 0 and 240 min were stimulated with the above agonists for 5 min and lysed for determination of phosphorylation of intracellular intermediates Src, Lyn, Erk1/2, Akt, p38 MAPK, and NFkB (Multiplex, Biorad). Results/ Findings: Prothrombin time significantly increased over time (18.6 ± 0.3, 20.8 ± 0.5, 21.6 ± 0.5, 21.3 ± 0.3, and 21.9 ± 0.6 sec). The ability of ADP to stimulate platelet aggregation was increased at 30 and 60 min after trauma/ hemorrhage, but was significantly reduced by 2 and 4 hr. Arachidonic acid stimulation was also significantly lower 2 and 4 hr after trauma/hemorrhage. Collagen and thrombin (PAR4) stimulation was significantly elevated 2-4 hr after trauma/hemorrhage. The velocity at which aggregation occurred was significantly elevated for all agonist over the 4-hr period, except ASPI, which did not change. Polytrauma and hemorrhage elevated phosphorylation of NFkB. Stimulation of platelets with the ADP, collagen, arachidonic acid, and PAR4 also tended to increase the intracellular levels of phosphorylated

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NFkB and p38MAPK. ADP increases the intracellular levels of phosphorylated Src, PAR4 stimulation increases the intracellular levels of phosphorylated Akt and Erk. Conclusion: Polytrauma and hemorrhage led to complex changes in platelet function, exemplified by both an elevation in platelet function by collagen and thrombin and a decrease in ADP and arachidonic acid by 4 hr. The change in platelet function may be related to changes in intracellular signaling via Src, Akt and Erk, NFkB, and p38MAPK. This deficit in platelet function seen after ADP and arachidonic acid stimulation may contribute to the development of aCOT. This project was funded by the US Army MRMC. Disclosure of Commercial Conflict of Interest A. P. Cap: Nothing to disclose; D. Darlington: Nothing to disclose; X. Wu: Nothing to disclose Disclosure of Grants Conflict of Interest A. P. Cap: Nothing to disclose; D. Darlington: Nothing to disclose; X. Wu: Nothing to disclose

S10-010B Full-length Nucleotide Sequence of SLC44A2 Alleles Encoding Human Neutrophil Antigen-3 (HNA-3) in African Americans and Caucasians K Srivastava1, Q Chen2, S C Ardinski1, K Lam1, M J Huvard1, P Schmid1, W A Flegel1. 1Department of Transfusion Medicine, NIH, Bethesda, MD, United States; 2The Jiangsu Province Blood Center, Nanjing, China Background/Case Studies: HNA-3a alloantibodies can cause severe transfusion-related acute lung injury (TRALI). The frequency of the single nucleotide polymorphisms (SNPs) indicative of the two clinically relevant HNA-3a/b antigens is known in many populations. In the present study, we determined the full-length nucleotide sequence of the SLC44A2 gene encoding HNA-3. Study Design/Methods: A method was devised to determine the full-length coding sequence and adjacent intron sequences from genomic DNA by 8 PCR amplifications for all 22 SLC44A2 exons. Samples from 200 African American, 96 Caucasian, 2 Hispanic, and 4 Asian blood donors were analyzed. Results/Findings: A total of 10 SNPs were detected in the SLC44A2 coding sequence. The noncoding sequences harbored 28 SNPs (1 in 5′UTR; 23 in the introns; and 4 in the 3′UTR). No SNP indicative of a nonfunctional allele was detected. The nucleotide sequences of more than 20 SLC44A2 alleles (haplotypes) harboring SNPs in different amplicons were confirmed. There may be up to 66 haplotypes among the 604 chromosomes screened. Conclusion: We found 38 SNPs, including 1 novel SNP, in 8192 nucleotides covering the full-length SLC44A2 gene among 302 blood donors. Population frequencies of these SNPs were established for African Americans and Caucasians. Because alleles encoding HNA-3b are more common than nonfunctional SLC44A2 alleles, we conclude that African American donors are less likely to form HNA-3a antibodies compared to Caucasians. Disclosure of Commercial Conflict of Interest S. C. Ardinski: Nothing to disclose; Q. Chen: Nothing to disclose; W. A. Flegel: Nothing to disclose; M. J. Huvard: Nothing to disclose; K. Lam: Nothing to disclose; P. Schmid: No Answer; K. Srivastava: Nothing to disclose Disclosure of Grants Conflict of Interest S. C. Ardinski: Nothing to disclose; Q. Chen: Nothing to disclose; W. A. Flegel: Nothing to disclose; M. J. Huvard: Nothing to disclose; K. Lam: Nothing to disclose; P. Schmid: No Answer; K. Srivastava: Nothing to disclose

S11-010B Experimental Study on the Four Cases of Fetal And Neonatal Alloimmune Thrombocytopenia Mediated with Anti-CD36 Antibodies G Wu1,2, Y Zhou1,2, L Li1,2, W Shen1,2, Z Zhong1,2, J Liu1,2. 1Nanning Institute of Transfusion Medicine, Nanning, China; 2Nanning Blood Center, Nanning, China Background/Case Studies: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is one of the causes of serious bleeding in the neonatal period. The accurate diagnosis of FNAIT is important for effective treatment and for preventive intervention in subsequent pregnancies. CD36, also called glycoprotein IV or IIIb, is an 88-kD cell surface protein. We report four Chinese cases of FNAIT associated with maternal isoimmunization against

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CD36 and identified the isoforms as a cause of CD36 deficiency. Study Design/Methods: The platelet count and clinical symptoms for the four full-term newborns were clinically monitored. Circulating maternal antibodies were characterized with the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay and the flow cytometric (FCM) procedure. CD36 expression on platelets and monocytes of the parents of the four newborns were analyzed by FCM. For the confirmation of CD36 deficiency, the CD36 gene of the four patients was sequenced as genomic analysis, and their mutations of the CD36 genes were further verified by DNA-based polymerase chain reaction (PCR) sequence-specific primers (SSP) assay. Results/Findings: The clinical thrombocytopenia of the four newborns was clear. The mothers of the four infants were identified as Type I CD36 deficiency, while the fathers had normal CD36 expression on their platelets and monocytes. The MAIPA assay confirmed the presence of maternal anti CD36 isoantibodies, which were against the platelets of the newborns’ fathers. CD36 mutations identified in the four mothers were 380C > T (2 cases), 1163A > T (1 case), and 329-330 del AC (1 case), respectively. Conclusion: four cases of FNAIT were [proven to be caused approved that caused by anti-CD36 antibodies. Because Chinese populations have a higher proportion of CD36 deficiency, isoimmunization against CD36 should be considered in patients with apparent alloimmune platelet disorders not explained by immunization against HPA and HLA. Such cases should receive targeted diagnosis and treatment. Disclosure of Commercial Conflict of Interest L. Li: No Answer; J. Liu: No Answer; W. Shen: Nothing to disclose; G. Wu: Nothing to disclose; Z. Zhong: Nothing to disclose; Y. Zhou: Nothing to disclose Disclosure of Grants Conflict of Interest L. Li: No Answer; J. Liu: No Answer; W. Shen: Nothing to disclose; G. Wu: Nothing to disclose; Z. Zhong: Nothing to disclose; Y. Zhou: Nothing to disclose

S12-010B Candida albicans Triggers Inflammatory Responses of Human Platelets through Dectin-1 Q Chen1, M Li2. 1Jiangsu Province Blood Center, Nanjing, China; 2 Department of Medical Mycology, Institute of Dermatology, Chinese Academy of Medical Science & Peking Union Medical College, Nanjing, China Background/Case Studies: It is becoming increasingly clear that platelets play important roles in both innate and adaptive immune responses. It has been demonstrated that Dectin-1 was the principal C-type lectin patternrecognition receptor to recognize β-glucan from the cell wall of C. albicans and to induce immune responses. The role of platelets in the innate immune against the infection of C. albicans and related molecular mechanisms remains unclear. In this study, we sought to clarify whether C. albicans could induce inflammatory responses in human platelets via Dectin-1 and to determine the underlying mechanisms. Study Design/Methods: Human platelets were challenged with heat-killed C. albicans in vitro. The mRNA expression of Dectin-1 and proinflammatory cytokine (TNF-α, IL-6) was assayed by real-time reverse-transcription polymerase chain reaction (RT-PCR). The secretion of TNF-α and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to determine the protein level of Dectin-1. Western blotting was used to analyze NFκB p65 and phosphorylation of p38MAPK. Results/Findings: Exposure of human platelets to heat-killed C. albicans led to increased gene expression and secretion of proinflammatory cytokines (IL-6, TNF-α). C. albicans up-regulated the mRNA and protein levels of Dectin-1. Human platelets challenged with C. albicans resulted in the activation of NF-κB and p38MAPK. Dectin-1-neutralizing antibody and dexamethasone (NFκB, p38MAPK inhibitors) blocked the C. albicans -induced gene expression of IL-6 and TNF-α. Conclusion: These data suggest that the inflammatory responses against invasive C. albicans infection may depend on the Dectin1-NF-κB and p38MAPK signaling pathway. Disclosure of Commercial Conflict of Interest Q. Chen: Nothing to disclose; M. Li: Nothing to disclose Disclosure of Grants Conflict of Interest Q. Chen: Nothing to disclose; M. Li: Nothing to disclose

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ABSTRACT SUPPLEMENT

Cellular Therapies

S13-010C Development and Validation of a Real-time Polymerase Chain Reaction (RT-PCR) to Detect Human Cytomegalovirus (CMV) viremia in Cord Blood (CB)- Derived Cell Therapy Products. M S Albano1, R Ciubotariu1, L Dobrila1, M Tarnawski1, C Watanabe1, M M-Madry1, M Deleon1, A Scaradavou1, P-Rubinstein1. 1National Cord Blood Program, New York Blood Center, New York, NY, United States Background/Case Studies: CMV infection of immunosuppressed patients remains one of the leading causes of morbidity and mortality. In addition to donor screening for CMV to prevent transmission through HPC transplantation, the upcoming field of cell therapy requires screening of the respective sample sources with validated assays. In the CB setting, donor evaluation requires screening of the maternal peripheral blood (PB) for infectious disease markers. However, positivity for CMV total antibody (CMV-Ab) does not necessarily reflect viremia in the newborn or CB. Currently, there are no FDA-approved assays for testing CMV viremia in CB. The goals of this study were: 1) to design an RT-PCR assay to detect CMV-DNA in CB and to validate it against the gold standard assay, newborn saliva culture; and 2) to determine the incidence of CMV viremia among CB units banked at the National Cord Blood Program (NCBP), the viral load of the infected CB units, and their relationship with the serological status and PB viral load of the mother at the time of delivery. Study Design/Methods: The assay, designed as a multiplex RT-PCR, targets the CMV glycoprotein B and a housekeeping gene (internal control). Using a validated CMV standard, the linear reportable range, sensitivity, and precision were evaluated. Results/Findings: The limit of detection was 0.77 vc/uL [95% CI 0.62-1.18; n = 144; Probit analysis]. Inter- and intra-assay precision had a range of 1.28% to 12% (overall CV% 3.81), as evaluated by two operators over three days of testing. The assay had no cross-reactivity against eight viral species of similar genetic structure and was reactive with different CMV strains. Clinical performance was evaluated retrospectively in 90 CB samples (30 with CMVpositive saliva culture, 30 from newborns of mothers with CMV-Ab, and 30 from controls). Sensitivity was 96.6 % (95% CI 92-100%) and specificity was 100%. Since 1993, the NCBP has stored 67,011 CB units. CMV-Ab was detected in the maternal PB of 35,591 donors (53.1%), among whom a subset of 3,689 cases (10.4%) had neonatal DNA retrospectively tested for CMV-DNA with RT-PCR. Of those, 47 were positive (1.27%), with an average viral load of 208.44 vc/uL (range 0.02-2290). Only 15% of the corresponding mothers had CMV-DNA in their PB at the time of delivery. The viral load did not correlate with that of the newborn. None of these infants showed clinical signs of CMV congenital infection at birth. Conclusion: The presence of maternal CMV-Ab was a poor indicator of infection in the CB unit. It is important that most cases of CB CMV viremia would have been missed by the maternal blood screening for CMV-DNA at the time of delivery. RT-PCR is a useful, precise, and rapid screening tool to prevent transmission of CMV through CB transplantation or CB-derived cell therapy. Disclosure of Commercial Conflict of Interest M. S. Albano: Nothing to disclose; R. Ciubotariu: Nothing to disclose; M. Deleon: Nothing to disclose; L. Dobrila: Nothing to disclose; M. M-Madry: No Answer; P. -. Rubinstein: Nothing to disclose; A. Scaradavou: Nothing to disclose; M. Tarnawski: No Answer; C. Watanabe: Nothing to disclose Disclosure of Grants Conflict of Interest M. S. Albano: Nothing to disclose; R. Ciubotariu: Nothing to disclose; M. Deleon: Nothing to disclose; L. Dobrila: Nothing to disclose; M. M-Madry: No Answer; P. -. Rubinstein: Nothing to disclose; A. Scaradavou: Nothing to disclose; M. Tarnawski: No Answer; C. Watanabe: Nothing to disclose S14-010C The Tolerogenic Splenic Dendritic Cells from the Mice Treated with Induced regulatory T Cells are Responsible for Suppressing Collagen-Induced Arthritis J Yang1,2, H Zou2, H Fan1. 1Shanghai Blood Center, Shanghai, China; 2 Division of Rheumatology, Huashan Hospital, Shanghai, China Background/Case Studies: As is well known, Foxp3+ regulatory T cells play a crucial role in maintaining immune tolerance. It was reported that TGF-β-induced regulatory T cells (iTregs) could retain Foxp3 expression and immune suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppressed immune response, especially the interplay between iTregs and dendritic cells (DCs) in vivo,

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remained incompletely understood. In this study, whether splenic DCs were involved in iTreg-based suppression and how these DCs further inhibited CIA were determined. Study Design/Methods: In vitro, iTregs were induced by TGF-β and adoptively transferred into established CIA mice. After 7 days, splenic CD11c+DCs, termed “DCiTreg,” were isolated. The phenotype, the expression of cytokines and function-associated molecules, the immunogenicity, and the suppression of CD4/CD8 Tcell differentiation of DCiTreg were assessed. To determine the suppression in vivo, 5×105 of DCiTreg were re-infused into the new CIA mice. Clinical and histopathologic scores and cytokine and anti-CII antibody secretion in serum were analyzed. Additionally, the role of IDO in the inhibitory effect of DCiTreg was determined by 1-MT blocking in vitro and in CIA mice. Results/Findings: After iTregs were adoptively transferred, isolated DCiTreg exhibited a series of tolerogenic characteristics. Compared with splenic DCs isolated from CIA mice (DCCIA), DCiTreg expressed obviously lower levels of MHC molecule (IA-IE) and co-stimulatory molecules (CD80, CD86 and CD40). And IL-12p40 and IL-6 production by DCiTreg was negligible, while high levels of IL-10 and TGF-β were expressed; an especially enhanced level of IDO by DCiTreg was detected. In the proliferation assay, DCiTreg showed a poor ability to expand effector T cells and had effective inhibitory potency. Meanwhile, after DCiTreg was re-infused, a remarkable anti-arthritic activity was found, along with improved clinical scores and histological endpoints. Also, serological levels of TNF-α, IL-6, IL-17, and anti-CII antibodies were significantly low and TGF-β production was high in the DCiTreg-treated group. Conversely, DCCIA could not suppress CIA completely. Moreover, DCiTreg lost the inhibitory ability on CIA when 1-MT pretreatment was used. Conclusion: These findings suggested that iTregs could inhibit CIA via tolerogenic splenic DC formation. These tolerogenic splenic DCs could, further, effectively dampen the severity and progression of CIA in an IDO-dependent manner, which was associated with modulation of inflammatory cytokine and anti-CII antibody secretion. Thus, the potential therapeutic effects of iTreg in CIA and rheumatic arthritis are likely to be maintained, even enlarged by the effects on DCs in vivo. Disclosure of Commercial Conflict of Interest H. Fan: No Answer; J. Yang: Nothing to disclose; H. Zou: No Answer Disclosure of Grants Conflict of Interest H. Fan: No Answer; J. Yang: Nothing to disclose; H. Zou: No Answer S15-010C Therapeutic Effect of Tacrolimus-Modified Tolerogenic Dendritic Cells in a Mouse Model of Autoimmune Arthritis Y Ren1, H Fan1. 1Shanghai Blood Center, Shanghai, China Background/Case Studies: Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). tDCs can be generated in vitro by a variety of methods, including genetic or pharmacological modification. It is reported that tDCs modified by tacrolimus had tolerogenic characteristics in vitro. The aims of this study were to determine the effects of tacrolimus-modulated tDCs in mice with collagen-induced arthritis (CIA) and to test the responses of cells. Study Design/Methods: tDCs were generated by treating monocyte-derived DCs with tacrolimus. CIA was modeled in D1 mice by immunization with type II collagen (CII), who received one or three intravenous injections of tDCs (1×104 to 1×106) when onset of CIA was confirmed. Arthritis severity was monitored for up to fourweeks after treatment. Histological differences in CIA mice treated with or without tDCs were assessed by staining with hematoxylin and safranin O-fast green on day 56. Then CII antibody in CIA mice was quantified by ELISA. Finally, the in vivo effect of tDCs on splenic CD4 T cell populations was determined by intracellular cytokine staining and flow cytometry. Results/Findings: Mice vaccinated with 1×106 tDCs demonstrated the lowest arthritis scores, as compared with mice vaccinated with other doses or the untreated control CIA mice. Additionally, joints of untreated mice were severely damaged, with a large number of infiltrated cells in the synovium, together with disruption of the cartilage surface. In contrast, CIA mice treated with 106 tDCs showed a preserved architecture in the joints with few infiltrated cells and no apparent damage,. whereas mice vaccinated with lower doses of tDCs showed a moderate decrease in destruction of the cartilage. Moreover, serum levels of CII-specific immunoglobulin in mice treated with all doses of tDC were markedly lower at 56 days as compared with untreated CIA mice. And the levels of anti-CII antibodies were also significantly lower in CIA mice treated with 1×106 tDCs than in CIA mice treated with 1×105 and 1×104 tDCs. The improvement in CIA disease activity was associated with polarization of the Th1/Th17 balance. The frequency of CD4+IL-17+T cells (Th17 cells) decreased in the CIA mice after the 105 and 106 tDCs treatment.

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The percentage of CD4+IFN-γ+T cells (Th1 cells) in CIA mice treated with 105 and 106 tDCs was significantly increased as compared to those in CIA control mice, whereas tDC treatment had no detectable effect on Th2 cells or Treg cells. Conclusion: Tacrolimus-modified tDCs reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulation of inhibition of anti-CII secretion and CD4 T subset polarization.

Answer; H. Okazaki: Kyowa Hakko Kirin Pharma,Grants or Research SupportGrifols,Consulting or Board of Director Fees; T. Sato: Nothing to disclose; H. N. Tsuno: No Answer; N. Watanabe-Okochi: Nothing to disclose; Y. Yatomi: No Answer; N. Yoshikawa: No Answer; T. Yoshizato: Nothing to disclose

Disclosure of Commercial Conflict of Interest

S17-010C Validation of Microbial Testing in Cord Blood at Canadian Blood Services S Ramirez-Arcos1, Y Kou1, H M Perkins1, L Yang1, M Taha1, M A den Admirant1, M Halpenny1, H Elmoazzen1. 1Canadian Blood Services, Ottawa, ON, Canada

H. Fan: No Answer; Y. Ren: Nothing to disclose Disclosure of Grants Conflict of Interest H. Fan: No Answer; Y. Ren: Nothing to disclose S16-010C The High CD34+ Cell Count in Premobilization Peripheral Blood Predicts an Earlier Start of Leukapheresis and a Sufficient CD34+ Cell Yield in Autologous Hematopoietic Stem Cell Transplantation Patients T Sato1,2, N Watanabe-Okochi1,2, Y Masamoto1,2, T Yoshizato1, T Ikeda1, M Matsuhashi1, J Iino1, Y Mishima1, N Yoshikawa3, A Masuda3, Y Yatomi3, H N Tsuno1, H Okazaki1. 1Department of Transfusion Medicine, The University of Tokyo Hospital, Tokyo, Japan; 2Department of Hematology & Oncology, The University of Tokyo Hospital, Tokyo, Japan; 3Department of Clinical Laboratory, The University of Tokyo Hospital, Tokyo, Japan Background/Case Studies: The dose of reinfused stem cells holds the key for successful autologous hematopoietic stem cell transplantation (autoHSCT). To achieve a greater peripheral blood stem cell (PBSC) yield, chemotherapy is combined with G-CSF at the expense of higher morbidity compared to G-CSF alone. However, in auto-PBSC donors, a sizable population fails to mobilize sufficient PBSCs, due to prior therapy. Study Design/ Methods: To assess predictors of adequate PBSC (CD34+ cell) yields, we performed a retrospective analysis of clinical leukapheresis records. Thirtyone cases with hematological diseases who underwent chemomobilization in our institution during 2012-13 were retrieved. The chemotherapy regimen depended on the underlying diseases, and filgrastim was administered at 10 μg/kg/d. The start of leukapheresis was decided by a comprehensive evaluation of lab parameters, including leukocyte and differential, reticulocyte, and lactate dehydrogenase (LDH) every morning, from day 4 post G-CSF. Leukapheresis was conducted using the AS204 device (Fresenius), by processing 2× patient’s total blood volume, in a total of 50 sessions. The correlation of the premobilization (Mob; day 0), pre- and post-apheresis (Aph) PB, and harvest CD34+ counts with the patient’s basic data was assessed. Results/Findings: By the univariate analysis, pre-Aph PB CD34+, LDH on the apheresis day, and male gender were found to correlate with CD34+ yield (P < 0.001, P < 0.001, P = 0.015, resp.). By the multivariate analysis, pre-Aph CD34+ was found to be the variable strongly associated with CD34+ yield (P < 0.001). The threshold of 20 cells/μL for pre-Aph PB CD34+ was determined as an important value in predicting a minimum yield of 1 × 106 CD34+ cells/kg/Aph. Only 3.8% of subjects with pre-Aph PB CD34+ >20 did not achieve the minimum yield, whereas 62.5% of those with pre-Aph counts below the threshold had inadequate yields (P < 0.01). PreMob PB CD34+ correlated with CD34+ cell yield (P = 0.018). A threshold of 0.5 cells/μL for pre-Mob PB CD34+ could predict a minimum yield of 1 × 106 CD34+ cells/kg/Aph (P = 0.049). Of cases with pre-Mob PB CD34+ >0.5, 70% had the leukapheresis started on day 4, while 90% of those with pre-Mob counts below the threshold had the session started on day 5 or later (P < 0.01). Conclusion: Besides pre-Aph PB CD34+, the most reliable predictor of PBSC yield, pre-Mob PB CD34+ could support the earlier start of apheresis and predict a sufficient CD34+ yield in auto-HSCT patients. A pre-Mob PB CD34+ = 0.5 may be a parameter supportive of bone marrow recovery after chemotherapy enough to release more than 100 times PBSCs. Our results provide the basis for the effective prediction of adequate PBSC yields, allowing the more effective and economical tailored leukapheresis. Disclosure of Commercial Conflict of Interest J. Iino: No Answer; T. Ikeda: No Answer; Y. Masamoto: Nothing to disclose; A. Masuda: Nothing to disclose; M. Matsuhashi: No Answer; Y. Mishima: No Answer; H. Okazaki: Nothing to disclose; T. Sato: Nothing to disclose; H. N. Tsuno: No Answer; N. Watanabe-Okochi: Nothing to disclose; Y. Yatomi: No Answer; N. Yoshikawa: No Answer; T. Yoshizato: Nothing to disclose Disclosure of Grants Conflict of Interest J. Iino: No Answer; T. Ikeda: No Answer; Y. Masamoto: Nothing to disclose; A. Masuda: Nothing to disclose; M. Matsuhashi: No Answer; Y. Mishima: No

Background/Case Studies: International standards mandate sterility testing for cord blood (CB) products to prevent the transmission of microbial infections to immunocompromised transplant patients. The automated BacT/ ALERT culture system was validated to detect microbial contamination in CB units processed at the Canadian Blood Services National Public Cord Blood Bank. Study Design/Methods: A validation was developed in three phases. In Phase 1, CB units prepared with pentastarch were tested for sterility by inoculation of 8-10 ml of CB into aerobic and anaerobic BacT/ALERT culture bottles. The units were then spiked with ∼100 CFU/ml of the bacteria Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, and Bacteroides fragilis and the yeast Candida albicans. CB units were processed and the by-product plasma fraction was stored at room temperature for 24 h. Following storage, 8-10 ml of plasma was inoculated into BacT/ALERT culture bottles. In Phase 2, CB units prepared with pentastarch were processed and a mix of by-products (4 ml RBCs and 4 ml plasma) was inoculated into BacT/ALERT culture bottles. In Phase 3), the same experimental approach as in Phase 2 was followed, but CB units were prepared with hetastarch. In Phase 3, the mold Aspergillus niger was added as a test organism, and microbial concentrations in whole blood and the by-products were determined by serial dilutions and plating on blood agar. Results/Findings: During Phase 1, lack of bacterial growth was observed in units obtained from CB donors who were under antibiotic treatment. Thus, antibiotic-free CB units were used for the remaining validation test cases. Although all bacteria were detected in plasma, C. albicans was not always captured in this fraction. In Phases 2 and 3, all organisms were detected in the mix of by-products plasma and RBCs. Interestingly, data from Phase 3 showed higher concentrations of the fungi C. albicans and A. niger in the RBC fraction in comparison to the plasma by-product. Conclusion: The validation of microbial detection in CB units was successfully completed at Canadian Blood Services. The mix of by-products plasma and RBCs is an appropriate sample for sterility testing of CB. The BacT/ALERT system is able to detect microbial contamination in CB units prepared with both pentastarch and hetastarch. Occasional lack of growth of C. albicans in the plasma fraction during Phase 1 was likely due to its preferential segregation to cellular fractions. The clinical significance of the bactericidal or bacteriostatic effect of antibiotics present in CB merits further investigation. Disclosure of Commercial Conflict of Interest M. A. den Admirant: Nothing to disclose; H. Elmoazzen: Nothing to disclose; M. Halpenny: No Answer; Y. Kou: Nothing to disclose; H. M. Perkins: No Answer; S. Ramirez-Arcos: LightIntegra Technology Inc., Ownership or Partnership; M. Taha: Nothing to disclose; L. Yang: No Answer Disclosure of Grants Conflict of Interest M. A. den Admirant: Nothing to disclose; H. Elmoazzen: Nothing to disclose; M. Halpenny: No Answer; Y. Kou: Nothing to disclose; H. M. Perkins: No Answer; S. Ramirez-Arcos: LightIntegra Technology Inc., Grants or Research Support; M. Taha: Nothing to disclose; L. Yang: No Answer

S18-010C Culture of Rapamycin (Sirolimus)-Generated Allogeneic Th2 Cells with Alternative Protein Sources P Accooe1, V S Fellowes2, K M Byrne1, M Sabatino1, D Fowler2, D F Stroncek1. 1Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD, United States; 2National Cancer Institute, National Institutes of Health, Bethesda, MD, United States Background/Case Studies: Our cell processing facility performs numerous T cell cultures (TCCs) using either autologous or group AB heat-inactivated, filtered (AB HIF) plasma as protein sources. Recently, a commercial serum replacement additive (SRA) and an FDA-approved solvent detergent-treated (SDT) pooled plasma have become available. The purpose of this study was

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to culture Th2 rapamycin cells (T-rapa) using AB HIF, SRA, and SDT to determine culture success by expansion and phenotypic markers. Study Design/Methods: Three normal donor CD4+ T cells were polarized toward a predominately type 2 or anti-inflammatory cytokine profile. These cells were then cultured for 6 days in media with X-Vivo 20 (Lonza), 5% AB HIF, 1 uM sirolimus (Rapamune ® Oral Solution), 1000 IU/mL IL-4 (Schering Plough) and 20 IU/mL IL-2 (Chiron). Additional IL-2 and IL-4 were added on days 2 and 4 at the same concentrations. Similar protocol was repeated, with SRA or SDT used in place of AB HIF. Samples from all 3 sets were collected. Cell counts, viabilities, and phenotypes were determined. Based on initial findings, additional cultures were repeated using a new donor at lower concentrations of SRA. Results/Findings: On day 6, cells cultured in SDT were comparable to AB HIF in cell number and phenotype, but all cells in the SRA group died. All values from SDT cultures were within 5% of values for AB HIF cultures, with the exception of the Total Nucleated Cells (TNCs) for donor 16 (Table). There were large cell pellets in SRA at harvest, which were gone after final wash. Repeat cultures using various concentrations (0.5-5%) of SRA yielded differing results. Higher concentrations (3-5%) showed low cell numbers and poor viabilities, while 0.5% and 1% SRA resulted in acceptable viabilities (>85%); however, cell numbers were lower (approximately half) than those of control cells in 5% AB HIF. While

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viabilities and counts of cells cultured in lower SRA concentrations are still not optimal, the phenotypes were similar to those of the AB HIF. Conclusion: SDT is comparable to AB HIF for the culture and expansion of T-rapa cells. SRA as a protein source needs further investigation. Based on these findings, additional cultures are needed using lower concentrations (0.25% and 0.125%) of SRA. Once concentration is optimized, further functional studies of the three protein sources can be made based on cellular cytokine production (Luminex) and gene expression analysis. Disclosure of Commercial Conflict of Interest P. Accooe: Gibco/Life technologies, Other, Octaplas,Other; K. M. Byrne: Nothing to disclose; V. S. Fellowes: Gibco/Life Technologies, Other Octopharma, Other; D. Fowler: Nothing to disclose; M. Sabatino: No Answer; D. F. Stroncek: Life Technologies, Other Octapharma, Other Disclosure of Grants Conflict of Interest P. Accooe: Nothing to disclose; K. M. Byrne: Nothing to disclose; V. S. Fellowes: Nothing to disclose; D. Fowler: Nothing to disclose; M. Sabatino: No Answer; D. F. Stroncek: Nothing to disclose

TABLE. Results of T-rapa cultures CULTURE 1 Donor 14 14 15 15 16 16

Groups 5% AB HIF SDT 5% AB HIF SDT 5% AB HIF SDT

Day 0

Day 6

Day 6

Day 6

Day 6

Day 6

CULTURE 2

TNC x 106 TNC x 106 Viability CD3% CD3+4+% CD3+8+% Donor 45 45 38 38 45 45

122 123 72 72 176 137

89 93 82 84 91 95

98.2 98.3 96.7 96.3 99.1 98.9

99.5 99.3 95.5 95.6 99.7 99.5

0 0 0 0 0 0

Donor and Therapeutic Apheresis

17 17 17

Groups 5% ABHIF 1% SRA 0.5%SRA

Day 0

Day 6

Day 6

Day 6

Day 6

Day 6

TNC x 106 TNC x 106 Viability CD3% CD3+4+% CD3+8+% 50 50 50

122 56 76

95 89 86

93 88.4 85.9

92.4 87.8 85.5

0 0 0

Disclosure of Commercial Conflict of Interest G. J. Despotis: No Answer; J. J. O’Brien: Nothing to disclose; P. S. Shah: No Answer

S19-020A Characterization Of Respiratory Function In Guillain-Barré Syndrome Patients after Plasma Exchange J J O’Brien1, P S Shah1, G J Despotis1. 1Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, United States Background/Case Studies: Guillain-Barré Syndrome (GBS) is an acute autoimmune peripheral nervous system polyneuropathy, frequently triggered by an infection. GBS is characterized by ascending paralysis and weakness, beginning in the feet and hands and migrating toward the trunk. Dysfuction of the autonomic nervous system can occur and cause life-threatening complications of respiratory muscle weakness, requiring ventilatory assistance in 30% of patients. GBS can resolve with prompt treatment by intravenous immunoglobulins or plasmapheresis. We decided to evaluate the change in respiratory function in response to sequential plasma exchange (PEX) procedures in GBS patients. Study Design/Methods: Charts and computer databases were used to obtain clinical and laboratory data from 18 patients who received multiple plasma exchange procedures from January 2010 to December 2013. Baseline vital capacity and negative inspiratory force were compared against sequential post-plasma-exchange respiratory function. Data were analyzed using Excel software. Values were compared to criteria of VC T, c.455A>C, c.509T>C, and c.667T>G, indicating a new RHD*DIIIa-DOL hybrid. P2’s RBCs were group O D+ (see Table) C−E−c+e+ and reacted very weakly in the DAT. The patient’s EGA-treated RBCs gave reactions consistent with weak D type 1 with the partial RhD kit. Her plasma and an eluate from her RBCs contained anti-D. As the patient had been two months prior, it is not known whether the anti-D is allo- or autoantibody. DNA testing identified two alleles; RHD*weak D type 1 and RHD*509T>C (p.Met170Thr). The c.509C is one of two changes found in RHD*DOL, but it has not been reported alone. P3’s group O RBCs (see Table for D typing) were DAT−, C−E−c+e+ and DAK−, DW−, Go(a−), Rh32−, and BARC−. They reacted with only 1 of the 12 anti-D in the partial RhD kit; consistent with a DBT phenotype. DNA analysis revealed a new hybrid RHD*DV type 1-CE (6-9)-D with 667G and 697C that encode 223Val, 233Gln in exon 5 with exons 6-9 replaced with RHCE. Conclusion: We report three novel RHD. P1 has a hybrid (RHD*DIIIa-DOL) that encodes a DAK+ partial D phenotype associated with production of alloanti-D. P2 has a previously undescribed RHD*509T>C in trans to RHD*weak D type 1. P3 has a new hybrid allele, RHD*DV type 1-CE(6-9)-D, which encodes a D antigen that resembles the partial D, DBT. Disclosure of Commercial Conflict of Interest K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; G. Shakarian: No Answer; A. Smith: Nothing to disclose; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose Disclosure of Grants Conflict of Interest K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; G. Shakarian: No Answer; A. Smith: Nothing to disclose; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose

S34-020C A Weak D Phenotype Discovered in Nine Patients in Western Canada due to a Nucleotide 1187C>G (Pro396Arg) Change in RHD C M Westhoff4, J Hannon1, G Clarke1, J M Cote2, M Goldman2, D Lane5, S Vege4, C Lomas-Francis4, G Ochoa3. 1Canadian Blood Services, Edmonton, AB, Canada; 2Canadian Blood Services, Ottawa, ON, Canada; 3 Progenika, Medford, MA, United States; 4Immunohematology and Genomics, Long Island City, NY, United States; 5Canadian Blood Services, Winnepeg, MB, Canada Background/Case Studies: Alleles reported to encode a weak D phenotype now total more than 76 different RHD variants (http:// rhesusbase.atspace.com/). Some are more prevalent in specific ethnic or population groups. We investigate RHD in seven prenatal and two crossmatch samples with indeterminate RhD typing by automated testing. Three of the prenatal women had been previously reported as Rh negative, and two were unresolved on previous genotyping. Study Design/Methods: RBC typing was performed by automated testing (Galileo or NEO, Immucor Gamma) with Series 4 and 5 and by manual tube testing with Series 4 and Series 5, DBL Novaclone, Bio-Rad Seraclone, Ortho BioClone, and Immucor Gamma-clone. RBCs of three women were tested with the Albaclone advanced partial RhD typing kit. RHD zygosity was determined by hybrid box detection. Bloodchip (Progenika), RHD BeadChip (BioArray), and RHD

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exon-specific gene sequencing was done. Results/Findings: The Rh status was indeterminate for all samples by automated typing. RBCs were tested with anti-D from multiple manufacturers, and all reacted variably (mi/1+-2+) in direct testing and moderately (2+) in the IAT with anti-D reagents. The RBCs also reacted variably (1+-3+) with all the monoclonal anti-D in the Albaclone advanced partial RhD typing kit. DNA testing revealed that all samples were hemizygous for RHD. The RHD BeadChip was negative for variants detected by that assay, but Bloodchip testing suggested the presence of a variant in exon 9. RHD exon sequencing by two referral laboratories found a nucleotide change 1187C>G in exon that was predicted to encode amino acid change Pro396Arg. No other changes in RHD were found. Conclusion: The RHD nucleotide 1187C>G change encoding Pro396Arg has not been previously reported and appears to encode a weak (rather than partial D) phenotype based on reactivity with all monoclonal anti-D tested. All samples were R1r (DCe/ce), suggesting that this weak D allele is linked to RHCE*Ce. Five of the samples were among samples from 106,616 prenatal patients tested from northern Alberta, Canada, for an incidence of ∼1/ 20,000. These represent 7.6% (5/66) of D typing discrepancies sent for genotyping from these prenatal patients. One patient identified in the crossmatch laboratory in Winnipeg was of French-Canadian Metis background. The Pro396Arg is located in the 3′ intracellular COOH terminus of the RhD protein at the cytoplasmic interface. The large, highly positive, charged side chain of Arg might disrupt interaction of RhD protein with the underlying cytoskeleton, causing the reduced D expression on the RBCs. Although they are unlikely to be at risk for anti-D, in the absence of additional data, all these women were conservatively treated as Rh-negative for transfusion and candidates for RhIG. Disclosure of Commercial Conflict of Interest G. Clarke: Nothing to disclose; J. M. Cote: Nothing to disclose; M. Goldman: Nothing to disclose; J. Hannon: Nothing to disclose; D. Lane: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; G. Ochoa: No Answer; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose Disclosure of Grants Conflict of Interest G. Clarke: Nothing to disclose; J. M. Cote: Nothing to disclose; M. Goldman: Nothing to disclose; J. Hannon: Nothing to disclose; D. Lane: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; G. Ochoa: No Answer; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose S35-020C Automated RH Genetic Matching Applied to African American Donors and Patients with Sickle Cell Disease D F Friedman2, M A Keller1, S Vege3, T E Jackson2, S T Chou2, C M Westhoff3. 1National Molecular Laboratory, American Red Cross, Philadelphia, PA, United States; 2Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; 3Genomics Laboratory, New York Blood Center, New York City, NY, United States Background/Case Studies: RH gene variation is frequent in persons of African descent and contributes to Rh alloimmunization among patients (pts) with sickle cell disease (SCD). RH genotyping of pts with SCD and selected African American (AA) donors may help to improve transfusion therapy. An automated process to collect and match pt and donor RH genotypes for RBC matching is preferable to manual searches. We developed virtual matching algorithms with datasets from AA donors and SCD pts to select blood by RH genotype, in order to project whether such a matching strategy is feasible. Study Design/Methods: RHD and RHCE genes were characterized by RH BeadChip (BioArray Solutions, LLC), manual allele-specific polymerase chain reaction, and exon sequencing in 440 pts with SCD and 625 AA donors not selected on the basis of Rh. After harmonizing nomenclature and gene assignment algorithms between data sources, automated virtual donor matching for each pt was performed, considering RHD and RHCE separately and together. Customized scripts in a Filemaker Pro database counted exact genetic matches and used an “inclusive” criteria to select only donors with RH genes that would not encode Rh antigens absent in the pt. Matching ignored ABO and non-Rh RBC antibodies and included D- donors. A set of computer-generated matches was validated manually from the same donor lists. Results/Findings: Both donor and pt groups were 5.9% D-, and distributions of RHD and RHCE were similar. Of 24 RHD genes detected in both groups, 5 (RHD*01, *DAU0, *deleted D, *weak D 4.0, and *inactive RHDψ) accounted for 91% of donor alleles, and 6 (same 5 and RHD*DIIIaCE(4-7)-D) accounted for 92% of pt alleles. Of 21 RHCE genes, the 7 most common accounted for 92% of donor alleles and 90% of pt alleles. Match results are summarized in the Table. Of note, 74% of pts had a conventional RHD*01 allele and >350 donors by “inclusive” match. Compared to donor selection by serologic D type, genetic matching in these pts could avoid a

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20%-30% chance of exposure to a foreign RhD protein. The 26% of pts who lack conventional RHD*01 could avoid a 90% chance of exposure to a foreign RhD protein, but they would have fewer donors. Gene-based RHCE matching could reduce the chance of exposure to a foreign RhCE protein by 70%-90%, but that would limit donor numbers vs. standard phenotype matching for C and E. Conclusion: Automated selection of AA donors based on RHD, RHCE, or both would be feasible for most pts with SCD, given an adequate genotyped donor pool. Reduced exposure to foreign Rh proteins may decrease alloimmunization.

number of altered RHCE alleles identified in Caucasians increases, it is important to be aware that further molecular and serologic investigation may be needed.

Disclosure of Commercial Conflict of Interest

Disclosure of Grants Conflict of Interest

S. T. Chou: Nothing to disclose; D. F. Friedman: Nothing to disclose; T. E. Jackson: No Answer; M. A. Keller: Nothing to disclose; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose

J. Adamy: Nothing to disclose; J. Aronowitz: Nothing to disclose; Z. Hu: No Answer; K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; J. K. Slayten: No Answer; S. Vege: Nothing to disclose; D. A. Waxman: Nothing to disclose; C. M. Westhoff: Nothing to disclose

Disclosure of Grants Conflict of Interest S. T. Chou: Nothing to disclose; D. F. Friedman: Nothing to disclose; T. E. Jackson: No Answer; M. A. Keller: BIoArray Solutions LLC,Grants or Research Support; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose TABLE. Percentage of 440 pts with ≥25 or no matches by locus and criteria stringency Match by: Criteria Exact Inclusive

RHD ≥25 67% 100%

RHCE None 5.4% 0

≥25 42% 76%

None 4.7% 1%

RHD and RHCE ≥25 11% 59%

None 22% 1.6%

S36-020C Anti-hrB-like Reactivity Identified in a Caucasian Patient: Evidence That RHCE*Ce(890C) Encodes a Partial Phenotype S Vege1, J Adamy1, Z Hu1, J K Slayten2, J Aronowitz2, K Hue-Roye1, C Lomas-Francis1, D A Waxman2, C M Westhoff1. 1Immunohematology and Genomics Laboratory, New York Blood Center, Long Island City, NY, United States; 2Indiana Blood Center, Indianapolis, IN, United States Background/Case Studies: The number of new RHCE alleles identified continues to rise. Some, mainly found in people with black African ancestry, encode partial antigens and are associated with production of antibodies to high-prevalence Rh antigens. Many alleles were identified because of weak antigen expression; later, some were realized to be partial antigens, leaving patients at risk to make alloantibody. The presence of an antibody to a high-prevalence antigen, with possible Rh specificity, in a Caucasian woman led to a serological and RH molecular investigation. Case: A 60-year-old Caucasian female, admitted to hospital for endometrial bleeding was transfused with three crossmatch-compatible red blood cell (RBC) units. At the time, no unexpected antibodies were detected. Two months later, reactivity of the woman’s plasma was consistent with anti-C, -E, and an antibody to a high- prevalence antigen. Study Design/Methods: Serologic testing was performed by standard tube hemagglutination methods. The patient’s plasma (native and adsorbed with papain-treated, phenotypically similar rr RBCs) was tested by the IAT. DNA was isolated from WBCs for DNA testing. RNA was isolated from reticulocytes for RhCE-specific cDNA analysis. Results/Findings: Anti-E, reactive by albumin, papain, and PEG IAT was identified in the patient’s plasma. Also by papain and PEG IAT, the plasma reacted 2+ to 3+ with E-C+e+, weakly with C-E-e+, very weakly with hrB- and not at all with D-—RBCs, suggesting anti-C, anti-e, and/or anti-hrB. Anti-C was ruled out by PEG IAT with the patient’s plasma adsorbed with rr RBCs. The patient’s RBCs were negative in the DAT and typed C+E-c+e+; the reactivity with anti-C was marginally weaker than the control (3+ versus 4+). The RHCE and RHD BeadChip and manual PCR-RLFP assays predicted the patient’s genotype as RHD/D and RHCE*Ce/ce. RhCE-specific cDNA sequencing confirmed a conventional RHCE*ce and revealed an RHCE*Ce(809C) allele. The exon 6 c.890T>C (p.Leu297Pro) change was confirmed in DNA sequencing. Conclusion: The RHCE*Ce(809C) allele is a rare allele previously reported in a German donor with weaker-thanexpected C and e expression (Doscher et al, Transfusion 2009), but the clinical significance of the allele could not be determined. We show that the RhCe protein encoded by this allele expresses a partial phenotype, which, in this patient, allowed production of an alloantibody compatible only with D-—RBCs but had anti-hrB-like characteristics. It is more common knowledge that African Americans with unexpected Rh antibodies may have underlying partial alleles. However, in Caucasians, it is considered rare, and many of these antibodies may be presumed to be autoantibodies. As the

Disclosure of Commercial Conflict of Interest J. Adamy: Nothing to disclose; J. Aronowitz: Nothing to disclose; Z. Hu: No Answer; K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; J. K. Slayten: No Answer; S. Vege: Nothing to disclose; D. A. Waxman: Nothing to disclose; C. M. Westhoff: Nothing to disclose

Pediatric Transfusion Medicine

S37-020D Impact of Paternal KEL2 RBC Antigen Density on Maternal Alloimmunization and Fetal Outcomes in a Murine Model of HDFN S R Stowell2, N H Smith2, K R Girard-Pierce2, C M Arthur2, J C Zimring3, J E Hendrickson1. 1Lab Medicine, Yale University, New Haven, CT, United States; 2Pathology and Laboratory Medicine, Emory University, Atlanta, GA, United States; 3Research Institute, Puget Sound Blood Center, Seattle, WA, United States Background/Case Studies: RBC antigen copy number has been shown to be important in determining recipient immune responses in transfusion and in pregnancy [e.g., weak Rh(D) and Rh(D)]. Herein, we have utilized our recently developed transgenic KEL murine models, with RBC-specific expression of the human KEL2 antigen at 3 different copy numbers (low, medium, and high), to further investigate the impact of copy number on induction of maternal alloimmunization and fetal outcomes. Study Design/ Methods: Transgenic animals that express the human KEL2 antigen specifically on RBCs were generated. Three founder animals were characterized, including one with 300 copies (KEL2 low), one with 1000 copies (KEL2 medium), and one with 2000 copies (KEL2 high) of the antigen. C57BL/6 wild-type females, lacking the human KEL2 antigen altogether, were bred three times each with KEL2 low, KEL2 medium, or KEL2 high transgenic males. Maternal alloantibody responses as well as fetal outcomes were characterized. Results/Findings: Eleven of 12 wild-type females bred with KEL2 low males failed to make detectable anti-KEL glycoprotein alloantibodies after 3 pregnancies/deliveries, and all pups were healthy. As previously reported, the majority of wild-type females bred with KEL2 medium males made detectable alloantibodies after three pregnancies, with some KEL2-positive pups dying in utero, some born with severe anemia/hydrops fetalis, and some born healthy. Eleven of 13 wildtype females bred with KEL2 high males made detectable anti-KEL glycoprotein alloantibodies after 3 pregnancies, with anti-KEL titers higher than 1:100 measured in 8/11 of the alloimmunized mothers. The clinical consequences of the antibodies generated by these alloimmunized females bred with heterozygous KEL2 high males were dire: a mean 4.2% (s.d. 8.9) of pups born to alloimmunized mothers in the third litter were KEL2 antigen-positive, compared to 52% (s.d. 18) of pups born to control mothers. Conclusion: These studies demonstrate that KEL2 antigen copy number on paternally derived fetal RBCs impacts maternal alloimmunization and fetal outcomes. Lack of antibody induction after exposure to KEL2 RBCs with low copy number was predicted, given the known poor immunogenicity of weak Rh(D) RBCs in humans. However, the robust alloantibody responses observed in pregnancy to fetal KEL2 high RBCs was surprising, given ongoing experiments showing that KEL2 high RBCs are only weakly immunogenic in a transfusion setting (in the absence of systemic inflammation). The duration of RBC exposure, as well as the state of pregnancy itself, likely contributes to the observed differences. Ongoing studies are investigating these findings and are also exploring the possibility of utilizing RBCs with low antigen-copy number to induce non-responsiveness in a therapeutic manner. Disclosure of Commercial Conflict of Interest C. M. Arthur: No Answer; K. R. Girard-Pierce: No Answer; J. E. Hendrickson: Nothing to disclose; N. H. Smith: Nothing to disclose; S. R. Stowell: Nothing to disclose; J. C. Zimring: Nothing to disclose

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Disclosure of Grants Conflict of Interest C. M. Arthur: No Answer; K. R. Girard-Pierce: No Answer; J. E. Hendrickson: Terumo, Grants or Research Support; N. H. Smith: Nothing to disclose; S. R. Stowell: Nothing to disclose; J. C. Zimring: Immucor, Grants or Research Support S38-020D Microthromboelastography: Enabling Near-patient, Small-volume Diagnostics for Pediatric Transfusion Therapy R C Spero1, J K Fisher1, R Judith2, R M Taylor1,3, M Falvo2, R Superfine1,2. 1Rheomics, Chapel Hill, NC, United States; 2Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States; 3Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Background/Case Studies: Transfusion therapy is both essential and potentially dangerous. Outcomes can be improved and risks minimized by using a visco-hemostatic assay (VHA) to direct the type, amount, and duration of treatment. However, existing VHA devices are ill-suited for point-ofcare settings, because of size, mechanical sensitivity, training requirements, and the use of liquid reagents. Their utility in pediatric applications is limited by the large blood volumes required. Study Design/Methods: We present a VHA method that has no moving parts, uses dry reagents, and requires only 20 μL of sample. Numerous Actuated Surface-Attached Posts (ASAPs) are driven by a magnet, and their motion is detected by using an optical system. An image analysis algorithm drives the ASAP elements in fixed-amplitude deflection and produces results similar to those with existing VHAs, including clotting time, rate, stiffness, and lysis. The precision and accuracy of this measurement was evaluated relative to an existing VHA, the TEG5000. Samples were interference studies (saline dilution, streptokinase, and fibrinogen) on normal blood from healthy volunteers. Results/Findings: Using control specimens, we demonstrate precision similar to that with existing VHAs (N = 15, CV = 8%). Both in interference studies and with patient specimens, we show strong correlation with thromboelastography MA (N = 20, r = 0.90). Sensitivity to clotting time (R), rate (alpha), and lysis are also shown. Finally, we present a fully integrated device that requires no user calibration, has a sustained power draw under 20W, and is roughly 6″ x 4″ x 4″. Conclusion: ASAP-based devices can enable portable, mobile, visco-hemostatic assays that are ideal for near-patient, time-critical, pediatric, and small animal model applications. Disclosure of Commercial Conflict of Interest M. Falvo: No Answer; J. K. Fisher: No Answer; R. Judith: Nothing to disclose; R. C. Spero: Nothing to disclose; R. Superfine: No Answer; R. M. Taylor: No Answer Disclosure of Grants Conflict of Interest M. Falvo: No Answer; J. K. Fisher: No Answer; R. Judith: Nothing to disclose; R. C. Spero: Nothing to disclose; R. Superfine: No Answer; R. M. Taylor: No Answer Clot stiffness measured by ASAP shows strong correlation with TEG5000 MA in a blood dilution interference study. S39-020D Association Between Cumulative Iron Exposure and Bronchopulmonary Dysplasia in Very Low Birth Weight Infants R M Patel1, A Knezevic3, M T Hinkes4, J D Roback2, K A Easley3, C Josephson1,2. 1Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA, United States; 2Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, United States; 3Rollins School of Public Health, Biostatistics and Bioinformatics, Atlanta, GA, United States; 4Northside Hospital, Atlanta, GA, United States Background/Case Studies: Very low birth weight (VLBW) infants are at risk of iron overload from packed red blood cell (RBC) transfusions and enteral iron supplementation. Unbound iron may lead to oxidative stress and contribute to the development of bronchopulmonary dysplasia (BPD). The risk to BPD from greater amounts of cumulative supplemental iron exposure is unknown. The objective of this study was to test the hypothesis that VLBW infants exposed to a greater quantity of iron in the form of RBC transfusions and enteral supplementation will have a higher risk of BPD. Study Design/ Methods: A secondary, multicenter, observational cohort study used prospectively collected data from VLBW infants (A, 681A>A, 771C>T, 803G>C, and 829G>C in the 6th and 7th extrons of CisAB01/O02, while a 297A>G occurred in extron 6 and 467C>T, 803G>C, 526C>G, 657C>T, 703G>A, 796C>A, and 930G>A in extron 7 of CisAB01/B101. Conclusion: An inherited CisAB blood group family with 4 of 10 CisAB members in three generations was varified. Two genotypes of CisAB were identified as CisAB01/O02 and CisAB01/B101. Sequence variations in CisAB alleles contributed to the differential phenotyes. In clinical blood tranfusion practice, for CisAB blood group identified via serological blood grouping, a further genotypic analysis to acquire a detailed CisAB allelic information is de facto indispensable for donor blood sample management and transfusion safety.

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anti-A,B. No anti-A1 was detected in the plasma. Additional testing performed at a second IRL showed significant variability in tube agglutination strength with the use of four different anti-A and anti-A,B reagents, including those listed above, ranging from very weak to 2-3+ agglutination; results were generally stronger with anti-A,B than with anti-A. No agglutination with anti-B reagents was detected. A1 lectin testing with three different reagents was negative, and the patient was identified as A subgroup. By PCR-RFLP testing, the patient had an A1 allele and an O allele. Sequencing of ABO exons 6 and 7 identified an O02 allele (297G, 646A, 681A, 771T, and 829A) and a new A1 allele with a nucleotide 722G>C change encoding Arg241Pro. Conclusion: Licensed testing reagents may not detect some A or B subgroups. The finding of an ABO discrepancy may require further serologic and molecular testing to determine the correct blood type. Here, we identified a new A1 subgroup allele with 722G>C (Arg241Pro). Interestingly, while this change is novel, a change at this same position to a different nucleotide, 722G>A (Arg241Gln), has been reported in a sample from France on an A2 background associated with an Ax phenotype (ABO*AW.23) (Transfusion 2010;50:1471). This case is a reminder of the limitations of current monoclonal testing reagents and of the advantage of using a variety of reagents containing different antibody clones and DNA analysis for investigation of ABO discrepancies. Disclosure of Commercial Conflict of Interest B. Firetag: Nothing to disclose; Y. S. Hsu: Nothing to disclose; C. LomasFrancis: No Answer; E. C. Lomeli: Nothing to disclose; M. Moayeri: Nothing to disclose; A. Nambiar: Nothing to disclose; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose Disclosure of Grants Conflict of Interest B. Firetag: Nothing to disclose; Y. S. Hsu: Nothing to disclose; C. LomasFrancis: No Answer; E. C. Lomeli: Nothing to disclose; M. Moayeri: Nothing to disclose; A. Nambiar: Nothing to disclose; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose S75-030L Utility of Routine RBC Genotyping for RBC Alloantibody Problems G Ramsey1, J Zinni2, R D Sumugod2, P F Lindholm1. 1Pathology, Northwestern University, Chicago, IL, United States; 2Blood Bank, Northwestern Memorial Hospital, Chicago, IL, United States

Disclosure of Commercial Conflict of Interest Q. Cui: Nothing to disclose; Y. Liu: Nothing to disclose; H. Zhao: Nothing to disclose Disclosure of Grants Conflict of Interest Q. Cui: Nothing to disclose; Y. Liu: Nothing to disclose; H. Zhao: Nothing to disclose S74-030L A New Subgroup A Allele with 722G>C (Arg241Pro) Associated with Significant Variation in Detection by A Typing Reagents M Moayeri1, B Firetag1, E C Lomeli1, Y S Hsu1, A Nambiar1, C Lomas-Francis2, S Vege2, C M Westhoff2. 1Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, United States; 2Immunohematology and Genomics Laboratory, New York Blood Center, Long Island City, NY, United States Background/Case Studies: A or B subgroups may result in ABO typing discrepancy. Different ABO typing reagents and methods are commercially available, and each reagent may not detect all subgroups. We report a new A allele in an A subgroup patient with significant variability in ABO typing results, who was initially misidentified as O. Study Design/Methods: Serologic testing was performed by standard tube, automated gel (ORTHO ProVue with MTS gel card), or solid-phase (Immucor Galileo) technology. Typing reagents included Ortho BioClone, Immucor Series and Gammaclone, and Bio-Rad Seraclone. Genomic DNA from WBCs was used for duplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Vox Sang 1995;69:242) and ABO gene sequencing. Results/ Findings: A 31-year-old Hispanic woman was referred to our hospital at 27 wk gestation for management of twin-twin transfusion syndrome. ABO typing using MTS gel card revealed an ABO discrepancy with forward typing as O and back typing as A. Tube testing with Ortho BioClone anti-A showed variable agglutination (1-2+). rther investigation revealed that, 3.5 months earlier, the patient typed as O-positive by another lab using the Immucor Galileo platform (anti-A Series 1; back type 1+ with A1 and 3+ with B cells). Testing performed at a regional Immunohematology Reference Laboratory (IRL) showed 4+ reaction with both Ortho BioClone anti-A and Immucor

Background/Case Studies: Red blood cell (RBC) blood group genotyping usually is done by blood collection facilities for purposes of identifying reference-laboratory antibodies or rare donors. Our hospital transfusion service performs genotyping in all patients with RBC alloantibodies who have not had prior full serological phenotyping. Study Design/Methods: Antibody screens and panels were done by using solid-phase red cell adherence (Capture-R®, Immucor, Norcross, GA), augmented by polyethylene glycol and low-ionic-strength saline testing. RBC genotyping utilized HEA BeadChipTM (Immucor BioArray, Warren, NJ). Genotypings with predicted possible Rh VS+ phenotype were inspected for possible partial e status due to homozygosity or hemizygosity (E+) of RhCE 245Val. In selected cases, more-detailed genotyping was obtained (American Red Cross, Philadelphia, PA). We retrospectively reviewed seven consecutive months of testing to identify patients with uncommon findings in genotyping. Results/Findings: In seven months, we performed 27,235 antibody screens, of which 8.8% were reactive. Genotyping was performed in 346 patients with alloantibody reactivity. In most patients, genotyping correlated as expected with the antibodies identified. However, 18 patients (5.2%) had uncommon or unusual genotype findings of significance for current or potential future antibody identification. In 10 of 18 cases (56%), the genotyping and predicted phenotyping of concern involved the same blood group as the patient’s antibody(ies): anti-Jkb with a novel JK*B silencing gene†; anti-Jka with predicted Jk(a+w) phenotype†; anti-Fyb with homozygous GATA-1 promoter mutations; anti-c and c+; anti-C and C+; anti-C and partial C; partial C plus possible partial e; possible partial e; k-; and Co(a-) (†detailed genotyping). The other eight patients (44%) did not have Rh, Dombrock, or MNS antibodies, but had predicted phenotypes of Jo(a-) and partial C; partial C and e; partial e (2); partial C (3); and U+var. Seven of 18 (39%) had been transfused in the previous 3 months. Eleven patients were African American (AA), 5 Caucasian, 1 Hispanic, and 1 Asian. In four other patients not included above, genotyping uncovered incorrectly identified phenotypes or antibodies. Conclusion: In our transfusion service, 5% of all patients with RBC alloantibodies had clinically pertinent uncommon or unusual findings revealed by genotyping. In half of these cases, the genotypings of interest were in the same blood groups as their antibodies. The other half had findings indicating risk of antibodies to unexpected (C, e) and/or highfrequency (U, Joa) antigens. In several cases, recent RBC transfusions

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would have complicated serological typing. Sixty-one percent of the patients with uncommon or unusual genotypes were AA. Routine genotyping in patients with RBC alloantibodies can reveal important information for current or future antibody problems.

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Disclosure of Grants Conflict of Interest L. J. Eltringham-Smith: Nothing to disclose; M. D. Lambourne: No Answer; X. Lei: No Answer; H. Ni: No Answer; E. L. Pryzdial: No Answer; A. Reheman: No Answer; W. P. Sheffield: Nothing to disclose

Disclosure of Commercial Conflict of Interest P. F. Lindholm: Immucor BioArray, Other; Immucor, Other; G. Ramsey: Nothing to disclose; R. D. Sumugod: No Answer; J. Zinni: Nothing to disclose Disclosure of Grants Conflict of Interest P. F. Lindholm: Nothing to disclose; G. Ramsey: Nothing to disclose; R. D. Sumugod: No Answer; J. Zinni: Nothing to disclose

S77-040A Systems Biology Analysis of Stored Red Blood Cells in SAGM Reveals Three Distinct Metabolic States G Paglia3, A Bordbar2, K Wichuk3, M Magnúsdóttir3, S Palsson2, Rolfsson3, M B Hansen5, E Sigurjónsson1,4, S Gudmundsson1, B Palsson3,2. 1The Blood Bank, Landspitali—University Hospital, Reykjavik, Iceland; 2Arbeseus, San Diego, CA, United States; 3Center for Systems Biology, University of Iceland, Reykjavik, Iceland; 4School of Science and Engineering, Reykjavik University, Reykjavik, Iceland; 5 Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark

Components and Component Processing II

S76-040A Transfused Plasma Lacking Factor VIII (FVIII) but not Fibrinogen Restores Hemostasis in Coagulopathic Mice L J Eltringham-Smith5, X Lei3,4, A Reheman4, M D Lambourne5, E L Pryzdial1,6, H Ni3,4, W P Sheffield2,5. 1Centre for Innovation, CBS, Vancouver, BC, Canada; 2Centre for Innovation, CBS, Hamilton, ON, Canada; 3Centre for Innovation, CBS, Toronto, ON, Canada; 4St. Michael’s Hospital, Toronto, ON, Canada; 5Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada; 6Centre for Blood Research, University of British Columbia, Vancouver, ON, Canada Background/Case Studies: The evidence supporting plasma transfusion as a means to restore hemostatic control and prevent or treat bleeding is weak, leading to uncertainties as to which proteins in plasma affect its therapeutic quality. Some regulators focus on coagulation factor VIII (FVIII) activity, but whether this measure reflects the overall efficacy of transfusable plasma is questionable. A mouse model of coagulopathy in which bleeding outcomes were responsive to plasma transfusion was developed to address the relative contributions of FVIII and fibrinogen to plasma quality. Study Design/Methods: Anesthetized mice were rendered coagulopathic by four rounds of exchange of whole blood for washed red cells in 5% human albumin solution (HAS) in a Blood Exchange-induced Coagulopathy Approach (BECA). Prior to tail transection, BECA mice were transfused with HAS, wild-type murine fresh-frozen plasma (WT mFFP) or mFFP from FVIII -/- or fibrinogen (Fg) -/- knockout mice. Shed blood was quantified by spectrophotometry. BECA mice were also subjected to laserinduced arteriolar injury, and intravascular thrombus formation was assessed by intravital microscopy and quantified as the platelet fluorescence intensity (PMFI). Results/Findings: Compared to untreated mice, BECA mice exhibited reduced platelet counts (278 ± 130 vs. 825 ± 280 X 109/L) and reduced hemoglobin (78 ± 9 vs. 134 ± 8 g/L) (n = 6 to 8 ± SD, p < 0.0004 for both). Compared to pre-procedure levels, BECA mice exhibited a five-fold reduction in plasma proteins (e.g., 20 ± 6 % Fg, p = 0.0008) and a 36% increase in prothrombin times (from 10 ± 1 to 13 ± 3 s, p = 0.004). Following tail transection, BECA mice lost nine-fold more blood than control mice (188 ± 96 μL versus 31 ± 30 μL). BECA mice transfused with 5% HAS lost 270 ± 170 μL of blood, an amount reduced by either WT (70 ± 50 μL, p < 0.001) or FVIII -/- (80 ± 60 μL, p < 0.01), but not Fg -/(210 ± 70 μL, p > 0.05), mFFP transfusion (n = 15 ± SD). Substituting murine plasma thawed and refrigerated for five days for mFFP did not diminish its efficacy (compare blood losses of 80 ± 40 μl versus 80 ± 50 μl; p > 0.05). BECA mice formed similar-sized intravascular clots in response to laser injury of the cremaster muscle arteriole when transfused with HAS or Fg -/- FFP; in contrast, average PMFI increased 3.5- to 4.0-fold in BECA mice transfused with WT or FVIII -/- FFP (p < 0.05). Conclusion: The content of Fg, but not of FVIII, was a relevant determinant of the anti-hemorrhagic efficacy of plasma transfusion in coagulopathic mice. The mice’s residual FVIII levels appeared sufficient to support hemostasis without FVIII supplementation when all other factors in plasma were provided. Thawed plasma retained its efficacy, consistent with Fg’s greater stability compared to FVIII. The regulatory focus on FVIII as a plasma quality marker may be misplaced. Disclosure of Commercial Conflict of Interest L. J. Eltringham-Smith: Nothing to disclose; M. D. Lambourne: No Answer; X. Lei: No Answer; H. Ni: No Answer; E. L. Pryzdial: No Answer; A. Reheman: No Answer; W. P. Sheffield: Nothing to disclose

Background/Case Studies: The global metabolic changes that stored red blood cells (RBCs) undergo are still not fully understood. The following methods were used to better understand the metabolic decay process: 1) global metabolite profiling of the endo- and exo-metabolomes using mass spectrometry, 2) multiple deep-coverage proteomic data sets integrated with the global reconstruction of the human metabolic network, yielding a highly validated and comprehensive RBC-specific metabolic map, 3) multivariate statistics, and 4) novel constraint-based methods (COBRA tools) for systems biology analysis. The computational approaches were used to analyze the high-throughput measurements of saline-adenine-glucose-mannitol (SAGM) media and intracellular RBC samples from units during standard storage. Study Design/Methods: Twenty RBC units were stored in SAGM at 4°C in standard blood bank conditions. A 5-mL sample was taken for metabolite profiling and quality control/quality assurance (QC/QA) measurements at 14 time points across 46 days. In total, 135 variables were tracked, including 80 intracellular metabolites, 40 extracellular metabolites, and 15 physiological and QC/QA measurements. A multi-step data analysis workflow composed of statistical and systems biology methods was deployed to analyze the ∼40,000 collected data points. Results/Findings: Over the 42 days of storage, 46 extracellular metabolites and 53 intracellular metabolites significantly changed (p < 0.05, Sidak Correction, effect size >50%). With the use of principal component analysis, three distinct metabolic states were identified. RBC metabolic decay is nonlinear, with transitions on days 11 and 18 of storage. By examining the principal components and metabolic modeling of reaction fluxes through pathways, the three states were characterized (Table). Conclusion: Systems biology approaches enable analysis of timecourse, deep-coverage, high-throughput data sets to reveal nonlinear metabolic shifts. The three metabolic phenotypes are distinct with differential metabolic pathway usage. These results have potential implications for RBC storage. Clinical studies that assess deferential posttransfusion outcomes of SAGM units based on these phases are warranted. Disclosure of Commercial Conflict of Interest A. Bordbar: No Answer; S. Gudmundsson: No Answer; M. B. Hansen: No Answer; M. Magnúsdóttir: No Answer; G. Paglia: No Answer; B. . Palsson: No Answer; S. Palsson: Nothing to disclose; . Rolfsson: No Answer; . E. Sigurjónsson: No Answer; K. Wichuk: No Answer Disclosure of Grants Conflict of Interest A. Bordbar: No Answer; S. Gudmundsson: No Answer; M. B. Hansen: No Answer; M. Magnúsdóttir: No Answer; G. Paglia: No Answer; B. . Palsson: No Answer; S. Palsson: Nothing to disclose; . Rolfsson: No Answer; . E. Sigurjónsson: No Answer; K. Wichuk: No Answer

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TABLE. Metabolic Pathway Activity Changes during the Three Storage States Metabolic subsystem/pathway

Representative Measured Metabolites

Phase I Activity

Upper Glycolysis Lower Glycolysis Adenine Metabolism Purine Degradation Glutathione Usage Glutathione Breakdown SAM Metabolism

Fructose-6-phosphate Phosphoglycerates ATP and Adenine Hypoxanthine and Inosine Oxidized Glutathione Cysteinyl-glycine, total glutathione SAM and Homocysteine

High High High Negligible High Negligible High (Increases)

Phase II Activity Low Low Negligible Negligible Low High Low

Phase III Activity Low Low Negligible High Low High High (Decreases)

SAM, S-adenosyl methionine; ATP, adenosine triphosphate. “Increases” indicates that metabolite pools are building up, and “Decreases” indicates the opposite. S78-040A Identification of Steap3 as a Candidate Gene Associated with Altered Red Blood Cell (RBC) Storage in a Mouse Model K de Wolski2, H Waterman2, P C Thomson4, J D Gorham1, M S Ranson1, H L Howie2, A Munday2, J Johnsen2,5, J C Zimring2,3. 1Pathology, Geisel School of Medicine at Dartmouth, Lebanon, NH, United States; 2Research Institute, Puget Sound Blood Center, Seattle, WA, United States; 3 Laboratory Medicine, University of Washington, Seattle, WA, United States; 4Veterinary Science, University of Sydney, Sydney, NSW, Australia; 5Hematology, University of Washington, Seattle, WA, United States Background/Case Studies: There is substantial donor-to-donor variability in the poststorage survival of transfused RBCs in humans. RBCs from different strains of inbred mice also store differently; RBCs from C57BL/6 (B6) mice store well, whereas RBCs from FVB/NJ (FVB) mice store poorly (24-hr posttransfusion recoveries). We hypothesized that the differences in RBC storage observed between inbred mouse strains are heritable and could be mapped by using mouse genetic tools. Study Design/Methods: B6 and FVB mice were crossed to generate F1 mice, which were then intercrossed to generate 156 F2 animals. RBC storage phenotypes were defined by storing individual donor RBCs for 7 days, followed by transfusion into F1 recipients and calculation of 24-hour recoveries. Each mouse was surveyed with a Mouse Diversity Genotyping Array containing 1400 informative single-nucleotide polymorphisms (SNPs), and Quantitative Trait Loci (QTL) analysis was performed. Progeny testing and fine mapping were done by backcrossing “poor-storing” F2 mice to B6 for three generations and testing offspring for RBC storage phenotypes and SNP genotypes. Results/ Findings: RBCs from F2 mice had a Gaussian distribution of recoveries, with a range that exceeded parental strains on both extremes. QTL analysis identified a 149-Mb region on chromosome 1 (rs13475827 to rs13476300), which was associated with RBC storage with extreme statistical significance (maximum peak p = 2.1 x 10−31). Fine mapping refined this to a 9.5-Mb candidate region containing 64 genes, 35 of which encoded proteins. Filtering for coding genes with nonsynonymous B6-FVB SNPs identified five genes (Gli2, Steap3, Ccdc93, Rab3gap1, and Tli). Of these, Steap3 is known to function in erythroid cells, where it is the primary ferrireductase converting Fe3+ to Fe2+, both mitigating oxidative stress and allowing transferrin-dependent iron uptake. The identified Steap3 B6-FVB variant, p.A350V, lies within the conserved ferric reductase superfamily domain. Conclusion: We have identified a genetic region on chromosome 1 that is strongly associated with RBC storage in mice. Within this region, a nonsynonymous SNP in Steap3 is a strong candidate gene. Steap3 has previously been implicated in RBC phenotypes, as mice lacking Steap3 exhibit a profound anemia. Moreover, a human family has been reported with a nonsense mutation in the human Steap3 orthologue and a congenital hypochromic anemia. We hypothesize that Steap3 p.A350V is a hypomorphic variant that adversely impacts RBC storage biology. Additional studies of the effect of p.A350V on Steap3 function and RBC storage are ongoing. In addition to identifying a novel genetic locus associated with stored RBC survival, these studies suggest that polymorphisms in STEAP3 or related proteins could contribute to human blood donor variability. Disclosure of Commercial Conflict of Interest K. de Wolski: Nothing to disclose; J. D. Gorham: Nothing to disclose; H. L. Howie: Nothing to disclose; J. Johnsen: Nothing to disclose; A. Munday: Nothing to disclose; M. S. Ranson: Nothing to disclose; P. C. Thomson: Nothing to disclose; H. Waterman: No Answer; J. C. Zimring: Nothing to disclose Disclosure of Grants Conflict of Interest K. de Wolski: Immucor, Grants or Research Support; Terumo, Grants or Research Support; J. D. Gorham: Nothing to disclose; H. L. Howie: Immucor,

Grants or Research Support; J. Johnsen: Agilent, Grants or Research Support; Biogen Idec, Grants or Research Support; A. Munday: Nothing to disclose; M. S. Ranson: Nothing to disclose; P. C. Thomson: Nothing to disclose; H. Waterman: No Answer; J. C. Zimring: Immucor, Grants or Research Support

S79-040A Loss of Protein Tyr-phosphorylation During in vitro Storage of Human Erythrocytes: Impact on RBC Morphology M Prudent1, B Rappaz2, R Hamelin3, J Delobel1, M Müller2, P Marquet4, M Moniatte3, G Turcatti2, J Tissot1, N Lion1. 1Unité de Recherche et Développement, Service Régional Vaudois de Transfusion Sanguine, Lausanne, Switzerland; 2Biomolecular Screening Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 3Proteomics Core Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 4Center for Psychiatric Neuroscience, Department of Psychiatry DP-CHUV, Prilly-Lausanne, Switzerland Background/Case Studies: Clinical studies feed the debates on the age of stored red blood cells (RBCs) in transfusion medicine and the impact of that age on patients. As revealed by in vitro studies, RBC, stored as erythrocyte concentrates (ECs) at 4°C in saline adenine glucose mannitol (SAGM) solution up to 42 days, suffer from storage lesions that deteriorate the metabolism, proteins, release of oxygen, and so forth. In particular, the RBC deformability decreases gradually over the 42-day period as does the adenosine triphosphate (ATP) content required for membrane fluctuation. The deformability is affected by phosphorylation of cytoskeleton and membrane proteins that modulates protein-protein interactions. The present study characterizes the effect of storage on tyrosine phosphorylation (pY) and therefore on RBCs. Study Design/Methods: RBCs (rejuvenated or not) stemming from ECs (n = 3) at various storage times ( 0.05). However, ex vivo treatment of SRBCs with NO increased the red cell velocity of SRBCs as compared to untreated SRBCs (102.7 ± 5.4 vs. 94.2 ± 2.9 μm/s, respectively; p < 0.05 but p > 0.01). Conclusion: Ex vivo exposure of SRBCs to NO prior to transfusion prevents pulmonary hypertension during SRBC transfusion in sheep. Exposure of stored RBCs to 300 ppm NO did not alter the lifespan of transfused SRBCs, but there was increased SRBC velocity thru a microfluidic obstacle course (p < 0.05), which suggests improved erythrocyte membrane deformability. Disclosure of Commercial Conflict of Interest S. E Du: No Answer; A. Beloiartsev: No Answer; M. Dao: No Answer; S. Muenster: Nothing to disclose; W. M. Zapol: No Answer Disclosure of Grants Conflict of Interest S. E Du: No Answer; A. Beloiartsev: No Answer; M. Dao: No Answer; S. Muenster: Nothing to disclose; W. M. Zapol: No Answer

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S81-040A New RBC Additive Solution Reduces Storage Hemolysis while Maintaining in vivo Performance K Radwanski2, L J Dumont1, L Herschel1, S L Waters1, Z M Szczepiorkowski1, A H Siegel1, K Min2. 1Geisel School of Medicine at Dartmouth, Lebanon, NH, United States; 2Fresenius Kabi USA, Lake Zurich, IL, United States Background/Case Studies: A new red blood cell (RBC) storage solution, E-Sol 5, has been shown in preclinical in vitro studies to promote improved maintenance of adenosine triphosphate (ATP) and 2,3-diphospholycerate (2,3-DPG) while reducing microvesicle release and hemolysis when compared to a standard FDA-approved RBC storage solution. E-Sol 5 leverages low chloride ion concentration, hypotonicity (with respect to cell penetrable solution components), and alkaline pH to promote these effects. The objective of this study was to evaluate E-Sol 5’s in vitro characteristics and autologous in vivo RBC recovery in healthy subjects and compare these findings to FDA performance criteria. Study Design/Methods: Whole blood units were collected into citrate phosphate dextrose (CPD) from 12 healthy subjects utilizing 500-mL collection sets (4R3340E, Fenwal Inc. Lake Zurich, IL), leukocyte-reduced by using the integral whole-blood filter, centrifuged at 6035g x 10 min, and separated into plasma and packed RBCs. E-Sol 5 was provided in two separate connected containers: Part A (55 mL) and Part B (50 mL). The two solutions were combined into one bag immediately before addition to the red cell concentrate. RBCs in E-Sol 5 were stored in DEHPPVC storage containers for up to 42 days without mixing under standard blood bank conditions of 1-6°C within 8 hours of WB collection. A standard panel of in vitro tests was performed on Days 0 and 42. In vivo 24-h 51Cr RBC recovery was measured in n = 10 units on Day 42. Results/Findings: All units contained 75% with the lower 95% CL of 81.6% and 79.9%. Conclusion: This study confirms the previously reported in vitro performance of E-Sol 5, while also demonstrating that 42-day-stored E-Sol 5 RBCs meet the FDA criteria for licensure for hemolysis and 24-hour in vivo recovery. Disclosure of Commercial Conflict of Interest L. J. Dumont: APT, Consulting or Board of Director Fees; APT, Grants or Research Support; Fresenius-Kabi, Grants or Research Support; L. Herschel: Fenwal, Inc, Grants or Research Support; Advanced Preservation Technologies, Grants or Research Support; K. Min: No Answer; K. Radwanski: Nothing to disclose; A. H. Siegel: Fresensius-Kabi, Other; Z. M. Szczepiorkowski: No Answer; S. L. Waters: No Answer Disclosure of Grants Conflict of Interest L. J. Dumont: APT, Grants or Research Support; EryDel, Grants or Research Support; Fresenius-Kabi, Grants or Research Support; CitraLab, Grants or Research Support; APT, Consulting or Board of Director Fees; New Health Sciences, Consulting or Board of Director Fees; Cytosorbents, Grants or Research Support; Haemonetics, Grants or Research Support; L. Herschel: Haemonetics Corporation, Grants or Research Support; Fenwal, Inc, Grants or Research Support; Citra Labs, Grants or Research Support; Advanced Preservation Technologies, Grants or Research Support; EryDel, Grants or Research Support; CytoSorbants, Grants or Research Support; K. Min: No Answer; K. Radwanski: Employee of Fresenius Kabi, Other; A. H. Siegel: Fresensius-Kabi, Other; Z. M. Szczepiorkowski: No Answer; S. L. Waters: No Answer

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TABLE. In vitro measures of E-Sol 5 RBCs stored for 42 days (n = 12)

Volume (mL) Automated Hct (%) Spun Hct (%) Hgb (g) pH (37C) Potassium (mM) Hemolysis (%) ATP (μmol/g Hgb) RBC Morphology (%)

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Day 0

Day 42

Mean ± sd, range

Mean ± sd, range

303 ± 17 279-347 62 ± 1 60-64 68 ± 1 66-70 61 ± 5 56-75 6.88 ± 0.05 6.79-6.98 all units < 1.5

259 ± 16 239-297 61 ± 2 59-65 62 ± 1 60-65 53 ± 5 47-64 6.36 ± 0.05 6.28-6.45 59 ± 5 49-69 0.19 ± 0.08 0.11-0.38 3.2 ± 0.6 2.4-4.5 69.4 ± 5.6 58.3-76.0

0.04 ± 0.01 0.02-0.06 3.8 ± 0.5 3.0-4.5 99.9 ± 0.2 99.4-100

Hct, hematocrit; Hgb, hemoglobin.

Recipient Non-Adverse Infectious Events

S82-040B Risk of TRALI from Apheresis Platelets A F Eder1, B A Dy1, R J Benjamin1. 1American Red Cross, Rockville, MD, United States Background/Case Studies: Blood centers have significantly reduced the risk of transfusion-related acute lung injury (TRALI) from plasma components by relying predominantly on male donors. Mitigation is more challenging for apheresis platelets (APs) because of the predicted effects on

component availability. A large blood system preferentially recruits male AP donors and has tested first-time female AP donors who report prior pregnancy for HLA antibodies since 2010. We compared TRALI with AP to that with other components and assessed the risk associated with male or female donors. Study Design/Methods: Cases of TRALI and possible TRALI (herein, TRALI) reported to the blood center’s national hemovigilance program due to APs and RBCs in 2006-2012 and male donor-predominant plasma in 2008-2012 were calculated as rates per 106 distributed units, and cases with female donor(s) were compared to cases with only male donors. Mixed-component cases (i.e., AP and RBC units) were excluded. The blood center distributed 5.56 million AP units (>70% male donors; excludes PAS platelets), 8.07 million plasma units (>95% male donors), and 43.2 million RBC units (54% male donors). Results/Findings: The overall rate of TRALI was 6.7 per 106 distributed AP units (Table), which was higher than the rate for male-predominant plasma [3.8 per 106 units; OR 95% CI, 1.7 (1.1-2.8)] and RBCs [2.0 per 106 units; OR 95% CI 3.3 (2.3-4.9)]. The difference was not significant for reported fatalities. TRALI was more likely to involve or implicate female donors than male donors of all components, but the rate was highest with female donors of plasma, followed by AP (Table). HLA antibodies were identified in involved female donors in 62% AP, 71% plasma, and 49% RBC cases. Four female AP donors had a specific HNA antibody as well as HLA antibodies; none had an isolated HNA antibody. Conclusion: The overall TRALI rate is higher for APs than for male-predominant plasma or RBCs, although the difference was not significant for reported fatalities. Plasma units from female donors still pose the highest risk, possibly related to component volume, donor characteristics, or recipient susceptibility to TRALI. The risk of TRALI with AP is approximately five-fold greater with female donors than male donors. A strategy to test female AP donors who report prior pregnancy and to defer those with HLA antibodies may reduce the risk of TRALI by at least 60% and prevent most cases from HNA antibodies. Disclosure of Commercial Conflict of Interest R. J. Benjamin: Nothing to disclose; B. A. Dy: Nothing to disclose; A. F. Eder: Nothing to disclose Disclosure of Grants Conflict of Interest R. J. Benjamin: Nothing to disclose; B. A. Dy: Nothing to disclose; A. F. Eder: Nothing to disclose

TABLE.

Involved Component

TRALI Cases

All Cases

Cases with Male Donor(s)

Apheresis Platelets

Number Rate [95% CI] Fatalities (Rate)

37 6.7 [4.5-8.8] 4 (0.7) 31 3.8 [2.5-5.2] 3 (0.4) 86 2.0 [1.6-2.4] 8 (0.2)

11 2.8 [1.1-4.5] 1 (0.3) 6 0.8 [0.2-1.4] 1 (0.1) 17 0.7 [0.4-1.1] 1 (0.04)

Plasma

RBC

Number Rate [95% CI] Fatalities (Rate) Number Rate [95% CI] Fatalities (Rate)

S83-040B Transfusion-Associated Circulatory Overload: Hints from the Hemodynamics? F Khawaja1, P Wagholikor1, L Clifford1, G A Wilson1, G Ognjen1, D J Kor1. 1 Mayo Clinic, Rochester, MN, United States Background/Case Studies: The prevailing theory of transfusion-associated circulatory overload (TACO) pathogenesis is volume overload with cardiogenic pulmonary edema (CPE). Notably, numerous reports have outlined a hypertensive response in those who develop TACO, suggesting a potential role for vasoactive substances in the implicated blood components. To further characterize this cardiovascular response, we aimed to compare the blood pressure profiles observed in patients who develop TACO with those seen in CPE in the absence of transfusion therapies. Study Design/

Cases with Female Donor(s) 26 15.2 [9.4-21.1] 3 (1.8) 25 65.1 [39.6-90.7] 2 (5.2) 69 3.5 [2.7-4.3] 7 (0.4)

Female vs. Male; p-value p < 0.00001 p = 0.05 (NS) p < 0.00001 p < 0.00001 p < 0.00001 p = 0.02

Methods: This is a case-control study evaluating the blood pressure profiles seen in TACO (cases) as compared with those seen in CPE unrelated to transfusion therapies (controls). Cases and controls were identified from previous investigations performed at a single academic medical center. TACO diagnoses had been previously adjudicated as part of an NHLBIsponsored prospective cohort investigation. Consistent with the National Healthcare Safety Network definition of TACO, at least one blood component was required to have been transfused in the six-hour interval preceding the onset of respiratory insufficiency. The CPE cohort had previously been identified by using a validated electronic surveillance tool. To be included in the CPE cohort, no blood products were allowed in the six-hour interval preceding the first chest film noting pulmonary edema (PE). The present investigation was restricted to patients ≥18 years of age. Changes in systolic (ΔSBP), diastolic (ΔDBP), and mean arterial blood pressures (ΔMAP) from

52A

ABSTRACT SUPPLEMENT

baseline to the onset of PE (TACO or CPE) were assessed. Baseline blood pressures were defined as the blood pressure recorded just before the transfusion episode(s) associated with the onset of PE (TACO) or the blood pressure closest to the six-hour time point preceding the onset of PE in the CHF cohort. The maximum blood pressures recorded during this interval were then identified allowing calculation of the change from baseline. Wilcoxon rank-sum tests were used to compare the change in blood pressure between groups. Results/Findings: A total of 100 TACO cases and 115 CPE controls met our inclusion criteria and had blood pressure recordings available for evaluation. Changes in the blood pressure parameters are presented in the table below. Conclusion: The results of this investigation suggest that the blood pressure response seen in TACO exceeds the blood pressure response seen in CPE not related to transfusion therapies. This may support the existence of vasoactive substances within the donor product contributing to the development of TACO. Disclosure of Commercial Conflict of Interest L. Clifford: Nothing to disclose; F. Khawaja: Nothing to disclose; D. J. Kor: Nothing to disclose; G. Ognjen: No Answer; P. Wagholikor: No Answer; G. A. Wilson: Nothing to disclose

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intensity until 21 d. Antibody titers declined thereafter but were detectable to 8 mo. This IgG1 antibody was BioRBC-specific and was not neutralized by free biotin. There were no detectable clinical or laboratory RBC abnormalities and no effect on biotin plasma or urine levels. Screening for spontaneous antibodies to BioRBCs identified only 2 out of 659. In contrast to the BioRBC-induced antibodies, these were IgM and were neutralized by free biotin. Conclusion: Re-exposure to BioRBCs in a subject with a history of weak BioRBC antibodies resulted in a strong anamnestic antibody response associated with a profound shortening of BioRBC RCS but no other clinical or laboratory effects. We speculate that the longer survival of the lowest BioRBC density relative to the three higher densities indicates a threshold in removal of BioRBCs. Future studies using BioRBCs biotinylated at even lower biotin densities may overcome the immunogenicity issues currently associated with BioRBC transfusion. The physiological relevance of the low prevalence of spontaneous antibodies to BioRBCs remains undefined. Additional data are needed to clarify these clinical and laboratory findings— especially for subjects in whom multiple BioRBC studies are anticipated. Disclosure of Commercial Conflict of Interest

L. Clifford: Nothing to disclose; F. Khawaja: Nothing to disclose; D. J. Kor: Nothing to disclose; G. Ognjen: No Answer; P. Wagholikor: No Answer; G. A. Wilson: Nothing to disclose

J. A. Cancelas: No Answer; R. Franco: Nothing to disclose; C. Geisen: Cerus Corporation, Concord, CA, USA, Grants or Research Support; D. M. Mock: Nothing to disclose; D. Nalbant: No Answer; A. K. North: Cerus Corporation, Stocks or Bonds; R. L. Schmidt: Nothing to disclose; S. R. Stowell: Nothing to disclose; R. G. Strauss: Nothing to disclose; J. A. Widness: Nothing to disclose

TABLE.

Disclosure of Grants Conflict of Interest

Disclosure of Grants Conflict of Interest

Parameter ΔSBP (mmHg)* ΔDBP(mmHg)* ΔMAP(mmHg)*

TACO

CPE

P-value

35.4 (20.3-52.8) 18.1 (8.65-29.0) 22.5 (5.2-38.4)

20.4 (1.1-35.9) 15.2 (6.0-28.3) 15.1 (7.4-33.7)

P < 0.05† P = 0.187 P < 0.05†

* Median (25%-75% interquartile range). † Statistically significant result. S84-040B Shortened Survival of Biotin-labeled Red Blood Cells (BioRBCs) Following a Second BioRBC Transfusion in an Adult with a Previous BioRBC Antibody Response S R Stowell7, A K North2, R Franco6, J A Cancelas9, D M Mock4,5, R G Strauss8,3, C Geisen1, R L Schmidt3, D Nalbant3, J A Widness3. 1 Goethe University Frankfurt/Main, German Red Cross Blood Centre Baden-Württemberg-Hessen, Institute of Transfusion Medicine and Immunohematology, Frankfurt/Main, Germany; 2Cerus Corporation, Concord, CA, United States; 3Pediatrics, University of Iowa, Iowa City, IA, United States; 4Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States; 5Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, United States; 6Internal Medicine, University of Cincinnati, Cincinnati, OH, United States; 7Laboratory and Transfusion Medicine, Emory University, Atlanta, GA, United States; 8Pathology, University of Iowa, Iowa City, IA, United States; 9Hoxworth Blood Center & Department of Pediatrics, The University of Cincinnati, Cincinnati, OH, United States Background/Case Studies: Biotinylated allogeneic or autologous red blood cells (BioRBCs) are used to determine in vivo RBC volume, 24-h posttransfusion recovery (PTR24), and long-term RBC survival (RCS), expressed as time to 50% survival (T50). Individual RBC samples biotinylated to distinct densities permit measures concurrently of multiple BioRBC populations. Although ∼15% of adults receiving autologous BioRBCs develop antibodies to BioRBCs (Transfusion 2012;52:1596), there are no data to document consequences of BioRBC re-exposure in immunized subjects. Study Design/Methods: A healthy 64-year-old female subject developed a transient, weak BioRBC antibody response 3 mo following her first autologous multidensity BioRBC exposure (Transfusion 2011;51:148). We now report the clinical and laboratory responses following her re-exposure 3 y later to the same transfusion of BioRBC (6, 18, 54, and 162 μg/mL BioRBCs) in which 2.4% of her RBCs were labeled. BioRBC antibodies were serially studied by IgG gel card testing (Ortho Clinical Diagnostics). Also evaluated were her RCS, hematology, and biotin nutritional status. To assess specificity, we also determined the prevalence of anti-BioRBC antibodies in adults without prior exposure to BioRBC. Results/Findings: Following BioRBC re-exposure, PTR24 was unchanged, but a dose-dependent accelerated removal of BioRBCs was observed: T50 for 162 μg/mL t (HGVS designation c.336+3t) is 95% conserved and is predicted to cause aberrant exon splicing; it either silences or severely reduces e antigen expression not detectable by adsorption elution. The sample was not informative for c expression because of RHCE*cE in trans. Both samples were found in Caucasians of Italian ancestry. Disclosure of Commercial Conflict of Interest P. Bonomo: No Answer; G. Cattaneo: No Answer; K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; C. Paccapelo: No Answer; S. Travali: No Answer; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose Disclosure of Grants Conflict of Interest P. Bonomo: No Answer; G. Cattaneo: No Answer; K. Hue-Roye: Nothing to disclose; C. Lomas-Francis: Nothing to disclose; C. Paccapelo: No Answer; S. Travali: No Answer; S. Vege: Nothing to disclose; C. M. Westhoff: Nothing to disclose

SP233 Providing Units for Patients with Anti-E and Antibodies to hrB, hrS, or e Variants: An Update C Flickinger1, M A Keller1, G Meny2, S J Nance1. 1American Red Cross, Philadelphia, PA, United States; 2UT Health Science Center at San Antonio, San Antonio, TX, United States Background/Case Studies: A national rare-donor program has provided rare units for patients since 1998. Rare donors are defined as those whose RBCs lack a high-incidence antigen or multiple common antigens. The program is challenged to provide units for patients with antibodies to hrB and hrS. Since 2007, donors are screened by a medium-throughput genotyping platform; the RH locus of possible hrB- and e variant donors are further characterized by using lab-developed tests. Donors predicted to be E- hrBand/or hrS- or having two variant RHCE alleles were entered into a database, which has been used by the program since 2008 to provide genotypematched units for respective patients. This report provides an update on the program’s provision of units for such patients. Study Design/Methods: A retrospective analysis of provided units for E- hrB-, E- hrS-, E- hrB- hrS-, and E- e variant requests from 2009 to 2013 was performed. Requests are considered filled if any units of the desired genotype were provided. Results/ Findings: Since 2007, 248 E- hrB-, 5 E- hrS-, 6 E- hrB- hrS-, and 287 E- e variant donors have been identified. From January 2009 to December 2013, the program received 127 requests for E- units negative for hrB, hrS, or both or for e variant units for 60 different patients. E- hrB- units were provided for 73 of 91 requests (80.2%) for 15 different E- hrB- genotypes: E- hrS- units for 1 of 7 requests (14.2%) for 3 different E- hrS- genotypes; no units for 2 requests for E- hrB- hrS- (0%) and E- e variant units for 21 of 27 requests (77.7%) for 5 different E- e variant genotypes (Table). Conclusion: With ongoing molecular screening of donors and consequent interrogation of RHD and RHCE genes, the provision of units for patients with anti-E and antibodies to hrB or to e variants improved from 2009 to 2013, with fill rates ranging from 73.9% to 78.3%, respectively. However, filling such requests can be complicated by ABO and by the need for donors with homozygous alleles or for those lacking additional antigens. In addition, providing E- hrSor E- hrB- hrS- units requires increased screening of donors of African descent. Identification, recruitment, and retention of such donors are crucial to providing units for current and future patient needs. Disclosure of Commercial Conflict of Interest C. Flickinger: Nothing to disclose; M. A. Keller: Nothing to disclose; G. Meny: Nothing to disclose; S. J. Nance: Nothing to disclose Disclosure of Grants Conflict of Interest C. Flickinger: Nothing to disclose; M. A. Keller: BIoArray Solutions LLC,Grants or Research Support; G. Meny: Nothing to disclose; S. J. Nance: Nothing to disclose

TABLE. Number and Haplotypes of E- hrB-; E- hrS-; E- hrB- hrS-; and E- e Variant Requests From January 2009 to December 2013 Request

#

RHD RHCE Haplotype

E- hrB-

38 21 9

DIIIa-ceS/ (C)ceS Dce733G/ (C)ceS (C)ceS/ (C)ceS WeakD 4.0-ce48C733G/ Dce733G Dce733G/ Dce48C, 733G DAR-ceAR/ DAR-ceAR DAU0-ceMO/ DAU0-ceMO Dce733G/ Dce733G DAU0-ce48C/ DAU0-254G

6 3

E- hrSE- hrB- hrSE- e Variant

5 2 23 1

Requests Filled # (%)

#

RHD RHCE Haplotype

Requests Filled # (%)

#

32 (84.2%) 20 (95.2%) 6 (66.6%)

2 2 2

DceS / (C)ceS DIIIa-ceS/ DIIIa-ceS DIIIa-ceS/ceS

1 (50%) 1 (50%) 1 (50%)

1 1 1

6 (100%)

2

DIIIa-ceS/ Dce733G

1 (50%)

1

2 (66.6%)

1

DceS/ DcE907delC

0 (0%)

0 (0%) 0 (0%) 21 (91.3%) 0 (0%)

1

DceEK/ DAU0-ceEK

1 1

Dce48C/ Dce733G DIVa-ceTI / DAU0-ceMO

RHD RHCE Haplotype

Requests Filled # (%) 0 (0%) 1 (100%) 1 (100%)

1

DceS/ Dce733G,1006T DceS/ Dce48C,733G (C)ceS/Dce733G, 1006T (C)ce733G,1006T/ Dce733G ce254G/ce254G

1 (100%)

1

DAR-ceAR/ DOL-ceBI

0 (0%)

0 (0%) 0 (0%)

1

Weak D4.0-ceCF/Dce733G

0 (0%)

1 (100%) 0 (0%)

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SP234 Genomic Analysis of RH Alleles on Samples with Weak or Discrepant Results in the RhD Typing M A Mota1, M R Dezan1, K B Torres1, T H Costa1, E P Bastos1, L D Santos1, M G Aravechia1, L Castilho2, J M Kutner1. 1Hemotherapy, Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hemocentro, Unicamp, Campinas, Brazil Background/Case Studies: Background: D is the most clinically significant antigen of the RH blood group system, and it is one of the most important in transfusion medicine and in pregnancy. Many variants of D have been described, which are classified according to their phenotype and molecular variation in weak D, partial D, and Del. The variability of D antigen is caused by different molecular mechanisms: missense mutations, gene conversions between RHD and RHCE, and splice site mutations. Among European subjects, about 0.2% to 1% carry aberrant RHD alleles. Mistyping of these individuals may potentially lead to anti-D alloimmunization. The purpose of this study was to use a high-throughput method to identify weak and partial D on samples with weak or discrepant results in the RhD typing. Study Design/Methods: We investigated 64 samples that had weak or discrepant results by hemagglutination with 2 anti-D monoclonal antibodies (IgM and IgG). The genomic DNA was extracted from whole blood, collected from patients and donors, by QIAamp DNA Blood mini-kit using EpMotion instrument (Eppendorf, Germany) according to the manufacturer’s instructions. The DNA concentration was evaluated by using NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). The determination of D allele was performed by using the RHD BeadChip™ (BioArray, Immucor). In all samples classified as partial D, we also performed the determination of CE allele by using the RHCE BeadChip™ (BioArray, Immucor). Results/Findings: Forty-seven samples were characterized as D variants: 30 as partial D (15 RHD*DAR, 5 as weak partial RH*D 4.0, 3 as RHD*DVI, 1 as RHD*DFR-2, 1 as RHD*DBT-2, 5 as RHD*DIVa-2), and 17 as weak RH*D (6 weak D type 3, 6 weak D type 1, 5 weak D type 2). Three samples were RHD compound heterozygous [1 RHD*DAR/RHD*DIVa-2, 2 RHD*DAR/RHD*DIIIa, 2 RHD*DIVa-2/RHD*DIIIa, and 1 RHD*DVI/DIIIa-CE(4-7)-D)]. Combinations between RHD and RHCE variants were also found in the studied samples: RHD*DAR with RHCE*ceAR (n = 10) and RHD*DIVa-2 with RHCE*ceTI (n = 2). We also identified one sample with an altered RHD in combination with a variant RHCE in compound heterozygosity (RHD*DAR/ RHCE*ceAR/(C)ces). Conclusion: Because of the high RH allelic diversity found in the Brazilian population, determination of an accurate Rh phenotype often requires DNA testing in conjunction with serologic testing. RHD and RHCE microarray platforms allow the performance of a highthroughput identification of weak and partial D that cannot be characterized by serology. Knowledge of the RH variants in populations of African ancestry can be used to assist in efforts to evaluate and reduce the risk of red cell alloimmunization in individuals with sickle cell disease. Disclosure of Commercial Conflict of Interest M. G. Aravechia: No Answer; E. P. Bastos: No Answer; L. Castilho: No Answer; T. H. Costa: No Answer; M. R. Dezan: Nothing to disclose; J. M. Kutner: No Answer; M. A. Mota: Nothing to disclose; L. D. Santos: No Answer; K. B. Torres: Nothing to disclose Disclosure of Grants Conflict of Interest M. G. Aravechia: No Answer; E. P. Bastos: No Answer; L. Castilho: No Answer; T. H. Costa: No Answer; M. R. Dezan: Nothing to disclose; J. M. Kutner: No Answer; M. A. Mota: Nothing to disclose; L. D. Santos: No Answer; K. B. Torres: Nothing to disclose

SP235 A New Weak RHD Allele in North Africans N Robitaille2, J Constanzo-Yanez4, C Éthier3, J Lavoie1, M St-Louis1. 1 R&D, Hema-Quebec, Quebec, QC, Canada; 2Pediatrics Hematology, CHU-Ste-Justine, Montreal, QC, Canada; 3Immunohematology Reference Laboratory, Héma-Québec, Quebec, QC, Canada; 4Immunohematology Reference Laboratory, Héma-Québec, Montreal, QC, Canada Background/Case Studies: Posttransfusion blood samples from a 2-y-old North African girl were referred for D antigen abnormal results with acute myeloid leukemia M5. She received 18 D- RBC units before blood samples were sent to us. This young girl recently immigrated to Canada from Belgium, where she had been typed D+. Study Design/Methods: Serology work-up was done by following approved techniques by the submitting hospital and at Héma-Québec. RHD genotyping was performed, and RHD

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exons were sequenced to determine if a variant was present. A family study was also undertaken. Results/Findings: The D phenotype done at the hospital was 1+ (immediate-spin and weak D testing), and a weaker D antigen reaction was noted. However, at that time, the transfusion history from Belgium was unknown. This result could not be confirmed by HémaQuébec, due to recent transfusions. Antibody screen was found negative. RHD PCR-SSP genotyping results indicated a normal RHD (exons 3, 4, 5, 6, 7, and 9 + RHD*Ψ). Several intronic polymorphisms were observed, as well as a missense mutation at nucleotide 717C > A (exon 5, Phe239Leu, located on the fourth extracellular loop close to the membrane). RHD mRNA from the child’s father was analyzed and had the same 717C > A hemizygote polymorphism. The mother was typed RHD- negative. The three siblings also had the same variant. D phenotyping was performed on the siblings. The reaction was weaker-than-normal D at 3+ for one individual and 4+ for the other two. The close proximity to the external membrane might cause a disturbance in the protein structure, resulting in the slight reduction of the D expression. Conclusion: Since the implementation of nucleotide-based analyses, several variants have been found in all blood group systems. These variants could be problematic in transfusion medicine by introducing new antigens in blood donors and reducing others in blood recipients. Disclosure of Commercial Conflict of Interest J. Constanzo-Yanez: Nothing to disclose; J. Lavoie: Nothing to disclose; N. Robitaille: Nothing to disclose; M. St-Louis: Nothing to disclose; C. Éthier: Nothing to disclose Disclosure of Grants Conflict of Interest J. Constanzo-Yanez: Nothing to disclose; J. Lavoie: Nothing to disclose; N. Robitaille: Eli Lilly,Grants or Research Support; M. St-Louis: Nothing to disclose; C. Éthier: Nothing to disclose

SP236 A Case of Changing Rh Phenotype: First Reported Case of RHD*DAU-2 Allele in an Arab Patient F Anwar1, M A Abdelaal1, F A Khalifa2, D J Lewis1, E M Mensinger1, Z Farzal3. 1Pathology & Laboratory Medicine, King Abdulaziz Medical City, Jeddah, Saudi Arabia; 2Obstetrics and Gynecology, King Abdulaziz Medical City, Jeddah, Saudi Arabia; 3UT Southwestern Medical Center, Dallas, TX, United States Background/Case Studies: The D antigen is a very significant blood group clinically. Almost all blood banks rely on serological testing to detect the D antigen. However, the serological distinction between D+ and D- red blood cells is not always unequivocal, which can have clinical implications for obstetric patients and transfusion recipients. The D antigen includes category D, partial D, and weak D types, which are important because anti-D alloimmunization can occur in some, but not all, patients whoexpress a variant RHD allele. The molecular basis is generally a single or multiple missense gene mutation(s) in which parts of the RHD gene are replaced by the respective segment of the RHCE gene. We report a first case of RHD*DAU-2 allele in an Arab patient. Study Design/Methods: The patient was a Saudi female, gravida 4 para 3. In 2013, a type and screen on the patient with a historical blood type of O-negative typed as O-positive. Review of all previous testing indicated the patient had originally typed as O-positive, then O-negative, and now O-positive (antibody screen-negative). Investigation was initiated, at first suspecting that two patients had shared the same medical card (and, thus, the same medical record number). An interview with the patient and her spouse confirmed that that was not the case. The next step was to review all previous ABO Rh typings performed (Table). Additional testing was performed on the current specimen, and finally an additional sample was requested and sent to a reference laboratory, where DNA was extracted and genomic DNA sequencing was performed for exons 1, 2, and 3 to 10 of the RHD gene. Results/Findings: DNA mutations indicated the variant RHD*DAU-2 at Exon 2: 209G > A (R70Q), Exon 7: 998G > A, (S333N), and Exon 8: 1136C > T (T379M). Conclusion: DAU alleles have been known to be a major cause of D antigen variability and anti-D immunization in patients of African descent. Due to the proximity of Africa to Saudi Arabia and to the centuries-old relationships with African peoples, the extent and characterization of RH phylogeny in the Arab population in Saudi Arabia and the region are unknown. Most anti-D reagents do not agglutinate DAU-2; however, in our samples, 4 of 10 were agglutinated by the anti-D reagents. Because anti-D immunization may occur in carriers of the DAU allele, molecular characterization provides a valid option for evaluating the clinical relevance in transfusion recipients and obstetric patients.

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ABSTRACT SUPPLEMENT

Disclosure of Commercial Conflict of Interest M. A. Abdelaal: Nothing to disclose; F. Anwar: Nothing to disclose; Z. Farzal: No Answer; F. A. Khalifa: No Answer; D. J. Lewis: Nothing to disclose; E. M. Mensinger: Nothing to disclose Disclosure of Grants Conflict of Interest M. A. Abdelaal: Nothing to disclose; F. Anwar: Nothing to disclose; Z. Farzal: No Answer; F. A. Khalifa: No Answer; D. J. Lewis: Nothing to disclose; E. M. Mensinger: Nothing to disclose TABLE. ABO Rh Serology Results of the Patient Time Line

Times Tested

ABO Rh

Method

1996-1998 2000-2005 2013*

2 6 2

O-positive O-negative O-positive

Tube Gel Card Gel Card

* Previous results were reviewed, and molecular testing was initiated.

SP237 Noninvasive Prenatal RHD Genotyping by Analysis of Circulant-Free Fetal DNA from Maternal Plasma F Truglio1, C Paccapelo1, S Scognamiglio1, M Villa1, N Revelli1, M Marconi1. 1Immunohematology Reference Laboratory, Blood Transfusion Centre, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy Background/Case Studies: Anti-D alloimmunization in pregnant women is the most common cause of hemolytic disease of the fetus and newborn (HDFN), but the introduction of immune prophylaxis in RhD-negative pregnant women has drastically reduced it. Information on the fetal RhD genotype can be useful to avoid unnecessary treatment of RhD-negative women. The discovery of circulant-free fetal DNA (cffDNA) in maternal plasma has made noninvasive fetal RHD genotyping possible. The purpose of this study was to validate a method to extract cffDNA from maternal plasma for the prenatal and noninvasive determination of the fetal RHD status. Study Design/Methods: We analyzed samples from 50 consecutive RhD-negative pregnant women between 15 and 17 wk of gestation who have an RhDpositive partner and were subject to prenatal diagnosis. The genomic DNA was extracted from 800 μl of maternal plasma that was separated by QIAamp DNA Blood micro kit (Qiagen, Valencia, CA, USA). To determine the fetal RHD genotype, we used Taqman primers and probes to detect exons 4, 5, and 10 of RHD gene by Real-Time PCR (Applied Biosystems, Foster City, CA). As internal controls, we used primers/probes sets to SRY and CCR5. Fetal genotyping results were compared with the RHD genotype determined by commercial PCR-SSP kits (Partial D-TYPE, BAG Health Care GmbH, Lich, Germany) by using DNA extracted from amniocentesis samples and with RhD phenotype from newborn cord blood samples obtained at birth. Results/Findings: Of 50 samples, 26 were RHD-positive and 21 RHDnegative. Among 26 samples, 2 were DNB variants, 1 was a DVII variant, and the remaining samples showed a normal RHD genotype with PCR-SSP analysis. All 26 samples were RhD-positive at birth. The 21 RHD-negative samples were concordant with PCR-SSP analysis and phenotype at birth Three samples were inconclusive, due to discordant results with exon 5 probe, while PCR-SSP analysis and phenotype at birth were RhD- negative. Conclusion: Noninvasive fetal RHD genotyping from maternal plasma is a useful tool for the management of RhD-negative pregnant women, and it can avoid the need for invasive procedures and reduce wastage of anti-RhDIg. Disclosure of Commercial Conflict of Interest M. Marconi: Nothing to disclose; C. Paccapelo: No Answer; N. Revelli: Nothing to disclose; S. Scognamiglio: Nothing to disclose; F. Truglio: Nothing to disclose; M. Villa: Nothing to disclose Disclosure of Grants Conflict of Interest M. Marconi: Nothing to disclose; C. Paccapelo: No Answer; N. Revelli: Nothing to disclose; S. Scognamiglio: Nothing to disclose; F. Truglio: Nothing to disclose; M. Villa: Nothing to disclose

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SP239 Two New RH Hybrid Genes and Their Potential Role in Pregnancy J A Crowley1, T Horn1, C Carson2, J Christensen3, J Plafker2, M A Keller1. 1 Biomedical Services, National Molecular Laboratory, American Red Cross, Philadelphia, PA, United States; 2Newark Beth Israel Medical Center, Newark, NJ, United States; 3New York-Penn Region Reference Laboratory, American Red Cross, West Henrietta, NY, United States Background/Case Studies: Case 1 is a 47-y-old Caucasian female of consanguineous origin who was scheduled for cervical laminectomy. The patient reported stillborn births of one infant in 1989 and of twins in 1992; she received prophylactic Rh Immune globulin with each pregnancy. RBCs typed D− C+ E− c− e+ with a positive preoperative antibody screen. The IRL identified a broadly reactive antibody in MTS IgG gel (gel). The patient’s plasma was reactive in gel when tested against two phenotypically similar panel cells and nonreactive against Rh null cells. Case 2 is a 26-y-old black female whose red cells typed D-negative with Series 4 and 5 anti-D at immediate-spin or by Immucor Galileo ECHO and 3+ to 4+ at antiglobulin phase. She was antibody screen negative by solid-phase testing. Her obstetric history is G3P2012. Both live births were Group O, D 4+, and DAT negative. Study Design/Methods: DNA was extracted by using standard methods. Genotyping involved RHD zygosity by presence-absence PCR and PCR-RFLP assays and the RHD and RHCE BeadChips (BioArray Solutions). Genomic DNA sequence analysis and cDNA plasmid cloning and sequence analysis were also performed. Results/Findings: Case 1: The RHD BeadChip identified c.186T, 410T, and 455C with low signal for markers in exons 4-7, which is consistent with homozygosity for RHD*DIIIa-CE(47)-D. The RHCE BeadChip detected c.48C, 307T, 733G, and 1006T. cDNA analysis confirmed RHD*DIIIa-CE(4-7)-D, which is known to express an altered C antigen, and identified a hybrid RHCE*CE-D(2-3)-CE. This novel RHCE-RHD-RHCE hybrid contains exons 2 and 3 from the RHD*DIIIa allele, including p.103S and RHCE gene regions containing c.48C in exon 1, c.676G, c.733G in exon 5, and c.1006T in exon 7. It was not possible to deduce whether the novel RHCE-RHD-RHCE hybrid encodes a C antigen and whether it is weakened or altered. Case 2: Zygosity testing determined the patent to be hemizygous for RHD. Genotyping via RHD BeadChip detected c.667G (p.223V), c.697C (p.233Q) with low signal for markers in exons 7-9. RHD exon 7 sequencing showed RHCE sequences in exon 7. RHD cDNA analysis showed that exons 7-9 were derived from RHCE. This allele is most similar to RHD*04.05 (DIV type 5). With this genotype information, the blood bank considers her D-negative for purposes of transfusion and a candidate for RhIgG prophylaxis. Conclusion: There is much genetic exchange between RHD and RHCE genes. The two cases described involve novel RH hybrid genes detected by initial use of RHD and RHCE BeadChip arrays and by follow-up of unexpected genotype results with cDNA sequence analysis. Both cases were women of child-bearing age, and, in Case 1, there is an association with failed pregnancies. Pregnant women with anti-D or D typing discrepancies can benefit from RHD genotyping to determine their risk of alloimmunization and their candidacy for RhIg prophylaxis. Disclosure of Commercial Conflict of Interest C. Carson: Nothing to disclose; J. Christensen: Nothing to disclose; J. A. Crowley: No Answer; T. Horn: No Answer; M. A. Keller: Nothing to disclose; J. Plafker: Nothing to disclose Disclosure of Grants Conflict of Interest C. Carson: Nothing to disclose; J. Christensen: Nothing to disclose; J. A. Crowley: No Answer; T. Horn: No Answer; M. A. Keller: BIoArray Solutions LLC, Grants or Research Support; J. Plafker: Nothing to disclose SP240 RH Variants in Patients with Sickle Cell Disease (SCD) Associated with Clinically Significant Antibodies L Castilho1, A C Gaspardi1, E Sippert1, J Pellegrino1, S Gilli1. 1 Hemocentro, Unicamp, Campinas, Brazil Background/Case Studies: RH genotyping has revealed that many patients with sickle cell disease (SCD) carry alleles encoding partial Rh antigens, but little clinical or biological evidence related to alloimmunization and hemolytic transfusion reaction is presented for all of the RH variant alleles. The identification of alloantibodies in patients with SCD carrying RH variants is important, and a complete characterization of the Rh variants producing alloantibodies is required, because alloantibodies in those patients may be confused with autoantibodies. In this study, we characterized variant RH alleles in patients with SCD who made antibodies to Rh antigens despite antigen-positive status and we evaluated the clinical

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significance of the antibodies produced. Study Design/Methods: We studied DNA samples from 18 African Brazilian patients with SCD (homozygous for hemoglobin S) with multiple transfusions, who were receiving RBC units that had been antigen-matched for RhD, C, E, c, e and K and that made one or more antibodies with Rh specificities. RHD and RHCE BeadChip from BioArray Solutions (Warren, NJ, USA) and/or amplification and sequencing of exons were used to identify the RH variants. Serologic features of all Rh antibodies in antigen-positive patients were analyzed, and the clinical significance of the antibodies was evaluated by retrospective analysis of the hemoglobin (Hb) levels before and after transfusion and determined by changes from the patient’s baseline values on pretransfusion Hb and the percentage (%) of HbS levels at the time of antibody detection. Results/ Findings: RHD variants were found in 4/18 patients, RHCE variants in 5/18 and RHD, and RHCE allele combinations in 4/18 patients. The Rh antibodies associated with altered D, C, and e antigens in 13 patients due to homozygous or compound-heterozygous RHD and RHCE variant alleles were shown to be clinically significant, as verified by a worsened anemia and/or rise in HbS when the patients were transfused with the corresponding antigens. Conclusion: Reports such as this one on the clinical significance of antibodies produced in individuals carrying Rh variants support the development of strategies to match RH to avoid Rh alloimmunization and the risk of hemolytic transfusion reactions and/or poor transfusion outcomes. Disclosure of Commercial Conflict of Interest L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; S. Gilli: Nothing to disclose; J. Pellegrino: Nothing to disclose; E. Sippert: Nothing to disclose Disclosure of Grants Conflict of Interest L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; S. Gilli: Nothing to disclose; J. Pellegrino: Nothing to disclose; E. Sippert: Nothing to disclose

SP241 Proposal to Shorten Molecular Investigation by using Serology to Identify RhD Variants C Arnoni1, J G Muniz1, R D Person1, T A de Paula1, D Gazito1, L Castilho2, F Latini1. 1Colsan, São Paulo, Brazil; 2Unicamp, Campinas, Brazil Background/Case Studies: RhD variant detection in blood donors and patients is a problem frequently encountered during routine donor and patient typing. The distinction between partial D and weak D is important for optimized management of D-negative RBC units and to prevent anti-D hemolytic disease of the fetus and newborn. Currently, RHD genotyping is shown to be an excellent tool to distinguish weak D and partial D; however, this investigation may be expensive and complex. Accordingly, in order to shorten and reduce the cost of the molecular RHD investigation, we determined a correlation among the score results obtained in the D typing performed in Immucor NEO® instrument, the associated RH haplotype, and the RhD variants classified by molecular analysis. Study Design/Methods: A total of 70 samples with different scores of agglutination in the D typing performed in Immucor NEO® using an anti-D IgM (RUM-1) and a blend (D175+D415) were selected for this study. All samples were analyzed by molecular assays using PCR-RFLP, Multiplex-PCR, and sequencing to identify possible D variants. Results/Findings: The results are summarized in the Table. Score results are expressed in median ± standard deviation. We observed that variants from the same cluster, such as DAR, weak D type 4.0, and weak D type 4.2.2, presented similar median score results. In addition, variants associated with Ce present a greater variation between score values. Conclusion: Our results suggest that molecular investigation could be driven by the haplotype association and score results obtained in NEO®. The use of this strategy can reduce the time and cost of molecular analysis. Disclosure of Commercial Conflict of Interest C. Arnoni: Nothing to disclose; L. Castilho: No Answer; T. A. de Paula: Nothing to disclose; D. Gazito: No Answer; F. Latini: No Answer; J. G. Muniz: Nothing to disclose; R. D. Person: Nothing to disclose Disclosure of Grants Conflict of Interest C. Arnoni: Nothing to disclose; L. Castilho: No Answer; T. A. de Paula: Nothing to disclose; D. Gazito: No Answer; F. Latini: No Answer; J. G. Muniz: Nothing to disclose; R. D. Person: Nothing to disclose

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TABLE. Score results of D variants and haplotype association Associated haplotype cDe

CDe

cDE

D variants (N) Weak D type Weak D type DAR (3) DMH (2) DIIIa (1) DAU2 (1) DOL2 (1) Weak D type Weak D type Weak D type Weak D type DIIIc (1) Weak D type

4.0 (7) 4.2.2 (28)

1 (4) 3 (11) 11 (1) 38 (5) 2 (5)

IgM

Blend

87 ± 4.8 71 ± 9 81 ± 4.7 86 ± 3.5 22 92 92 48 ± 15.6 75 ± 6.7 7 10 ± 1.4 35 15 ± 1.2

87 ± 5.7 76 ± 11.1 83 ± 1.7 78 ± 10.6 27 95 94 55 ± 10.8 83 ± 5.6 7 10 ± 2.8 65 22 ± 3.3

SP242 Evaluation of ID-Core XT, a New Molecular System for the Identification of Red Blood Cell Antigens by using Luminex Technology E Sippert1, A C Gaspardi1, L Castilho1. 1Hemocentro, Unicamp, Campinas, Brazil Background/Case Studies: The development of high-throughput red blood cell genotyping platforms, which combines both DNA analysis and complex bio-informatic software, offers the opportunity to perform large-scale testing on numerous antigens simultaneous, thus allowing an accurate selection of donor units to facilitate matching of donor RBCs to recipient blood type. Commercial kits are becoming available, and their accuracy must be evaluated in populations of different ethnic backgrounds to avoid false-positive or false-negative results due to the presence of unrecognized alleles. ID-Core XT is a recently developed molecular assay that analyzes 29 SNPs determining 37 blood group antigens. Study Design/Methods: A total of 229 Brazilian donor and patient DNA samples in a concentration range varying between 20 ng and 100 ng, previously genotyped by laboratory developed tests (LDT) or HEA BeadChip™ (Bioarray, Immucor), were tested in the ID-Core XT (Progenika, Grifols, Spain) that uses purified human genomic DNA to type blood group allelic variants with the Luminex® xMAP technology. Each probe is designed to hybridize to a complementary region that specifies a SNP and that is attached to a unique colored Luminex microsphere. The Luminex instrument quantifies the relative amounts of labeled PCR product hybridizing to each Luminex microsphere, and the analysis software (BIDS-XT) automatically imports data from the Luminex instrument. The comparison between the methods was performed by using the Chi-square test or Fisher’s exact test, when appropriate, using a 2 × 2 contingency table. Discrepancies found were to be solved by using phenotyping, genotyping, and sequencing on a new collected sample. Results/Findings: A reproducibility of 100%, a repetition rate of 0%, and a 1.7% incidence of no call or invalid was observed. Results obtained by ID-Core XT analysis agreed with those obtained by previous genotyping of 219 (95.6%) samples. Four samples presented discrepancies for VS and V between ID-Core XT and HEA BeadChip™; one sample was discrepant for K between ID-Core XT and LDT; one sample for Jsa, Fyb, and M was concordant with LDT but discordant with HEA BeadChip™; and four samples are suspected of new variants. All discrepant samples are under investigation. Conclusion: This new molecular assay is easy to perform, uses a low concentration of DNA, and has powerful software for interpreting the genotype results and for predicting the phenotypes, which allows traceability of the results. The reproducibility, the performance, and the high level of concordance provided by this new Luminex-based genotyping test in combination with other existing technologies demonstrate that this assay can be effectively applied to populations with a highly diverse genetic background, and that it has enormous potential to be used for large-scale RBC genotyping. Disclosure of Commercial Conflict of Interest L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; E. Sippert: Nothing to disclose Disclosure of Grants Conflict of Interest L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; E.Sippert: Nothing to disclose

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SP243 Genotyping with ID-Core+: Follow-up after 2 Years in Clinical Routine L Gustafsson1, A I Johnsson1, L Hannu1, A T Wikman1. 1Clinical Immunology and Transfusion Medicin, Karolinska University hospital, Stockholm, Sweden Background/Case Studies: Our recommendations for genotyping of blood group antigens are: multitransfused patients due to difficulties with serologic typing, in the investigation of antibodies, test cell donors to improve the antibody identification diagnostics, to provide prophylactic antigen-matched blood in chronic transfusions. The first aim of the study was to evaluate how the recommendations for genotyping were implemented and in what diagnoses of the patients they were implemented. The second aim of the study was evaluate the results of genotyping compared to phenotyping. Study Design/Methods: Whole-blood samples from 141 patients previously phenotyped to various extents were analyzed according to a request or assessment by the laboratory. In addition, 22 samples from test cell donors phenotyped for Rh (CcEe), Kell, Duffy, and Kidd were genotyped. The samples were stored at −30oC until DNA preparation, which was done by utilizing the MagNA Pure LC instrument (Roche); DNA concentration was adjusted to 25 μg/mL. The genomic typing was done by using ID-Core+ (PROGENIKA Espana, Derio), an identification panel for 23 red blood cell (RBC) antigens analyzed through Luminex xMAP® technology. Results/ Findings: Sixty-nine females and 72 males with the following diagnoses were genotyped: thalassemia major, 25; sickle-cell disease, 24; AIHA/direct antiglobulin (DAT)-positive, 9; spherocytosis, 4; and request for blood transfusion with unknown diagnosis, 5. Other indications were aplastic anemia, Wiscott Aldrich syndrome, hematopoietic stem cell transplantation, and unidentified antibodies. In 14/141 patient samples, at least one discrepant result was found (C,c, E, Fya Fyb, Jkb, and k). Twelve of these patients were multitransfused. One had no previous blood transfusions. In one case, the genotyping corresponded with phenotyping in 2/3 repeated analyses. In 40/141 samples, the patients were DAT-positive, and complete phenotyping could not be performed with indirect antiglobulin technique (IAT). Ten of 141 patients were phenotyped Fy(a−b−) but were genotyped FYGATA FYB/FYB. Seven had sickle cell anemia, and three were pregnant. Two of 141 phenotyped Fy(a−b−) were genotyped FYA/FYA. Conclusion: The majority of genotyped patients had chronic hematological diagnoses following our local recommendations. In 10%, the genotype was different from a previous phenotype. In most cases, the discrepancies were in the Rh system (C,E) and Duffy. The majority of discrepancies were due to previous blood transfusions. Genotyping improves our ability to provide antigen-matched red blood cell transfusions for DAT-positive, Duffy-negative FYGATA, and multitransfused patients. The effects of genotyping and RBC matching on transfusion intervals and frequency of antibody investigations and antibodies detected remain to be studied. Disclosure of Commercial Conflict of Interest L. Gustafsson: Nothing to disclose; L. Hannu: No Answer; A. I. Johnsson: No Answer; A. T. Wikman: Nothing to disclose Disclosure of Grants Conflict of Interest L. Gustafsson: Nothing to disclose; L. Hannu: No Answer; A. I. Johnsson: No Answer; A. T. Wikman: Nothing to disclose SP244 Evaluation of the OpenArray® Real-Time PCR Platform for RBC and Platelet Genotyping J V Bianchi1, E C Sabino1, C L Dinardo2, V Niewiadonski1, A Mendrone2, M A Mota3. 1University of Sao Paulo, Sao PAulo, Brazil; 2 Imunohematology, Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil; 3Imunohematology, Hospital Israelita Albert Einsten, São Paulo, Brazil Background/Case Studies: Red blood cell (RBC) DNA typing is especially necessary in the care of chronically transfused patients, mainly those with sickle cell disease (SCD). Recently, a new microarray platform (OpenArray® Real-Time PCR) that allows the development of customized assays was launched by Applied Biosystems. The objective of this study was to evaluate the accuracy of one customized platform in genotyping RBC and platelet (PLT) antigens. Study Design/Methods: We have chosen 31 SNPs encoding 25 RBC antigens (S > s; Lua > Lub; K > k; Kpa > Kpb; Jsa > Jsb; Fya > Fyb; Fy null; Fyx; Jka > Jkb; Dia > Dib; Wra > Wrb; Yta > Ytb; Doa > Dob; Hy; Joa; Coa > Cob; Kx; Tca > Tcb; Dra; Cra; Kna > Knb; McCa > McCa; Sla > Vil; JMH) and 6 PLT antigens (HPA-1; HPA-2; HPA-3; HPA-4; HPA-5; HPA-15). A total of 68 previously genotyped controls (HEA— Immucor®) were evaluated using OpenArray® for RBC antigens and 51

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previously genotyped controls (HEA—Immucor®) were evaluated for PLT antigens. In parallel, 242 random-donor samples were simultaneously tested by using both the Openarray® method and the Luminex ID Core-XT and ID Core-HPA method. DNA was extracted by using the Qiagen DNA kit and quantified by using a spectrophotometer (Nanodrop, Thermo Scientific, EUA). DNA concentration was set for 50 ng/μl. Results/Findings: Among 10,164 tested alleles (5082 SNPs), 22 alleles showed discrepant results, which represented 99.8% agreement in the results. In comparison to control samples, the discrepant results identified concerned the alleles GPB*s, JK*A, DO*A, and HPA-15 at a rate of 3.8%, 3.4%, 1.5% and 2.1%, respectively. Ten samples were tested in triplicate in different plates, with 100% of accuracy. Conclusion: Our results suggest that the OpenArray® platform is suitable for genotyping the most transfusion-relevant RBC and PLT antigens. This platform can be used in the screening of rare blood donors and in preventing alloimmunization. Considering its low cost, OpenArray® represents a very cost-efficient tool in molecular immunohematology. Disclosure of Commercial Conflict of Interest J. V. Bianchi: No Answer; C. L. Dinardo: Nothing to disclose; A. Mendrone: Nothing to disclose; M. A. Mota: Nothing to disclose; V. Niewiadonski: No Answer; E. C. Sabino: Nothing to disclose Disclosure of Grants Conflict of Interest J. V. Bianchi: No Answer; C. L. Dinardo: Nothing to disclose; A. Mendrone: Nothing to disclose; M. A. Mota: Nothing to disclose; V. Niewiadonski: No Answer; E. C. Sabino: Nothing to disclose SP245 Molecular Genotyping and Modification of Pretransfusion Testing Will Decrease Cost and Turnaround Time (TAT) for Patients Requiring Complex Antibody Workups W S Crews2, B Parsons2, L J Sutor2,1, P Wynn2. 1University of Texas Southwestern Medical Center, Dallas, TX, United States; 2Reference and Transfusion, Carter BloodCare, Bedford, TX, United States Background/Case Studies: Molecular genotyping (MG) is used as an aid for antibody identification and investigation of discrepant results and as an alternative to serologic typing for determining antigen-negative requirements. Genotyping can be particularly helpful when a patient has a positive direct antiglobulin test (DAT) and cannot be phenotyped; if a patient has been recently transfused and needs an adsorption; in the case of a sensitized sickle cell disease patient; or if a patient has an autoantibody or multiple alloantibodies. Molecular genotyping also alleviates the manpower it takes to perform elutions, adsorptions, and other labor-intensive serological testing. Study Design/Methods: MG has been available at our blood center since 2008. From January 2012 to January 2014, we evaluated patients who could benefit from a modified pretransfusion workup. A molecular genotype, DAT, and ABO type were included in the modified workup. All patients had warm autoimmune hemolytic anemia with underlying alloantibodies. Each patient underwent initial serological workups that included antibody screens, positive DAT, elution, and adsorption. Each patient then had MG performed by using ID CORE+ (Progenika Biopharma, Derio, Spain). If the patient required transfusion on subsequent admissions, an ABO type and a DAT were performed; if there was no change from previous results or strength of reaction, no further serological testing was performed, and antigen-matched units were issued in lieu of performing a crossmatch. Results/Findings: The findings for 10 patients were evaluated. All 10 patients had a significant decrease in TAT and total savings. The TAT for initial complete serological workup before MG was performed ranged from 4 to 18 h. The average decrease in TAT was 6.1 h, and the average total savings were $7,900. No transfusion reactions were reported on any of the transfused antigen-matched RBC units. See the Table for a summary of the results. Conclusion: Including MG in pretransfusion testing of patients who require complex workups can complement serological testing and safely allow the omission of expensive and labor-intensive serological testing on subsequent hospital admissions This process will help reduce the costs and turnaround time of blood product availability. Disclosure of Commercial Conflict of Interest W. S. Crews: Nothing to disclose; B. Parsons: Nothing to disclose; L. J. Sutor: Nothing to disclose; P. Wynn: Nothing to disclose Disclosure of Grants Conflict of Interest W. S. Crews: Nothing to disclose; B. Parsons: Nothing to disclose; L. J. Sutor: Nothing to disclose; P. Wynn: Nothing to disclose

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TABLE. Patient 1 2 3 4 5 6 7 8 9 10

Cost of serological workup ($)

TAT w/o modified workup (h)

TAT with modified workup (h)

Subsequent Hospital Admissions

Ag-Matched RBC units transfused

Total savings using modified workup ($)

900 1,100 1,200 1,100 1,400 1,100 1,100 1,200 1,200 1,400

4 6 9 6 8 6 12 9 7 18

1 2 2 2 3 3 2 3 2 4

4 6 16 3 3 3 14 5 2 11

9 14 35 6 6 7 33 11 5 24

3,600 6,600 19,200 3,300 4,200 3,300 15,400 6,000 2,400 15,400

SP246 A Pair of cis-linked Single-Nucleotide Polymorphisms of GYPB Leading to S-allele Silencing R Lapadat2, E A Fadeyi1, J Gottschall3, G A Denomme3. 1Wake Forest School of Medicine, Winston-Salem, NC, United States; 2BloodCenter of Wisconsin, Milwaukee, WI, United States; 3Diagnostic Laboratories, BloodCenter of Wisconsin, Milwaukee, WI, United States Background/Case Studies: Glycophorin B (GYPB) is a type I transmembrane protein on RBCs that expresses the S/s antigens of the MNS blood group system. The S/s antigens are encoded by mutually exclusive alleles. Three common phenotypes are described: S+S+, S+s+, and s+s+. The absence of S/s antigens on RBCs is associated either with the loss of the high-frequency U antigen or with the weakened expression of U (Uw+). The nucleotide changes responsible for these phenotypes are rare, but higher frequencies have been reported in African Americans (1%) and West Africans (37%). We describe an unusual GYPB*03 allele found in an African American female, characterized by two S-linked SNPs that silence the expression of the S antigen. Study Design/Methods: Blood was obtained from the proband. RBC phenotypes were established by using standard tube agglutination. DNA was extracted from whole blood by using the QIASymphony workstation. Red cell genotyping was performed by using genomic DNA and PCR with fluorogenic hydrolysis probes. PCR amplification of GYPB exon 5 and the flanking regions was performed with sequencespecific primers (PCR-SSP). The amplicon was sequenced in forward and reverse directions by using the Big Dye terminator sequencing strategy. Results/Findings: The proband had an S-s+ phenotype. Genomic DNA analysis revealed a GYPB*03/GYPB*04 genotype, with GYPB*03N [C230T] and GYPB*03N [IVS5+5t] SNPs identified by genotyping and confirmed by sequence analysis. The C230T mutation causes partial skipping of exon 5, due to the activation of a cryptic 3′ site that results from a C > G transversion at position nt230. The IVS5+5t mutation results from a G > T point mutation at position +5 of intron 5, which leads to skipping of the entire exon and loss of the transmembrane domain of GYPB. Both mutations are S-linked silencing polymorphisms. A combination of these two polymorphisms with an S-s+ phenotype and GYPB*03/GYPB*04 genotype has not been reported. Conclusion: Our molecular results indicate that the GYPB*03N [230T] and GYPB*03N [IVS5+5t] SNPs are linked in cis to the GYPB*03 allele. The observation that the patient’s RBCs had an S-s+ phenotype confirms the cis arrangement of the SNPs. Disclosure of Commercial Conflict of Interest G. A. Denomme: Nothing to disclose; E. A. Fadeyi: No Answer; J. Gottschall: Nothing to disclose; R. Lapadat: Nothing to disclose Disclosure of Grants Conflict of Interest G. A. Denomme: Nothing to disclose; E. A. Fadeyi: No Answer; J. Gottschall: Nothing to disclose; R. Lapadat: Nothing to disclose SP247 Applying Molecular Assays to Enhance Immunohematology and Guide Selection of Antigen-Matched Red Blood Cell (RBC) Components for Transfusion A C Gaspardi1, E Sippert1, C Carvalho1, J Pellegrino1, L Castilho1. 1 Hemocentro, Unicamp, Campinas, Brazil Background/Case Studies: Molecular testing is a rapidly advancing field in transfusion medicine, and it is proving to be a powerful adjunct tool to serology, with potential advantages for identifying Rh variants and rare blood and for finding antigen matches for chronically transfused patients. Genetic

analysis of blood group variants within a given population increases the accuracy of prediction of red cell phenotype by genotype determination. Given that genotyping can convey much more information about the expression of some complex antigens, studies are important to show that phenotyped transfusions based on genotyping results are superior. Study Design/Methods: We performed molecular analysis in 148 transfused patient samples with invalid or discrepant results in serologic tests, and we evaluated the use of DNA testing to guide selection of compatible donor units to prevent alloimmunization and delayed hemolytic transfusion reaction (DHTR). Genomic DNA was prepared from samples obtained from patients’ submitted blood group genotype. Genotyping was performed by using laboratory-developed tests (LDTs), the HEA BeadChip, the RHD BeadChip, and the RHCE BeadChip (Bioarray, Immucor). Results/Findings: Of the cohort, 105 patients required extended genotyping, and 43 required RHD and RHCE genotyping. Of this latter group, six patients had produced anti-D that included 2 RHD*DVI and 4 RHD*DAR; three had produced anti-C that included RHD*DIIIa-CE(4-7)-D hybrid alleles; seven had produced anti-e that included 5 RHCE*ceS, 1 RHCE*ceAR, and 1 RHCE*ceMO; and one patient had produced anti-hrS and genotype homozygous RHCE*ceAR. All patients were transfused with compatible donor units based on the genotype results, and they benefited from the received transfusions, as shown by good in vivo survival. The patients were followed by 1 y, and no other alloantibodies were detected. Conclusion: This study provides evidence that the information gained from molecular typing of transfused patients improves the ability to find highly antigen-matched RBC components for transfusion support. This step decreases the risk of DHTR and prevents alloimmunization. Disclosure of Commercial Conflict of Interest C. Carvalho: No Answer; L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; J. Pellegrino: Nothing to disclose; E. Sippert: Nothing to disclose Disclosure of Grants Conflict of Interest C. Carvalho: No Answer; L. Castilho: Nothing to disclose; A. C. Gaspardi: Nothing to disclose; J. Pellegrino: Nothing to disclose; E. Sippert: Nothing to disclose SP248 The Ongoing Need for Rare Blood Donors C Flickinger1, G Meny2, S J Nance1. 1American Red Cross, Philadelphia, PA, United States; 2UT Health Science Center at San Antonio, San Antonio, TX, United States Background/Case Studies: A large national rare-donor program has provided rare units for patients since 1998. Rare donors, per program definition, are those whose red cells lack a high-incidence antigen (HIA) or lack multiple common antigens (MCA). Program members screen and submit identified rare donors to the national database. Dwindling sources of antisera to HIA and the variant nature of certain antigens have made donor screening a challenge. The commercial availability of a medium-throughput genotyping array since 2007 has provided an efficient means of screening donor samples for MCA and HIA. Lab-developed tests and a recently released set of RH genotyping arrays have been used to identify rare hrB- and other e variant donors. Although molecular testing has improved the program’s fill rates, there are unfilled requests. This report assesses the frequency and phenotypes of unfilled requests. Study Design/Methods: A retrospective analysis of this program’s requests and fill rates for red cell requests in 2013 was performed. Results/Findings: Nine hundred twelve requests for rare red cell units were received in 2013; 403 were for units lacking MCA,and 509

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were for units lacking an HIA. Eleven of 403 MCA requests (2.7%) and 52 of 509 HIA requests (10.2%) were unfilled (no units provided); this represents an overall unfilled rate of 6.9%, as compared with unfilled rates of 7.2%, 6.4%, 5.1%, 4.6%, and 5.5% for 2008 to 2012, respectively. The Table shows the number and phenotypes of the unfilled requests in 2013. Conclusion: The use of molecular testing to identify rare donors has positively affected fill rates. However, some requests still cannot be filled, and each one of them represents a patient need. In addition, HIA requests can be complicated by ABO/Rh and/or the need for units lacking MCA. Filling these requests will require continued donor genotyping efforts to identify Vel-, At(a−), Lan-, Yt(a−), Jr(a−), Ge:-2, and Sc:-3 donors. The recent characterization of the genetic variation responsible for the Vel- and Jr- phenotypes should facilitate

these screening efforts. Equally important as identifying rare donors is increasing the donation rate of this population to ensure adequate availability of donor units. Disclosure of Commercial Conflict of Interest C. Flickinger: Nothing to disclose; G. Meny: Nothing to disclose; S. J. Nance: Nothing to disclose Disclosure of Grants Conflict of Interest C. Flickinger: Nothing to disclose; G. Meny: Nothing to disclose; S. J. Nance: Nothing to disclose

TABLE. Number and Phenotype of Unfilled Requests in 2013 Multiple Common Antigen-Negative

High-Incidence Antigen- Negative

#

Phenotype

#

Phenotype

#

Phenotype

1 1 1 1 1 1 1 1 1 1 1

rr, K-Fy(a-b-)Jk(b-)M-S-Vrr, K-Fy(a-)Jk(b-)S-V-Js(a-) rr, K-Fy(a-b-)Jk(b-)S-V-Js(a-) rr, K-Fy(a-b-)s-Js(a-) R2, K-Fy(a-)Jk(b-)M-SR2, K-Fy(a-b-)Jk(b-)SR2, K-Fy(a-)Jk(a-)S-Js(a-) R0, K-Fy(a-)Jk(b-)S-Js(a-) CMV neg R0, K-Fy(a-b-)Jk(b-)S-VS-Js(a-)Go(a-) R0, K-Fy(a-b-)Jk(b-)S-Le(a-)Js(a-) R1, K-Fy(a-b-)Jk(b-)S-V-VS-Kp(a-)Js(a-)Do(a-)

13 (4*,7†) 5 (2†) 4 (3†) 4 (1†) 4 (3†) 4 (2†) 3 (2†) 2 (1*) 2 (1*,2†)

UIE- hrBJk:-3 Velr’r’ At(a-) LanYt(a-)

2 (1*,2†) 2 (1*,1†) 1 (1†) 1 1 (1*) 1 (1*,1†) 1 1 (1*) 1

Jo(a-) Lu(a-b-) Js(b-) D-Di(b-) Kp(b-) Jr(a-) Ge:-2 Sc:-3

* D-negative required; † MCA required.

SP249 Hemo ID: A Robust and Flexible Panel for Blood Group Genotyping M Sykes1, S Keng1, M Shaw1, P Hunt1, B Patel1, C Vollmert1, A Nygren1. 1 Sequenom, San Diego, CA, United States

tion for molecular blood group genotyping. It offers high throughput, assays a wide range of antigens, and demonstrates both excellent reproducibility between analyses and excellent concordance with established methods.

Background/Case Studies: Immunohematology labs have access to a range of systems for blood group genotyping. Each system offers distinct benefits, such as the range of blood groups interrogated, precision, robustness, throughput, and cost. Here we describe the performance characteristics of a newly available research-use-only blood group-genotyping panel called Hemo ID. The Hemo ID panel consists of six independent modules, each containing a distinct set of assays that are used to interrogate 108 single-nucleotide variations and INDELs in the human genome. The six modules consist of 1) Kell, Kidd, Duffy; 2) MNS; 3) Rare Blood Groups; 4) RHD/CE Broad; 5) RHD Variant; and 6) HPA & HNA. The modular setup provides a flexible high-throughput solution that can easily adapt to newly discovered blood groups. In this work, we assessed the performance of the Hemo ID Panel in three ways: a feasibility study using an ethnically diverse set of samples, a reproducibility study involving the same set of samples analyzed at three different sites, and a challenge study using samples previously typed with an orthogonal technology (HEA BeadChip) and serology. Study Design/Methods: For the feasibility and reproducibility studies, 743 and 47 HapMap DNA samples,respectively, were used. For the challenge study, 22 samples were obtained from a regional blood donor center. These were previously typed by using both serotyping and the HEA BeadChip. For Hemo ID analysis, all samples were amplified by multiplex PCR, which was followed by single base extension and analysis by MALDITOF mass spectrometry. Hemo ID software was used to assign genotypes, predict phenotypes, and generate reports. For the reproducibility study, this process was repeated at three separate sites. Discordances between previously determined phenotypes and those predicted here were resolved by targeted sequencing. Results/Findings: Feasibility Study: We observed over 200 different predicted phenotypes across all six modules, including several rare phenotypes observed in A) associated with Bombay phenotype. This mutation creates a stop codon. Mutations detected in the FUT2 gene [461G > A (W154X) and 772G > A (G258S)] were evaluated as a polymorphism, due to the high allele frequencies observed inTurkey. However, one of these mutations (W154X) causes a “non-secretor” phenotype. The daughter of the proband was also identified with Bombay phenotype,

Red Blood Cells: Traditional Immunohematology SP254 Development of a Robust Method to Negate the Daratumumab Interference with Routine Blood Bank Testing C I Chapuy1, R T Nicholson1, M D Aguad1, J P Laubach2, B Chapuy4, P Doshi3, R M Kaufman1. 1Pathology, Brigham and Women’s Hospital, Boston, MA, United States; 2Hematologic Oncology, Dana-Farber Cancer Institute, Boston, MA, United States; 3Janssen, Inc., Spring House, PA, United States; 4Medicine, Dana-Farber Cancer Institute, Boston, MA, United States Background/Case Studies: Daratumumab (DARA), a promising novel therapy for multiple myeloma, is an IgG1 human monoclonal antibody (Ab) that recognizes CD38 on myeloma cells. On routine screening, we observed that five of five DARA-treated patients had a positive Ab screen and a plasma panagglutinin in solid-phase and tube testing. Because CD38 is expressed on red blood cells (RBCs), we hypothesized that the observed panreactivity was caused by binding of DARA to endogenous CD38 on reagent RBCs. We explored methods to negate the DARA interference by removing RBC surface CD38. Study Design/Methods: HL60 cells were stably transfected with a plasmid encoding human CD38. CD38negative HL60 cells were used as controls. Binding of DARA to HL60 cells or RBCs was assessed by flow cytometry using PE-labeled anti-human globulin. To remove cell surface CD38, HL60 cells or RBCs were preincubated with dithiothreitol (DTT) or trypsin. Routine blood bank serological (tube) methods were used to test samples from DARA-treated patients, as well as normal plasma samples spiked with DARA and/or alloAb (e.g., anti-E). For serological studies of DTT, reagent RBCs (3-5% cell suspension in PBS) were incubated with 0.2M DTT at 37oC for 30 min. Results/Findings: Normal plasma samples spiked with DARA (0.1-10 μg/ mL) and incubated with reagent RBCs recapitulated the interference observed with samples from DARA-treated patients. Flow cytometry confirmed DARA binding to RBCs and CD38+ HL60 transfectants, but not to CD38− controls. Eluates prepared from DARA-treated RBCs bound to CD38+ HL60 cells but not to CD38− controls. Preincubating CD38+ HL60 cells with DTT reduced DARA binding by 92%, while preincubating these cells with trypsin reduced DARA binding by 40%. Using DTT-treated RBCs allowed clinically significant Abs to be identified serologically in the presence of DARA (Table). The panreactivity of all DARA-treated patient samples was eliminated by using DTT-treated RBCs. Conclusion: DARA potently interferes with routine blood bank serological tests by directly binding to RBC CD38. DTT pretreatment of reagent RBCs is a robust method to negate the DARA interference, allowing the safe provision of RBC units to DARA-treated patients. Because DTT denatures Kell antigens, K- RBC units are provided to these patients. Disclosure of Commercial Conflict of Interest M. D. Aguad: No Answer; B. Chapuy: No Answer; C. I. Chapuy: No Answer; P. Doshi: No Answer; R. M. Kaufman: Janssen Pharmaceuticals, Other; J. P. Laubach: No Answer; R. T. Nicholson: No Answer Disclosure of Grants Conflict of Interest M. D. Aguad: No Answer; B. Chapuy: No Answer; C. I. Chapuy: No Answer; P. Doshi: No Answer; R. M. Kaufman: Nothing to disclose; J. P. Laubach: No Answer; R. T. Nicholson: No Answer

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TABLE. Representative experiment using DTT-treated RBCs to allow alloantibody detection in the presence of daratumumab Sample 1 2

Daratumumab

Plasma

Enhancement

Ab Screen Untreated RBCs

Ab Screen DTT-treated RBCs

DTT-treated RBC Panel Result

+ +

− AlloAb + AlloAb

Polyethylene glycol Polyethylene glycol

R1R1 1+ R2R2 1+ R1R1 1+ R2R2 1+

R1R1 0 R2R2 0 R1R1 0 R2R2 1+

Negative Anti-E

SP255 Total Loss of the A Antigen in Acute Myeloid Leukemia J Li1, J Sweeney1. 1Miriam Hospital, Providence, RI, United States Background/Case Studies: A decrease in the strength of the A and B antigens on red cells has previously been described in carcinomas and hematologic malignancies, especially leukemias. This decrease in strength is mostly seen as a poor prognostic marker, and it reflects decreased translation of the A or B glycosyltransferases in late-developing normoblasts. This weakening is usually not observed in routine front-typing, but, when observed, it appears as the failure of a subpopulation of red cells to agglutinate with either anti-A or anti-B, similar to a dual population. However, complete absence of the A or B antigen is very uncommon, and that raises issues regarding the ABO choice of blood components. We report a case of a Group A patient with acute myeloid ldeukemia who front-typed as Group O and back-typed as Group A when the leukemia was present but reverted to an A front-type when the disease went into remission. Study Design/ Methods: Serological testing was performed by using standard bank methods. An automated blood typing device was used (ProVue, OCD, Raritan, NJ) with column-agglutinating (gel) reagents. ABO genotyping was performed by polymerase chain reaction (Progenika, Cambridge, MA). Results/Findings: The patient was a 59-y-old male of Cambodian origin with a diagnosis of AML with monocytosis and eosinophilia. The WCC was 28 × 109/L with 39% blasts; hemoglobin 10.4 g/dL, and platelet count 512) and thermal amplitude of ≥30°C. Typical titer results in CAD are, for example, 37°C = 0, 30°C = 1, room temperature (RT) = 32, and 4°C = 1024. CAD frequently is not clinically severe. We report a 65-y-old man with lymphoma who presented with hemoglobin (Hb) of 7.6 g/dL. A prewarm (PW) antibody (ab) screen was negative, and 2 units were transfused. Three weeks later (day 1), the patient’s Hb was 5.5 g/dL. His direct antiglobulin test was positive for C3 only (3+), and his plasma contained a strong (4+) agglutinin reacting at RTand 37°C and by indirect antiglobulin test [PW ab screen = 1+]. Cold adsorptions (x3) removed the reactivity; no underlying alloantibodies were detected. He received prednisone (day 1), IVIG (days 1-5), and 3 RBC units (days 3-4). Further transfusion of 2 units on days 11 and 12 resulted in transfusion reactions with signs of intravascular hemolysis (LDH 1841, hemoglobinemia, hemoglobinuria). RBCs from both implicated units were compatible with coldadsorbed posttransfusion sera. Hemolysis was felt to be related to the cold autoantibody and not to the transfusions. On day 15, the patient’s Hb was 2.9 g/dL. The patient was treated with eculizumab (terminal complement inhibitor) and

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was transfused, with no further hemolytic transfusion reactions. Study Design/Methods: An autoimmune hemolytic anemia (AIHA) serum screen was performed with the patient’s day 2 serum, ±acidification and fresh normal sera (source of complement), against untreated (UT) andd ficintreated (FT) RBCs (10% v/v) at 20°C and 37°C (PW). The agglutinin titer and thermal amplitude were determined by incubating doubling dilutions of the patient’s days 2 and 10 sera (in saline) with test RBCs at 37°C (PW), 30°C (PW), RT, and 4°C. Results/Findings: In the AIHA serum screen, the patient’s serum (acidified, with added fresh complement) hemolyzed UT and FT RBCs at 20°C and 37°C. Due to in vivo hemoglobin in the patient’s serum, in vitro hemolysis was judged by changes in RBC button sizes. Titers with adult I RBCs were 37°C = 128, 30°C = 1024, 22°C = 16,000, and 4°C = 16,000. Titers with the patient’s “autologous” RBCs were weaker: 37°C = 0, 30°C = 1, 22°C = 64, and 4°C = 1024, possibly due to blocking by RBC-bound C3. ZZAP (dithiothreitol + enzyme, e.g., ficin) removes complement in addition to IgG from coated RBCs. The patient’s ZZAP-treated RBCs reacted 4+ with the his serum at 37°C PW. Conclusion: This patient had a high-thermal-amplitude cold agglutinin with unusually high titers. It is very unusual to see such strong reactions (agglutination and hemolysis) at 37°C or 30°C. It is also unusual for patients with CAD to have intravascular hemolysis. Treatment with a monoclonal antibody to complement (eculizumab) used for patients with paroxysmal nocturnal hemoglobinemia was helpful in this unusual case. Disclosure of Commercial Conflict of Interest P. A. Arndt: Nothing to disclose; G. Cecchi: No Answer; A. Shaieb: Nothing to disclose; S. A. Wise: Nothing to disclose Disclosure of Grants Conflict of Interest P. A. Arndt: Nothing to disclose; G. Cecchi: No Answer; A. Shaieb: Nothing to disclose; S. A. Wise: Nothing to disclose

SP258 Detection and Quantitation of RBC-bound Immunoglobulin and Complement in AIHA using Flow Cytometry G Choi1, S Kim1. 1Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine,, Seoul, Republic of Korea Background/Case Studies: The direct antiglobulin test (DAT) is a test to detect immunoglobulin, complement, or both bound to RBC surface antigens. A positive result on this test clinically implies an autoimmune, alloimmune or drug-induced immune-mediated hemolytic anemia (AIHA). DAT using the tube agglutination method or a microcolumn assay has several disadvantages, such as high false-negative results, subjective interpretation, and nonquantitation. Flow cytometry, on the other hand, has been used to detect and differentiate erythrocyte-bound antibodies that can be precisely and rapidly quantitated. In this study, a flow cytometric (FC) assay or detection and quantitation of erythrocyte membrane-bound immunoglobulins and complements was used, and the results were compared with those of the DAT. Study Design/Methods: DAT using gel technique (GT-DAT) and FC tests were performed on erythrocytes from 30 healthy nonanemic persons as negative controls and from 180 anemic patients with suspected immune-mediated hemolytic anemia. Reactive cells in FC were defined as cells with fluorescence intensity ≥mean fluorescence intensity (MFI) +2 SD of negative controls. Results/Findings: The FC and DAT were negative in all healthy persons. Of 180 anemic patients tested for erythrocyte-bound IgG, 138 were positive on the FC (senstivitiy:76%), and 78 were positive on the DAT (sensitivity:43%). Sixty showed a negative result on DAT but a positive result on FC, and the mean MFI was 16.3 ± 4.42. Conclusion: We concluded that the FC assay is a more sensitive test than the GT-DAT and that it helps in the serological diagnosis of Coombs-negative AIHA and quantitation. FC assays should be employed in assessing patients with strong clinical suspicion of AIHA, but who showed a negative result on DAT. Disclosure of Commercial Conflict of Interest G. Choi: Nothing to disclose; S. Kim: Nothing to disclose Disclosure of Grants Conflict of Interest G. Choi: Nothing to disclose; S. Kim: Nothing to disclose

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SP259 Reactivity of Reticulocyte-Enriched Cell Populations with Selected Commercial Antisera J G Angeles1, R M Leger1. 1American Red Cross Blood Services, Pomona, CA, United States Background/Case Studies: A reliable RBC phenotype is difficult to obtain when donor RBCs are present in a patient’s circulation after transfusion. Microhematocrit centrifugation is a commonly used technique to separate younger, less dense RBCs from mature RBCs (donors’ and patients’). It was reported in 1985, however, that harvested reticulocytes can give significantly weaker reactions than does whole blood with anti-E, -Ge, -Fya, -Jka, -c, and -e. Different reagents, such as monoclonal antibodies (MoAbs), are now used. We evaluated the reactivity of selected commercial antisera with reticulocyte-enriched RBCs separated by using microhematocrit centrifugation compared to reactivity with unseparated RBCs. Study Design/ Methods: EDTA samples from 27 patients with negative direct antiglobulin tests, low hemoglobin, and no recent transfusions were tested. Microhematocrit centrifugation was performed on ∼2 mL of washed RBCs. The top 5-mm fraction of RBCs was tested in parallel with unseparated RBCs by using 2 sources of commercial antisera for A, B, D, C, E, c, e, Fya, Jka, S, and P1 antigens. Antisera for ABO, Rh, 1 anti-Jka, and 1 anti-P1 were MoAbs. Reactions were graded; significant difference was determined by reactivity >1+ weaker for the reticulocytes. Reticulocytes suspended in an equal volume of autologous plasma were stained (1:1 ratio) with 1% New Methylene Blue for 15 min at room temperature to assess reticulocyte recovery. Smears were prepared by using the pull-apart technique; the reticulocytes were recorded as low (10/ field). Results/Findings: There was no significant difference in reactivity of reticulocytes compared to unseparated RBCs for 2 of 2 anti-A (# RBCs tested n = 13), -B (n = 3), -D (n = 23), -C (n = 15), -E (n = 6), -c (17), -S (n = 11); 1 MoAb anti-P1 (n = 25), 1 goat anti-P1 (n = 20); and for 1 of 2 anti-e (n = 27), -Fya (n = 16), and -Jka (n = 25). Reactivity was weaker >1+ with reticulocytes by using another anti-e (n = 2/25), -Fya (n = 4/16), and -Jka (n = 2/25). Goat anti-P1 did not react with 3/4 RBCs that reacted 1+ with MoAb anti-P1. Reticulocyte recovery was high on smears for 21/27 (78%) and moderate for 6/27 (22%) samples; about 200 RBCs/oil field were viewed. Conclusion: The moderate-to-high reticulocyte estimates indicated that young RBCs were tested, and separation was successful. MoAbs yielded superior results compared to the few polyclonal antisera tested in this study. The MoAb anti-e that reacted >1+ weaker with reticulocytes (±vs 3+ & 2+ vs 4+) contains a single clone; the other anti-e is a blended MoAb. The anti-S and -Fya were polyclonal. MoAb anti-P1 gave significantly stronger results with some RBCs than did goat anti-P1. When tested with most MoAb antisera in this study, younger RBCs separated by microhematocrit centrifugation did not show significantly weaker reactivity than the mixed population of RBCs. Caution should still be exercised when testing polyclonal and some single-clone MoAb reagents. Disclosure of Commercial Conflict of Interest J. G. Angeles: Nothing to disclose; R. M. Leger: Nothing to disclose Disclosure of Grants Conflict of Interest J. G. Angeles: Nothing to disclose; R. M. Leger: Nothing to disclose

SP260 Cord Blood Testing Using an Automated Column Agglutination Method S Chen2, S Krohto2, L D Roberts2, D Stachurski3, C Williams1, S Milbradt-Pohan1, J Sweeney2. 1Bio-Rad laboratories, Benicia, CA, United States; 2Miriam Hospital, Providence, RI, United States; 3 Pathology, Newport Hospital, Newport, RI, United States Background/Case Studies: A sample of umbilical cord red blood cells is tested for ABO and D, and a direct antiglobulin test (DAT) is performed when there is a suspicion of hemolytic disease of the newborn. On account of the latter test, cord blood testing is commonly performed by using manual tube methods. An automated method could improve efficiency and reduce errors associated with subjective interpretation of endpoints or with the manual entry of test results. Study Design/Methods: Cord samples were tested in parallel by using a manual tube method and an automated column agglutination system (IH-Cards with IH-1000 instrument, Bio-Rad, Dreieich, Germany) method (AutoCAT). The AutoCAT method used the following monoclonal blood grouping reagents in various card configurations: anti-A, -B, -A,B, -D(DVI+), -and D(DVI-). Gel cards containing anti-IgG and -C3d and anti-IgG only were used for the DAT. The anti-IgG card was also used in

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conjunction with anti-D Blend for the detection of D variant samples. Results/Findings: One hundred ninety-seven samples were tested, generating a total of 591 results. Concordant results were seen in 583/591 (99%). Fifty percent (4/8) of the discordant results involved equivocal instrument interpretations. Total discordance (positive/negative or negative/positive) was seen in only 4/591 results (0.47%). Analysis of these latter four discordant tests showed that, in all cases, the discordance was due to weak positive or negative agglutination reactions in the tube method. Two of the four samples were weak D samples that were not detected by the predicate anti-D reagent but were detected by the AutoCAT anti-D. Details are shown in the Table. Conclusion: The automated column agglutination system showed a high concordance with the predicate method and has the potential to apply automated testing into the traditionally manual area of cord blood testing.

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Disclosure of Commercial Conflict of Interest S. Chen: Nothing to disclose; S. Krohto: Bio Rad, Other; S. Milbradt-Pohan: Bio-Rad Laboratories, Inc., Other; L. D. Roberts: Haemonetics, Other; D. Stachurski: No Answer; J. Sweeney: Bio-Rad Laboratories, California, Grants or Research Support; C. Williams: Bio-Rad Laboratories, Inc., Other Disclosure of Grants Conflict of Interest S. Chen: Nothing to disclose; S. Krohto: Bio Rad, Other; S. Milbradt-Pohan: Nothing to disclose; L. D. Roberts: Nothing to disclose; D. Stachurski: No Answer; J. Sweeney: Nothing to disclose; C. Williams: Nothing to disclose

TABLE. Degree of Concordance of the Predicate (tube) and Test (AutoCAT) Methods CONCORDANCE %

CONCORDANCE %

PREDICATE

N=

ORIGINAL

AFTER DISCREPANCY RESOLUTION

Anti-A,B

Ortho Anti-A,B BioClone

100

99%

99%

Anti-A

Ortho Anti-A BioClone

197

99%

99%

Anti-B

Ortho Anti-B BioClone

197

99%

99%

Anti-D (DVI-) Anti-D (DVI+)

Ortho Anti-D BioClone Orhto Anti-D BioClone

100 97

100% 97%

100% 99%

IH Ant-D Blend

Ortho Anti-D BioClone Ortho Anti-IgG Ortho Anti-IgG Ortho Anti-IgG, -C3d

97

100%

100%

97 100

100% 98%

100% 99%

AUTOCAT REAGENT

DAT Anti-IgG DAT Anti-IgG, -C3d

REMARKS 1 sample dual population (EQV) with AutoCAT Anti-B and anti-A,B positive with predicate 1 sample weak A (EQV with AutoCAT/positive with predicate) 1 sample possible contamination 1 sample dual population (EQV) with AutoCAT Anti-B & anti-A,B positive with predicate 2 weak D samples detected by AutoCAT, not detected by predicate 1 sample possible contamination (EQV with AutoCAT/negative with predicate)

1 false-negative with AutoCAT 1 false-positive with predicate

EQV, Equivocal result. SP261 Clinical Outcomes of Gravid Patients with Nonspecific Antibodies Identified By Solid-Phase Testing During Routine Screening J Olson1, J M Pauli1, P Erdman1, M R George1. 1Penn State Hershey Medical Center, Hershey, PA, United States Background/Case Studies: The Immucor Echo antibody detection system is a very sensitive solid-phase red cell adherence assay (SPRCA). However, with this sensitivity, nonspecific antibodies are often identified. This situation leads to confusion for the obstetrics clinical team, as there are no guidelines for dealing with nonspecific antibodies of unknown clinical significance. Study Design/Methods: All gravid patients undergo antibody screening at their first- trimester obstetric visit. Any patient with nonspecific reactivity was approached and asked to give consent for additional testing. Data were collected prospectively after the initial first-trimester test. At the secondtrimester visit, an antibody screen and ID were performed by SPRCA, along with HLA antibody screening and identification and red cell genotyping. At delivery, an antibody screen and ID wwere performed by SPRCA on both the mother’s blood and the cord blood. At the 6-wk postnatal visit, a fourth antibody screen and ID were performed by SPRCA. A chart review of the baby’s initial health was performed to look for indications of obstetric complications, including hemolytic disease of the newborn, neonatal jaundice, need for intrauterine transfusion or phototherapy, and infant mortality. Results/Findings: Eight women with nonspecific first-trimester antibody screen reactivity were identified. Of these women, five had complete follow-up with results for all protocol testing. On average, the women were 30 y old, had had a total of 2.6 pregnancies, and had an ABO/Rh distribution consistent with the general population. Only four of the eight women had anti-HLA antibodies, with no definitive trends in reactivity. No pattern was noted in the patients’ red cell genotyping. Upon chart review, no common medical conditions were identified. Conclusion: There was no indication that nonspecific reactivity during a patient’s initial first-trimester antibody

screening causes hemolytic disease of the newborn or infant morbidity/ mortality. Although this is a small series of patients, it lends support to the lack of clinical significance of reactivity. There were no trends in HLA antibody reactivity in any of the patients, and no uniform RBC genotype results. This nonspecific reactivity was also not consistently present throughout any of the patients’ pregnancies, which lends credence to the theory that this reactivity may be an artifact of the testing system, and not clinically significant. More detailed serologic studies may be warranted to better characterize the source of nonspecific reactivity in the testing system; however, this series did not demonstrate any clinically significant impact in gravid patients. Disclosure of Commercial Conflict of Interest P. Erdman: Immucor Corporation, Grants or Research Support; M. R. George: Immucor, Inc., Grants or Research Support; J. Olson: Immucor, Grants or Research Support; J. M. Pauli: Immucor, Grants or Research Support Disclosure of Grants Conflict of Interest P. Erdman: Immucor Corporation, Grants or Research Support; M. R. George: Immucor, Inc, Grants or Research Support; J. Olson: Immucor, Grants or Research Support; J. M. Pauli: Immucor, Grants or Research Support

SP262 Should I Follow the Package Insert? E Nartowicz2, N Lang1, G Khalife1, J Minneman1. 1Community Blood Center, Dayton, OH, United States; 2Soin Medical Center, Beavercreek, OH, United States Background/Case Studies: Package inserts for the use of most FDAlicensed blood banking antisera address the issue of false-positive test

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results due to spontaneous agglutiniation of red cells by either the use of an inert control run in parallel with the test or the observation of at least one negative test result in a series of tests performed in the same manner. One manufacturer of monoclonal anti-Jka and anti-Jkb has a package insert that states if the patient has a positive direct antiglobulin test (DAT) and both antigen results are positive, the results should not be accepted. Assuming a Caucasian patient population, approximately 50% of patients with positive DATs would be expected to be Jk(a+b+) and would not have valid typings with these antisera. We challenged this limitation from the package insert to see whether Jka and Jkb typings could be validated with use of an inert control to detect spontaneous agglutination instead. Study Design/ Methods: Twenty-six samples from untransfused patients with a DAT of 1+ or greater were tested with the manufacturer’s monoclonal anti-Jka and anti-Jkb; these same samples were then treated with EDTA-glycine acid (EGA) to obtain a negative DAT and tested with AHG-reactive, licensed, polyclonal anti-Jka and anti-Jkb. In both methods, an inert control of 6% bovine serum albumin was tested in parallel, and in all instances was found to be negative. The results of the two sets of typings were then compared. Results/Findings: Eleven of the 26 samples typed as Jk(a+b+) by using the monoclonal source of anti-Jka and anti-Jkb. Nine of the 11 samples were confirmed as Jk(a+b+) by using EGA-treated cells and AHG-reactive anti-Jka and anti-Jkb. Two samples were negative for one of the Kidd antigens (1 Jka and 1 Jkb) when testing was repeated by using the EGA-treated cells and AHG-reactive antisera, which gives credence to the package insert. More interesting, perhaps, was the unexpected observation that five samples typed as negative for one of the Kidd antigens (2 for Jka and 3 for Jkb) by using the monoclonal antibodies, but they typed as Jk(a+b+) with the EGAtreated cells. This result calls the EGA treatment of the red cells into question. Conclusion: This was a study with a small sample size, but it led to a conclusion and an unexpected observation. Package inserts should be followed as written, and limitations for use must be observed. EGA treatment of red cells may be a cause of false-positive test results, even in light of a negative control for spontaneous agglutination. More investigation of this finding is warranted. As a result of this study, perhaps only antigen types obtained by validated molecular methods should be used with patients exhibiting a positive DAT.

assay (using anti-IgG-coated indicator cells lacking the anti-IgG4 component) also prevents detection of these antibodies in many cases. Many of these high-prevalence antibodies do not react with ficinpretreated cells. Tube/PEG testing prevented the detection of these antibodies in some patients. Conclusion: Simply switching to a different source of AHG may enable laboratories to deal with a majority of these so-called “HTLA-like” antibodies, which would translate to cost savings and prevent delays in transfusion. Other serologic strategies, many quite simple, may work as well to prevent detection of these “nuisance” antibodies and allow for the detection of clinically significant underlying alloantibodies. Disclosure of Commercial Conflict of Interest G. Khalife: No Answer; N. Lang: Nothing to disclose; J. Minneman: Nothing to disclose; G. C. Patch: Nothing to disclose; C. Todd: Nothing to disclose Disclosure of Grants Conflict of Interest G. Khalife: No Answer; N. Lang: Nothing to disclose; J. Minneman: Nothing to disclose; G. C. Patch: Nothing to disclose; C. Todd: Nothing to disclose TABLE. Success For Each Technique

Method Anti-IgG reagent lacking IgG4 component Solid-phase red cell adherence assay* Ficin-treated panel Tube/PEG enhancement; anti-IgG

Percent Successful

Percent Partially Successful

Percent Unsuccessful

Total Cases

80

11

9

121

63

11

26

27

68 36

11 12

21 52

91 33

* Only 3 years’ worth of data were available for the solid-phase assay review, resulting in fewer numbers to compare.

Disclosure of Commercial Conflict of Interest G. Khalife: No Answer; N. Lang: Nothing to disclose; J. Minneman: Nothing to disclose; E. Nartowicz: Nothing to disclose Disclosure of Grants Conflict of Interest G. Khalife: No Answer; N. Lang: Nothing to disclose; J. Minneman: Nothing to disclose; E. Nartowicz: Nothing to disclose

SP263 Serologic Strategies for Alloantibody Identification in the Face of an “HTLA-like” Antibody C Todd2, N Lang1, G C Patch1, G Khalife1, J Minneman1. 1Community Blood Center, Dayton, OH, United States; 2Veterans Administration Medical Center, Dayton, OH, United States Background/Case Studies: Laboratories choosing to investigate antibodies to high-prevalence antigens must have serologic strategies in place to detect underlying clinically significant antibodies. Several antibodies to highprevalence antigens fall into the category of “doubtful clinical significance” and are referred to in older textbooks as high-titer, low-antigen-density or high-titer, low-avidity (HTLA) antibodies. Strategies may be different when identifying underlying antibodies in the face of “HTLA-like” antibodies, due to their unique characteristics. Study Design/Methods: A retrospective review of all cases performed by a reference laboratory involving the identification of a “HTLA-like” antibody was performed for a 10-year period to identify what technique(s) was/were used to rule in or rule out the coexistence of other alloantibodies. Techniques used (after establishing the presence of the “HTLA-like” antibody) to rule in/rule out other antibodies included the use of anti-IgG lacking the anti-IgG4 component; PEG enhancement in tube testing; ficin-pretreated RBCs; and solid-phase red cell adherence assays. Techniques employed were evaluated as successful, partially successful, or not successful; statistics were then generated on the reviewed data. Results/Findings: A summary of our findings appears in the Table. The reference laboratory routinely performs all initial antibody identifications in tube using a LISS enhancement and a polyclonal antihuman globulin (AHG) reagent that contains all subcomponents of anti-IgG. Recognizing that many of these so-called “HTLA-like” antibodies are of the IgG4 subclass, the laboratory often repeats testing using a monoclonal anti-IgG reagent that lacks the IgG4 subcomponent. The use of a solid-phase red cell adherence

SP264 Comparative Study of the Upper and Lower Fractions of Centrifuged Red Blood Cells (RBCs) W A Beres1, S J Nance1. 1Technical Services, American Red Cross, Philadelphia, PA, United States Background/Case Studies: A comparative study was performed on two separate fractions of centrifuge-derived populations of RBCs. We hypothesized that older, heavier RBCs would be located at the bottom of a centrifuged tube and that these older RBCs would be more likely to have increased levels of immunoglobulin (Ig). Fractions obtained by centrifugation of whole-blood samples stored in EDTA from five healthy donors were evaluated by flow cytometry (FC) for RBC-bound IgG, IgA, and IgM on days 7, 14, and 21. Study Design/Methods: Samples from five healthy donors were stored in EDTA and tested on days 7, 14, and 21. On day 7, the plasma and visible white cells were removed; RBCs were mixed, and, centrifuged for 3 min; and 200-500 uL of RBCs were obtained from the top and bottom of each sample. On days 14 and 21, RBCs were mixed and centrifuged for 3 min, and 200-500 uL of RBCs were obtained from the top and bottom of each sample. Naive EDTA samples and Ig-coated RBCs were tested to determine FC settings and to control for validity and cross-reactivity. The samples were tested by FC with fluorescein isothiocyanate (FITC)-labeled anti-human IgG or -IgA (Jackson ImmunoResearch Lab, West Grove, PA) or FITC-labeled anti-human IgM (Life Technologies, Carlsbad, CA) at optimal dilutions in Dulbecco’s PBS containing 0.6% BSA. Fifty thousand RBCs acquired from each sample by the Becton Dickinson FACSCalibur™ (San Jose, CA) FC were analyzed. Normal ranges for RBC-bound Ig were determined by using the mean %M2 ±3 SD of normal donor samples. Results/ Findings: Results were unexpected, in that they were not distinctly different from top and bottom fractions, even over time. One of the five samples had elevated levels of RBC-bound IgG in the top and bottom fractions. Three samples had elevated levels of RBC-bound IgA in the top fraction on day 7, and one of the three had elevated bottom fractions levels on day 21. One sample had elevated RBC-bound IgM in the bottom fraction on day 7. Conclusion: No significant change was seen in fractions evaluated for RBC-bound IgG and IgM. Surprisingly, 60% of the top fractions had elevated levels of RBC-bound IgA on day 7, while the bottom fractions were negative. The IgA levels dropped into the normal range on days 14 and 21, most likely as a result of storage. In our experience with FC, RBC-bound IgA is rarely

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detected on donor RBCs, but, with these study data, that could possibly be due to sampling technique or sample age at testing. Unexpectedly, the comparison of the RBC fractions were not statistically different. The limited data suggest that sampling is less important than sample age if RBC-bound IgA is to be detected.

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Disclosure of Commercial Conflict of Interest W. A. Beres: Nothing to disclose; S. J. Nance: Nothing to disclose Disclosure of Grants Conflict of Interest W. A. Beres: Nothing to disclose; S. J. Nance: Nothing to disclose

TABLE. % RBC-bound IgG Top Mean Bottom Mean P value

% RBC-Bound IgA

% RBC-Bound IgM

Day 7

Day 14

Day 21

Day 7

Day 14

Day 21

Day 7

Day 14

Day 21

1.78 1.20 0.13

1.83 2.31 0.71

3.41 2.83 0.64

3.90 2.43 0.10

2.05 2.13 0.88

2.48 3.83 0.17

1.62 1.81 0.85

1.36 1.19 0.70

2.15 2.19 0.91

SP265 Serological Findings Related to Treatment with a Human Monoclonal Antibody (Daratumumab) in Patients with Advanced Plasma Cell Myeloma J L Hannon1, B Caruk1, G Clarke1. 1Medical Research and Innovation, Canadian Blood Services, Edmonton, AB, Canada Background/Case Studies: Targeted immunotherapy with monoclonal antibodies is being used with greater frequency to treat patients with advanced malignancies. Daratumumab is an IgG1 human monoclonal antibody (MoAb) directed against a unique epitope of CD38. Strong CD38 expression is typical of both normal plasma cells and myeloma cells. Daratumumab induces potent antibody- dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CD38positive malignant cells without damage to the marrow microenvironment. This MoAb is of interest to the transfusion service, because normal human erythrocytes express low levels of CD38, and the drug has been reported to interfere with the indirect antiglobulin test (IAT). Study Design/ Methods: Six patients with advanced plasma cell myeloma were started on Daratumumab between February 21 and March 27, 2014, as part of a clinical trial. The patients were 32 to 84 y old, and all were males. Pretreatment ABO/Rh typing and antibody screens (Ab screens) were performed. There were 2 O-positive, 2 A-positive, 1 A-negative, and 1-B negative. Ab screens were negative by solid-phase assay (SPA). Extended phenotyping was performed before treatment when possible. Genotyping was performed on all patients. Results/Findings: Two patients remain negative on Ab screens to date. One patient developed a weakly positive DAT at day 14 with a negative eluate and autocontrol. DAT and autocontrol remained negative on the other patient. Two patients developed panreactivity by SPA and PEG-IAT at day 1 and day 13. Reaction strengths varied from 1+ to 3+ by SPA and 1+ by PEG-IAT. DAT and autocontrol were negative on both. An eluate was negative on the latter. One patient was pan-reactive by SPA at day 14 and, at day 37, was reported to have an antibody of undetermined specificity. One patient died as a result of H1N1 influenza. Five of six patients required transfusion. Conclusion: As with Daratumumab, some MoAbs used for therapy in malignancy may recognize antigens expressed on human red cells and therefore have the potential to interfere with blood bank testing. These patients generally have advanced disease, which makes transfusion likely. Blood typing, extended phenotyping (beforereatment), and /or genotyping are recommended, in order to assist with selection of blood if transfusion becomes necessary. We are following our cohort with the intention of testing patients who come off therapy, to learn whether the serologic findings persist, but patients may be on these agents for several years. Disclosure of Commercial Conflict of Interest B. Caruk: Nothing to disclose; G. Clarke: Nothing to disclose; J. L. Hannon: Nothing to disclose Disclosure of Grants Conflict of Interest B. Caruk: Nothing to disclose; G. Clarke: Nothing to disclose; J. L. Hannon: Nothing to disclose

SP266 Use Of a Low-ionic-strength Solution to Remove Autoantibodies J Kosanke1, B Dickstein1, R Stephan1. 1American Red Cross Central Ohio, Columbus, OH, United States Background/Case Studies: Three methods for warm autoantibody removal have several references in the literature for performance, success, and limitations. The methods include pretreatment of red blood cells with enzymes, pretreatment with ZZAP, and the use of polyethylene glycol (PEG) during adsorption. A procedure with fewer published results is adsorption using low-ionic-strength solution (LISS). Our laboratory experienced two cases of known alloantibodies with 2+ saline-IgG reactivity that were nonreactive after a PEG adsorption. These findings prompted the laboratory to perform verification studies to demonstrate that a LISS adsorption is an effective method for autoantibody removal and could replace the laboratory’s standard PEG adsorption method. Assumptions were that 1) any adsorption method may not detect a weakly reactive alloantibody, based on previous in-house studies; 2) LISS-IgG is a method used for alloantibody exclusion when warm autoantibody is nonreactive by LISS-IgG in the neat plasma, so using the adsorbed LISS-plasma mixture is an acceptable method for alloantibody detection; and 3) efficacy of the LIS-adsorption equals removal of autoantibody after three adsorptions. Study Design/Methods: Ten previously analyzed samples with 2-3+ warm autoantibody reactivity by a salineIgG test were used. A mixture of one volume of plasma and one volume of LISS was added to one volume of untreated, hard-packed red blood cells (1:1:1 of plasma, LISS, red blood cells) in a 10-mL tube. The tube was incubated at 37°C for 15 min and centrifuged, and the LISS-plasma was harvested. A test for completion was performed after each adsorption: six drops of the LISS-plasma mixture were tested with one drop of a 3-4% cell suspension prepared from an aliquot of the same donor whose cells were used for the adsorption. The test was incubated at 37°C for 15 min prior to conversion to the anti-globulin test. For samples that were not adsorbed to completion after three adsorptions, a PEG method was also performed. Results/Findings: Eight samples were adsorbed to completion after the second LISS adsorption. One of 10 samples remained weakly reactive after the third adsorption. A PEG adsorption with this sample was also weakly reactive after three adsorptions. After four adsorptions, both methods were non-reactive. One of 10 samples reacted 2+ after the third LISS adsorption. A PEG adsorption removed the autoantibody after two adsorptions. Conclusion: Results were better than expected, as 80% of samples required only two adsorptions. The laboratory implemented the LISS adsorption as the standard procedure after the study was completed. Since this implementation, 10 additional samples with 2-3+ reactivity have been tested, and all samples were adsorbed to completion after the second adsorption. An allo-anti-E was also detected in one sample. Disclosure of Commercial Conflict of Interest B. Dickstein: Nothing to disclose; J. Kosanke: Nothing to disclose; R. Stephan: Nothing to disclose Disclosure of Grants Conflict of Interest B. Dickstein: Nothing to disclose; J. Kosanke: Nothing to disclose; R. Stephan: Nothing to disclose

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SP267 Different Prenatal Antibody Titration Methods May Produce Variable Results with Profound Clinical Implications M Oliveira1, D M Fountain1, L R Dunn-Alabanese2, W J Savage1, R M Kaufman1. 1Pathology, Brigham and Women, Boston, MA, United States; 2Obstetrics and Gynecology, Brigham and Women’s Hospital, Boston, MA, United States Background/Case Studies: Routine monitoring of sensitized pregnancies for hemolytic disease of the fetus and newborn (HDFN) includes following maternal antibody (Ab) titers. Titration methods vary among blood banks. We report the case of a 34-y-old G4P2 woman whose last pregnancy was complicated by an anti-C Ab. An outside hospital (OSH) informed the patient’s obstetrician that the anti-C had risen from untitratable to a titer of 64 at 37oC, which prompted plans to deliver the baby early at week 37. Follow-up testing in our laboratory revealed that the elevation in titer was mainly due to IgM and that early delivery was unwarranted. Study Design/ Methods: Ab screens were done on the TANGO device (Bio-Rad). RBC panels and Ab titrations were performed by using standard tube methods. Titration conditions used routinely in our hospital’s blood bank are incubation of test plasma and heterozygous RBCs at 37oC for 30 min; no enhancement; saline wash; anti-human globulin (AHG); and visual inspection for agglutination. Titration conditions used by the OSH are incubation of test plasma and homozygous RBCs at 37oC for 60 min; no enhancement; saline wash; AHG; and visual inspection. For tests using DTT, 0.01M DTT was added to plasma and incubated at 37oC for 30 min. Results/Findings: Titration results are summarized in the Table. On initial testing at our hospital, the patient’s Ab screen was positive, and the anti-C was confirmed by panel. However, the anti-C titer was only 1 at AHG phase (heterozygous C+c+ cells, 30-min incubation). The titer of 64 obtained at the OSH was replicated by incubating patient plasma with homozygous C+ cells for 60 min. DTT treatment of the patient’s plasma reduced the titer from 64 to 0. The patient’s obstetrician was advised that the previously reported titer of 64 was mainly due to IgM, and that the serological results did not suggest a risk for severe HDFN. Intracranial Doppler studies repeatedly showed no evidence of HDFN. The baby was delivered at 40 wk in good health. The baby’s RBCs typed as C+, but the DAT was negative, and there was no evidence of hemolytic anemia. Conclusion: The use of a very sensitive prenatal titration technique at an OSH led to the detection of a high-titer anti-C that was mainly IgM, which unnecessarily raised concerns about severe HDFN. Standardization of prenatal titration methods among laboratories would help to provide more reliable data to inform clinical decision-making. Disclosure of Commercial Conflict of Interest L. R. Dunn-Alabanese: No Answer; D. M. Fountain: Nothing to disclose; R. M. Kaufman: Janssen Pharmaceuticals, Other; M. Oliveira: Nothing to disclose; W. J. Savage: Nothing to disclose Disclosure of Grants Conflict of Interest L. R. Dunn-Alabanese: No Answer; D. M. Fountain: Nothing to disclose; R. M. Kaufman: Nothing to disclose; M. Oliveira: Nothing to disclose; W. J. Savage: Fresenius Kabi, Grants or Research Support TABLE. The anti-C titer measured on a maternal plasma sample varied widely depending on the technique used Anti-C titer

Blood Bank

RBC

Incubation

Patient plasma

37°C

AHG

OSH BWH

C+c− C+c+ C+c− C+c−

60 min at 37°C 30 min at 37°C 60 min at 37C 60 min at 37C

Untreated Untreated Untreated DTT-treated

64 NT 64 0

1 1 1 1

NT, not tested. SP268 Case Study: Identification of Probable Anti-Lu16 and its Clinical Significance T K Walters1, C K Hopkins2. 1Reference Laboratory, Amercian Red Cross, Columbia, SC, United States; 2Medical Director, American Red Cross, Columbia, SC, United States Background/Case Studies: The Lutheran System is a complex system comprising 20 antigens, including four antithetical pairs: Lua/Lub, Lu6/Lu9, Lu8/Lu14, and Aua/Aub. The Lu:16 antigen is classified as a high-prevalence antigen present in 100% of the population. The antigen was first reported in 1980 when three black women Lu(a+b−) were found to have a high-

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prevalence antigen in addition to anti-Lub. The antibody that defines Lu:16 is IgG, which is best reactive at the indirect antiglobulin phase (IAT), nonreactive with Lu(a-b-) cells, and resistant to treatment with ficin and dithiothreitol (DTT). It is unknot known whether anti-Lu16 is able to cause hemolytic disease of the fetus and newborn (HDFN) or a hemolytic transfusion reaction (HTR). Other antibodies within the Lutheran system generally have been implicated in mild delayed HTR and have not caused severe HDFN. Here we report a case of a multiply transfused patient with a probable Lu16 antibody and possible unrecognized delayed HTR (DHTR). Study Design/Methods: The patient was a 67-y-old black female with end-stage renal disease who had been transfused multiple times, with recent transfusion of 2 units of RBCs for a hemoglobin (Hb) of 8.3 g/dL. Her Hb increased to 11.3 g/dL 24 h after-transfusion and decreased to 9.8 g/dL 5 d after transfusion. A DHTR was not suspected. One month after transfusion, she tested O, Rh+ with a weakly positive direct antiglobulin test (DAT), with anti-IgG only. Results/ Findings: The patient’s serum was reactive w+−1+ with all panel cells tested with LISS-IAT and PEG-IAT, including phenotypically similar cells, but it was noneactive with EGA (EDTA glycine acid)-treated autologous cells. Reactivity was resistant to treatment with Ficin and 0.2M DTT. A selected panel of high-incidence antigen-negative cells was tested. A Lu (a+b−) cell remained w+ at PEG-IAT; however, three additional cells of Lu(a-b-) phenotype were nonreactive on PEG-IAT. Allogeneic adsorptions using PEG were performed to exclude other alloantibodies. Molecular characterization determined the patient to be Lu(a+b−); Lu:-16. A monocyte monolayer assay (MMA) was performed; it indicated that the probable anti-Lu16 may cause accelerated clearance of Lu:16-positive RBCs with a 20.3%-21.9% range of reactive monocytes (normal range 0-3%). Conclusion: Probable anti-Lu16 was identified in this patient’s sample using standard tube testing, and molecular genotyping confirmed the patient was Lu:-16. Given the MMA results indicating that the anti-Lu16 was clinically significant and given also the 1.5-g/dL drop in Hb 5 d after -transfusion and the weakly positive DAT one month posttransfusion, she may have experienced a clinically unrecognized DHTR secondary to the anti-Lu16. The patient has not received any subsequent transfusions to date. Disclosure of Commercial Conflict of Interest C. K. Hopkins: No Answer; T. K. Walters: Nothing to disclose Disclosure of Grants Conflict of Interest C. K. Hopkins: No Answer; T. K. Walters: Nothing to disclose

SP269 Manual Gel Testing: Technique Matters H A Winter1. 1Transfusion Services, University of California Davis Medical Center, Sacramento, CA, United States Background/Case Studies: Proper technique is important when performing manual gel testing. Delivery of the reagent red blood cells into the gel microtube columns below the upper reaction chamber can lead to decreased sensitivity and false-negative reactions. This variation in pipetting technique does not appear to be adequately addressed in the package inserts or gel testing user manuals. Study Design/Methods: A total of 20 plasma specimens were tested in parallel by using a manual gel antibody-screening test method. Nineteen of the 20 specimens were known to be positive, with antibody specificities from a variety of blood group systems. One plasma sample was known to be negative. Manual gel antibody screening tests were set up in parallel. Screening cells 1 and 2 were first pipetted vertically, allowing the reagent red cells to be delivered down into the neck of the gel microtube columns. The second set of antibody screening cells were pipetted at an angle, delivering the red cells so they were seated at the top of the gel microtube column in what is known as the upper reaction chamber. Plasma was then added to both sets of reagent antibody screening cells, incubated at 37°C for 15 min, and then centrifuged for 10 min in the appropriate centrifuge at preset conditions. Results/Findings: For the 19 positive samples, all demonstrated stronger reactions when the pipetting of the reagent antibody screening cells was delivered into the upper reaction chamber versus when the reagent red blood cells were delivered down into the neck of the gel microtube columns. The negative sample was completely negative with both methods of pipetting. In this study, 1 of 19 patients’ samples had a false-negative antibody screening test result, and 2 of the 19 specimens had a false-negative reaction in one of the two antibody screening cells, which may have affected proper antibody identification. Conclusion: When performing manual gel testing, it is important to use a pipetting technique in which the reagent red cells are delivered into the upper reaction chamber of the gel microtube columns and not allowed to flow down into the neck of the microtube column. This pipetting technique does not

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appear to be clearly addressed in the package inserts or the user manuals. When reagent red blood cells are delivered into the neck of the microtube column, not all of them are able to come in contact with the test plasma, which limits the degree of incubation of cells with plasma. This results in decreased sensitivity, leading to weaker or even false-negative reactions. Disclosure of Commercial Conflict of Interest H. A. Winter: Ortho Clinical Diagostics,Other Disclosure of Grants Conflict of Interest H. A. Winter: Nothing to disclose

SP270 The Study of Concomitant IgA and IgM on IgG-Coated Patient Red Blood Cells (RBCs) by Flow Cytometry (FC) W A Beres1, S J Nance1. 1Technical Services, American Red Cross, Philadelphia, PA, United States Background/Case Studies: In the testing of random samples, it was noted that, when IgG was present on the RBCs of patient’s samples, IgA and IgM was more likely to also be detected by FC. The instance of RBC-bound IgA and IgM when RBC-bound IgG is present was analyzed on study patients. Although the study was primarily focused on samples reported to be negative in direct antiglobulin testing (DAT), it also included samples of varying strength of DAT, up to 3+. Study Design/Methods: Patient’s blood samples were selected for FC analysis. Naive EDTA-anticoagulated samples and

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immunoglobulin-coated RBCs were tested to determine FC settings and control for validity and cross-reactivity. One hundred forty-four samples were tested by FC with fluorescein isothiocyanate (FITC)-labeled anti-human IgG and IgA (Jackson ImmunoResearch Lab, West Grove, PA) or FITC-labeled anti-human IgG, IgA, and IgM (Life Technologies, Carlsbad, CA) at optimal dilutions in Dulbecco’s PBS containing 0.6% BSA. Fifty thousand RBCs acquired from each sample by using the Becton Dickinson FACSCalibur™ (San Jose, CA) FC were analyzed. Normal ranges for RBC-bound Ig were determined by using the mean %M2 ±3 SD of normal donor samples. Results/Findings: One hundred forty-four patient blood samples were evaluated in this study: 65 were DAT-negative (by tube test with anti-IgG), 27 were microscopically positive, 38 were +w, 8 were 1+, 4 were 2+, and 2 were 3+. Overall, 55% had RBC-bound IgG detected by Tube-DAT and 64% by FC. Of the 92 samples with RBC-bound IgG detected by FC, 35.9% had only RBC-bound IgG, 8.7% also had RBC-bound IgA, 20.6% also had RBC-bound IgM, and 34.8% had both RBC-bound IgA and IgM detected by FC. Conclusion: Although the results were clearly not linear in this study, as the Tube DAT strength with anti-IgG increased, the percentage of samples with RBC-bound IgA and/or IgM also increased. And, although the data set was small, it was not expected that 34% of samples with RBC-bound IgG also had concomitant IgA and IgM. These cases are of interest for clinical correlation of hemolysis. Disclosure of Commercial Conflict of Interest W. A. Beres: Nothing to disclose; S. J. Nance: Nothing to disclose Disclosure of Grants Conflict of Interest W. A. Beres: Nothing to disclose; S. J. Nance: Nothing to disclose

TABLE. Patient Samples Tested (#) 144

RBC-Bound IgG

RBC-Bound IgA

RBC-Bound IgM

RBC-Bound IgG/IgA

RBC-Bound IgG/IgM

RBC-Bound IgA/IgM

RBC-Bound IgG/IgA/IgM

33

2

9

8

19

6

32

SP271 Assessment of the Bio-Rad IH System for Blood Grouping S Milbradt-Pohan1, C Williams1, P Szklarski2, K LaFary2, M Garis2, Y C Hawker2, Q Eichbaum2, P Young2,3. 1BioRad, Benicia, CA, United States; 2Pathology, Vanderbilt, Nashville, TN, United States; 3Pathology, Dept. Veterans Affiars, Nashville, TN, United States Background/Case Studies: Background The IH-1000 instrument is a fully automated analyzer for ABO+D testing, including the serum ABO and weak D testing, phenotyping (C, c, E, e, and K), detection and identification of unexpected antibodies, direct antiglobulin testing, and crossmatching using the principles of hemagglutination and gel filtration. The purpose of this study was to evaluate the performance of the Bio-Rad IH-System for blood grouping, including ABO/Rh, weak D, Rh and K phenotyping. Study Design/Methods: Patient samples and donor segments were tested on the IH-1000 and either the Immucor Galileo or by tube testing. Reagents evaluated included Anti-A, -B, -A,B, D(DVI-), -D Blend, -C, -c, -E, -e, and -K. Reagent Red Blood Cells included A1, B, and A2. The results were compared, and concordance with the reference method was determined. The details are provided in the Table. Results/Findings: The comparative method, number

of samples tested, percentage overall concordance, and explanations for discrepancies are listed in theTable. Discrepancy resolution included repeat testing, molecular testing, testing with third-party FDA licensed reagents, and/or investigating transfusion history. Conclusion: Conclusions The evaluation of the IH-1000 and associated reagents demonstrates the satisfactory performance with patient and donor samples for automated blood grouping testing in a transfusion service. The IH-1000 was able to correctly identify double populations of red cells in recently transfused patients. Disclosure of Commercial Conflict of Interest Q. Eichbaum: No Answer; M. Garis: No Answer; Y. C. Hawker: Nothing to disclose; K. LaFary: No Answer; S. Milbradt-Pohan: Bio-Rad Laboratories, Inc., Other; P. Szklarski: No Answer; C. Williams: Bio-Rad Laboratories, Inc., Other; P. Young: Alexion, Grants or Research Support Disclosure of Grants Conflict of Interest Q. Eichbaum: No Answer; M. Garis: No Answer; Y. C. Hawker: Nothing to disclose; K. LaFary: No Answer; S. Milbradt-Pohan: Nothing to disclose; P. Szklarski: No Answer; C. Williams: Nothing to disclose; P. Young: Alexion, Grants or Research Support

TABLE. OVERALL CONCORDANCE % IH-Reagent

Predicate

N=

ORIGINAL

AFTER DISCREPANCY RESOLUTION

Anti-A Anti-B Anti-A,B A1 Cells

Immucor Anti-A Series 1 Galileo Immucor Anti-B Series 3 Galileo Immucor Anti-A,B Series 4 Tube Immucor Referencells-2 Galileo

994 994 600 994

100% 100% 100% 99.8%

100% 100% 100% 100%

B Cells A2 Cells Anti-D(DVI-)

Immucor Referencells-2 Galileo Immucor Referencells-2 Tube Immucor Anti-D Series 4 Galileo—non-IAT

994 150 994

100% 100% 99.8%

100% 100% 100%

Anti-D Blend Anti-C Anti-c Anti-E Anti-e

Immucor Anti-D Series 4 Tube—IAT Bio-Rad Seraclone Anti-C Tube Bio-Rad Seraclone Anti-c Tube Bio-Rad Seraclone Anti-E Tube Bio-Rad Seraclone Anti-e Tube

316 130 130 130 140

100% 99.2% 100% 99.2% 99.3%

100% 100% 100% 100% 100%

Anti-K

Bio-Rad Seraclone Anti-KTube

140

99.3%

100%

REMARKS

1 sample = weak isoagglutinins, False-negative with IH-1000, repeat (+) 1 sample = (?) result with IH-1000, visual edit to negative 2 samples = weak D 1 sample = Correctly reported as double population (DP) by IH-1000 1 sample = correctly reported as DP by IH-1000 1 sample = correctly reported as DP by IH-1000 1 sample = correctly reported as e+ by IH-1000. Molecular testing supported IH-1000 result. 2 samples = correctly reported as DP by IH-1000

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SP272 Evaluation of Inconclusive Reactivity Detected in Antibody Screening and Panel Tests by Automated Solid-Phase Red Cell Adherence R Perry1, R S Shirey2, P M Ness2, K E King2. 1Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, MD, United States; 2 Pathology,Transfusion Medicine, Johns Hopkins Hospital, Baltimore, MD, United States Background/Case Studies: Although solid-phase red cell adherence (SPRCA) in conjunction with automation offers many advantages, a major disadvantage of this sensitive methodology is the increased detection of inconclusive reactivity in which no antibody specificity can be identified. We evaluated the frequency and nature of inconclusive reactivity detected by automated SPRCA in our patient population. Study Design/Methods: We reviewed the transfusion medicine records of patients presenting with inconclusive reactivity detected by automated SPRCA (Galileo®, Immucor, Inc., Norcross, GA) from April 19, 2013, to May 30, 2013. Our laboratory protocol calls for repeat panel studies, including an autocontrol using a microcolumn anti-IgG gel (Gel) method (MTS™, Ortho-Clinical Diagnostics, Raritan, NJ), when inconclusive reactivity is detected by SPRCA. Reactive panel cells and/or a positive autocontrol detected by Gel triggers further testing, which may include additional panel studies and utilization of test tube methods, direct antiglobulin tests, and eluate studies performed according to standard operating procedures. Samples that are repeatedly negative by Gel panels require no further testing. Results/Findings: Of 689 positive antibody screens by SPRCA, 74 (10.7%) showed inconclusive reactivity representing 46 index cases. The Table categorizes the inconclusive cases according to the percentage of cells that are reactive by SPRCA, with corresponding Gel panel results. Of the 46 index cases, 34 (73.9%) had negative Gel panels, and 12 (26.1%) showed inconclusive reactions by Gel method. Serologic workups on samples showing inconclusive reactions by both SPRCA and Gel methods suggested that the reactions were due to autoantibody activity. There were 3 (6.5%) cases in which SPRCA reactions were suspicious for the presence of alloantibody; following extensive serologic testing, which was supported by transfusion history and molecular-derived phenotypes, these cases were ultimately identified as anti-C, anti-Fya, and anti-Jka. Conclusion: Inconclusive reactivity by automated SPRCA occurs in approximately 10 of every 100 positive antibody screens in our large tertiarycare facility. More than 70% of these cases were considered false-positives by our laboratory protocol. However, in a small number of cases, clinically relevant alloantibodies were identified that were optimally reactive by automated SPRCA and would have been missed by other routine methods.

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Phenotyping in this patient population is often hindered by the presence of circulating donor cells or by a positive direct antiglobulin test (DAT). Several methods, such as EDTA glycine acid (EGA) treatment to remove IgG or separation of autologous cells by hypotonic wash or microhematocrit centrifugation (reticulocyte separation), are often used in these situations. This study aimed to determine the accuracy of each of these methods when compared to a phenotype predicted by the HEA BeadChip™ genotyping platform. Study Design/Methods: A total of 40 samples were phenotyped for Rh, Kell, MNS, Duffy, and Kidd antigens. Treatment methods for sample set were reticulocyte separation (n = 12), EGA treatment (n = 15), and hypotonic wash (n = 13). DNA was extracted by using standard methods, and genotyping was performed by using the HEA BeadChip™ platform (BioArray Solutions, LLC). Results/Findings: Three samples phenotyped after reticulocyte separation had discordant types with one antigen each: 1 sample phenotyped Fy(b-) and was predicted to be Fy(b+w) by HEA BeadChip™; 1 sample phenotyped N+ and was predicted to be N− by HEA BeadChip™; and 1 sample phenotyped K+ and was predicted to be K− by HEA BeadChip™. The EGA-treated sample set showed 1 discordant type, in which the sample phenotyped Fy(b-) and was predicted to be Fy(b+w) by HEA BeadChip™. The hyptonic wash-treated sample set showed two discordant types: one sample phenotyped Fy(b-) and was predicted to be Fy(b+) by HEA BeadChip™; and one sample phenotyped N− and was predicted to be N+ by HEA BeadChip™. Conclusion: A total of six discordant results were discovered in all 40 samples. Discordant types were most frequent in the reticulocyte separation sample set (n = 3), with each discordant result being a false-negative by phenotyping after red cell treatment. One discordant type was noted in the EGA- treated and two were noted in the hypotonic-treated sample sets. Two of the six total discordant samples (1 reticulocyte separation, 1 EGA treatment) had a missense mutation c.265C > T in the DARC gene, which encodes the Duffy antigens. This change causes marked weakening of the Fyb antigen, which can be weakly agglutinated by most commercial antisera, but may be missed by others. These findings suggest that caution should be taken when using phenotype results from treated red cells to confirm suspected antibody specificities or to provide extended matching of future transfusions. Utilization of genotyping to predict red cell antigen expression is preferable for these difficult patient samples. Disclosure of Commercial Conflict of Interest J. R. Hamilton: Nothing to disclose; V. Hare: Immucor/BioArray, Other; T. Horn: No Answer; M. A. Keller: Nothing to disclose; W. Kluver: No Answer; J. Kosanke: Nothing to disclose

Disclosure of Commercial Conflict of Interest

Disclosure of Grants Conflict of Interest

K. E. King: Nothing to disclose; P. M. Ness: Nothing to disclose; R. Perry: No Answer; R. S. Shirey: Nothing to disclose

J. R. Hamilton: Nothing to disclose; V. Hare: Nothing to disclose; T. Horn: No Answer; M. A. Keller: BIoArray Solutions LLC, Grants or Research Support; W. Kluver: No Answer; J. Kosanke: Nothing to disclose

Disclosure of Grants Conflict of Interest K. E. King: Nothing to disclose; P. M. Ness: Nothing to disclose; R. Perry: No Answer; R. S. Shirey: Nothing to disclose TABLE. Summary of inconclusive SPRCA results Percentage SPRCA Panel Cell Reactivity 18-49% 50-74% 75-100% TOTAL

Inconclusive SPRCA

Gelnegative

Gelinconclusive

19 4 23 46

12 4 18 34

7 0 5 12

SP273 Ability of Commonly Used RBC Phenotyping Pretreatments to Yield an Accurate Antigen Profile T Horn1, J R Hamilton2, V Hare4, J Kosanke3, W Kluver5, M A Keller1. 1 Biomedical Services, National Molecular Laboratory, American Red Cross, Philadelphia, PA, United States; 2Biomedical Services, Southeastern Michigan Region, American Red Cross, Detroit, MI, United States; 3Biomedical Services, Ohio-Michigan Region, American Red Cross, Columbus, OH, United States; 4Biomedical Services, Southern Region, American Red Cross, Douglasville, GA, United States; 5 Biomedical Services, North Central Region, American Red Cross, Madison, WI, United States Background/Case Studies: Red cell phenotyping is valuable for transfusion management of multiply transfused patients, as well as for determining the risk of forming alloantibodies. Patients with multiple alloantibodies may benefit from extended phenotype matching to avoid further sensitization.

SP274 Persistence of Anti-E in Solid-Phase Testing K V Mikesell1, T S Shah1, J W Davis1, R E Domen1, M R George1. 1 Department of Pathology, Penn State Hershey Medical Center, Hershey, PA, United States Background/Case Studies: Red cell antibodies have a propensity to wax and wane in strength, and they may, over months to years, become undetectable by standard antibody screen-testing methods. This is most commonly recognized with Kidd antibodies, but studies have shown it with other clinically significant red cell antibodies using tube testing (TT) and gel testing (GT). The introduction of automated solid-phase (SP) testing methods has increased the sensitivity of antibody detection; however, we have still observed that many antibodies become undetectable. In this study, we evaluated this phenomenon in patients with anti-E antibodies identified by SP. Study Design/Methods: We performed a 2-y retrospective evaluation of all positive antibody screens/panels due to anti-E on Immucor ECHO® SP (Immucor Inc.; Norcross, GA). Each patient with a positive screen for anti-E had all subsequent screens evaluated. We recorded the date and results of the antibody screen/panel. Any time a result went from positive to negative, we recorded the number of days between the two results. Results/ Findings: Since January 2012, we had 136 patients who had anti-E identified by SP. Of those 136 patients, 74 had at least one additional antibody screen performed after the positive result. Twenty-six of 74 (35%) patients had a result that was negative after the original positive result. Eleven of the 26 patients fluctuated between positive and negative results. In total, patients changed from a positive to a negative result 44 times. Thirty-two of these reversals occurred within 1 mo of the most recent positive result. Of those 32, 18 occurred within 1 wk of the most recent positive result. All in all,

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15 of the 26 patients developed a negative antibody screen within 1 wk of having a positive result for anti-E. Conclusion: Previous studies have shown that 38% of anti-E’s become undetectable, with a median persistence of 7 mo and a mean persistence of 20 mo with TT and GT. In our study, a similar percentage of patients had anti-E become undetectable with SP; however, 57% of the patients had no detection of anti-E within 1 wk of a positive result. Many of these patients were immunocompromised; however, IgG antibodies have a half-life of 25 to 30 d and should have persisted longer than a week. Some of the patients could have had a dilutional effect due to administration of blood components or other fluids; however, these results have made us question whether SP is detecting clinically insignificant levels of anti-E, an antibody against a cryptic antigen exposed during the SP manufacturing process, or nonspecific antibodies with apparent “E-like” specificity. Moving forward, we plan to review additional clinically significant antibodies discovered by SP to determine whether this trend continues. Disclosure of Commercial Conflict of Interest J. W. Davis: Nothing to disclose; R. E. Domen: Nothing to disclose; M. R. George: Immucor, Inc., Grants or Research Support; K. V. Mikesell: Nothing to disclose; T. S. Shah: No Answer Disclosure of Grants Conflict of Interest J. W. Davis: Nothing to disclose; R. E. Domen: Nothing to disclose; M. R. George: Immucor, Inc, Grants or Research Support; K. V. Mikesell: Nothing to disclose; T. S. Shah: No Answer

SP275 Anti-Rh17: Not So Rare At Our Institution S Slemp1, J K Slayten2, D A Waxman2. 1Pathology, Indiana University School of Medicine, Indianapolis, IN, United States; 2Indiana Blood Center, Indianapolis, IN, United States Background/Case Studies: Anti-Rh17 is a rare antibody produced after an immune stimulus by individuals who lack C/c and E/e antigens of the Rh blood group on their red cells (designated as D–). The antibody may cause severe hemolytic transfusion reactions or hemolytic disease of the fetus and newborn (HDFN). We present the case of a pregnant Hispanic woman with anti-Rh17 who delivered her third child without complications. This is the third case of anti-Rh17 from our institution in the past 8 y. Study Design/Methods: A retrospective chart review was performed. Standard manual methods were used to perform ABO/Rh, red cell phenotype, direct Coombs test (direct antiglobulin test; DAT), and crossmatch. The Immunohematology Reference Lab (IRL) performed antibody identificationby standard methods and with a red cell genotype using a beadchip microarray automated system. Results/ Findings: The patient, a 30-y-old Hispanic woman, blood group O, D-positive, G4P3, was known to our service since 2006. She originally presented during her second pregnancy; shewas found to have the Rh phenotype D– and had the corresponding high-incidence antibody, anti-Rh17, in her serum. She has never needed a blood transfusion and has had three children without complications during pregnancy and delivery. This patient is now the third patient with anti-Rh17 at our institution. The first patient, the sister of our current patient, was 20 y- old at presentation for delivery of her fourth child; she had a history of anti-Rh17, anti-e, and anti-c. She had had no prenatal care for the current pregnancy or past pregnancies. Shortly after delivery of her fourth child, the infant’s hemoglobin dropped and the bilirubin rose. No compatible blood was available for immediate provision of products, so incompatible units were successfully given, with the child surviving the episode of HDFN. The second patient, a 19-y-old Hispanic woman, presented during routine work-up of her third child and was also found to have anti-Rh17 antibody. All three children were delivered without complications. Conclusion: We present the third case of anti-Rh17 from our institution within the past 8 y. All women are Hispanic, Spanish-speaking only, with histories of multiple pregnancies. There has not been a population study documented in the literature linking the D– phenotype to the Hispanic population. With the rising population of the Hispanic community across the United States, the potential association between this population and the D– phenotype is important to acknowledge. It is also important to realize the impact that these patients could have in being recruited to become volunteer rare-blood type donors. Disclosure of Commercial Conflict of Interest J. K. Slayten: No Answer; S. Slemp: Nothing to disclose; D. A. Waxman: Nothing to disclose Disclosure of Grants Conflict of Interest J. K. Slayten: No Answer; S. Slemp: Nothing to disclose; D. A. Waxman: Nothing to disclose

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SP276 Sensitivity of the Common Red Cell Antibody Detection Methods in Detecting Rh-Immunoglobulin: A Comparison Study K V Mikesell1, M R George1, W Castellani1, R E Domen1, J H Herman2, J M Gould2, J W Davis1. 1Department of Pathology, Penn State Hershey Medical Center, Hershey, PA, United States; 2Department of Pathology, Thomas Jefferson University, Philadephia, PA, United States Background/Case Studies: Rhesus-immunoglobulin (RhIG) has had great success in protecting fetuses from potential harm, but it can often lead to confusing results in antibody panels. Little work has been done to demonstrate how long RhIG reactivity is detected in the mother after administration whencommon red cell antibody-detection methods are used. Study Design/ Methods: We collected pooled plasma from red cell antibody-negative patients. Six mL of the pooled plasma was spiked to a concentration of 50 ng/mL of RhIG preparation. Serial twofold dilutions were performed in a total of six additional tubes, representing six half-lives of the drug. This process was performed by using Rhophylac®, Rhogam®, HyperRHO®, and WinRHO®. Once the dilutions were made on the four RhIG preparations, we ran the samples on solid-phase testing (SP) with the Immucor ECHO® and Galileo®, Gel testing (GT) by Ortho®, and tube testing with polyethylene glycol. Results/Findings: The ECHO® detected all formulas of RhIG to 5 dilutions. The Galileo® detected all formulas of RhIG to 6 dilutions; however, nonspecificity was seen, and the antibody screening lot used for the testing was later recalled. TT and GT demonstrated detection at 3 to 4 dilutions in most RhIG preparations. Strength of reaction (SOR) was also evaluated. SP showed strong SOR (3+ or 4+) out to 4 dilutions in most RhIG. GT showed strong SOR out to 1 dilution with some preparations, while TT showed strong SOR at peak concentration in some preparations. Conclusion: SP was able to detect RhIG at 1.57 ng/mL, while GT and TT were able to detect RhIG only between 3.13 and 6.25 ng/mL. While SP was more sensitive, it also demonstrated issues with nonspecificity. GT and TT showed more reliable results; however, there was some variability between the different RhIG preparations. While SP showed a strong SOR for several dilutions, GT and TT did not. As a result, in GT and TT, if a strong SOR is seen after the first few weeks, the possibility of alloimmunization to anti-D should be considered. SP, however, would be less reliable is this situation. Using the data from this study and the drug information provided by the companies developing the different RhIG preparations, we are able to establish estimates of how long the different preparations would be detected by the different antibody screening methods. We feel this information may be beneficial to all blood banks that test samples from patients who have received RhIG. Disclosure of Commercial Conflict of Interest W. Castellani: Nothing to disclose; J. W. Davis: Nothing to disclose; R. E. Domen: Nothing to disclose; M. R. George: Immucor, Inc., Grants or Research Support; J. M. Gould: Nothing to disclose; J. H. Herman: Nothing to disclose; K. V. Mikesell: Nothing to disclose Disclosure of Grants Conflict of Interest W. Castellani: Abbott Diagnostics, Grants or Research Support; J. W. Davis: Nothing to disclose; R. E. Domen: Nothing to disclose; M. R. George: Immucor, Inc, Grants or Research Support; J. M. Gould: Nothing to disclose; J. H. Herman: Nothing to disclose; K. V. Mikesell: Nothing to disclose SP277 Drug-induced Hemolytic Anemia Presenting with Anti-e Specificity P J Lee1, B Patel2, J A Bailey1, J Jakway1, A Simard1, M Katharine1, M Greene1, R Weinstein1, V Chhibber3. 1Transfusion Medicine, UMass Memorial Medical Center, Worcester, MA, United States; 2Internal Medicine, Umass Memorial Medical Center, Worcester, MA, United States; 3 Transfusion Medicine, North Shore University Hospital, Manhasset, NY, United States Background/Case Studies: Hemolytic anemia can result from autoimmunity, alloimmunity, and drug-induced reactions, including drug-induced immune hemolytic anemia (DIIHA). Although DIIHA has been reported for approximately 130 drugs, the associated antibodies are usually panreactive without serologic specificity to RBC antigens. We report an unusual case of piperacillin/tazobactam-associated DIIHA that presented with anti-e specificity in a 48-y-old male with traumatic brain injury and chronic respiratory failure. The patient was admitted to the intensive care unit for sepsis due to pneumonia and was empirically treated with vancomycin and piperacillin/ tazobactam. Over the first 48 h, his hemoglobin decreased from 8.5 g/dL to 5.0 g/dL and he was transfused with 2 RBC units on the basis of his negative antibody screen at admission. His hemoglobin rose appropriately but returned to 5 g/dL within 24 h. Study Design/Methods: A posttransfusion

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specimen was collected and a type and screen was performed by using ID-MTS gel technology (Ortho CD, Raritan, NJ) as was a DAT by using standard serologic tube methods. A specimen was sent to a referral lab for evaluation for DIIHA, along with samples of the antibiotics from our pharmacy. LDH, bilirubin, and haptoglobin were also measured to check for the presence of hemolysis. Results/Findings: Hemolysis was suggested by the finding of a low haptoglobin (

Abstract Presentations from the AABB Annual Meeting, Philadelphia, PA, October 25-28, 2014.

Abstract Presentations from the AABB Annual Meeting, Philadelphia, PA, October 25-28, 2014. - PDF Download Free
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