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1.  Enhanced shear-induced platelet aggregation due to low-temperature storage 
Transfusion  2012;53(7):1520-1530.
BACKGROUND
Refrigeration of platelets (PLTs) offers an attractive alternative to the currently practiced storage at room temperature since it may mitigate problems associated with bacterial contamination and extend storage lifetime. Refrigeration causes a number of biophysical and biochemical changes in PLTs and decreases PLT circulation time in vivo. However, the effect of refrigeration on PLT hemostatic functions under physiologic and pathophysiologic shear conditions has not been adequately characterized.
STUDY DESIGN AND METHODS
Washed PLTs prepared from either fresh PLT-rich plasma (PRP) or PRP stored at 4°C for 2 days was mixed with exogenous von Willebrand factor (VWF) and fibrinogen and sheared in a cone-and-plate viscometer. PLT aggregation, activation, and VWF binding after shear and glycoprotein (GP) Ibα receptor expression and ristocetin-induced PLT agglutination were measured.
RESULTS
PLTs stored at 4°C for 2 days aggregated significantly more than fresh PLTs particularly at high shear rates (10,000/sec), and this increase was independent of PLT concentration or suspension viscosity. Further, refrigerated PLTs showed a greater increase in GP Ibα–dependent PLT activation under shear and also bound more VWF than fresh PLTs. However, the GP Ibα expression levels as measured by three different antibodies were significantly lower in refrigerated PLTs than in fresh PLTs, and refrigeration resulted in a modest decrease in ristocetin-induced PLT agglutination.
CONCLUSION
The combined results demonstrate that refrigeration increases PLT aggregation under high shear, but not static, conditions and also increases shear-induced VWF binding and PLT activation. Clinically, enhanced shear-induced PLT aggregation due to low temperature storage may be a beneficial strategy to prevent severe bleeding in trauma.
doi:10.1111/j.1537-2995.2012.03917.x
PMCID: PMC4321779  PMID: 23043289
2.  Primary hemostatic capacity of whole blood: a comprehensive analysis of pathogen reduction and refrigeration effects over time 
Transfusion  2013;53(0 1):137S-149S.
BACKGROUND
Whole blood (WB) has been used in combat since World War I as it is readily available and replaces every element of shed blood. Component therapy has become standard; however, recent military successes with WB resuscitation have revived the debate regarding wider WB use. Characterization of optimal WB storage is needed. We hypothesized that refrigeration preserves WB function and that a pathogen reduction technology (PRT) based on riboflavin and ultraviolet light has no deleterious effect over 21 days of storage.
STUDY DESIGN AND METHODS
WB units were stored for 21 days either at 4°C or 22°C. Half of each temperature group underwent PRT, yielding four final treatment groups (n = 8 each): CON 4 (WB at 4°C); CON 22 (WB at 22°C); PRT 4 (PRT WB at 4°C); and PRT 22 (PRT WB at 22°C). Testing was at baseline, Days 1–7, 10, 14, and 21. Assays included coagulation factors; platelet activation, aggregation, and adhesion; and thromboelastography (TEG).
RESULTS
Prothrombin time (PT) and partial thromboplastin time increased over time; refrigeration attenuated the effects on PT (p ≤ 0.009). Aggregation decreased over time (p ≤ 0.001); losses were attenuated by refrigeration (p ≤ 0.001). Refrigeration preserved TEG parameters (p ≤ 0.001) and PRT 4 samples remained within normal limits throughout the study. Refrigeration in combination with PRT inhibited fibrinolysis (p ≤ 0.001) and microparticle formation (p ≤ 0.031). Cold storage increased shear-induced platelet aggregation and ristocetin-induced platelet agglutination (p ≥ 0.032), as well as GPIb-expressing platelets (p ≤ 0.009).
CONCLUSION
The in vitro hemostatic function of WB is largely unaffected by PRT treatment and better preserved by cold storage over 21 days. Refrigerated PRT WB may be suitable for trauma resuscitation. Clinical studies are warranted.
doi:10.1111/trf.12048
PMCID: PMC4321786  PMID: 23301966
3.  Erythrocyte eNOS does not modulate red blood cell storage hemolysis 
Transfusion  2012;53(5):981-989.
Background
The red blood cell (RBC) endothelial nitric oxide synthase (eNOS) has been shown to regulate intrinsic erythrocyte rheological properties, such as membrane deformability, suggesting that a functional eNOS could be important in RBC viability and function during storage. This study examines the correlation between RBC eNOS deficiency and the propensity of RBCs to hemolyze under selected stress conditions including prolonged hypothermic storage.
Experimental design
Fresh or stored RBCs from normal and eNOS knock out (KO) mice or from healthy human volunteers were subjected to selected hemolytic stress conditions including mechanical stress hemolysis, osmotic stress hemolysis, oxidation stress hemolysis, and evaluated during standard storage in CPDA-1 solutions.
Results
Fresh RBCs from normal and eNOS KO mice demonstrated comparable susceptibility to hemolysis triggered by mechanical stress (mechanical fragility index = 6.5±0.5 in eNOS KO versus 6.4±0.4 for controls; n=8–9), osmotic stress, and oxidative stress. Additionally, RBCs from both mouse groups exhibited similar hemolytic profile at the end of 14-day hypothermic storage, analogous to 42 days of human RBC storage. Storage of human RBCs (28 days in CPDA-1) in the presence of NOS cofactors (L-arginine and tetrahydro-L-biopterin) or inhibitor (L-NMMA) did not affect cell recovery or hemolytic response to the selected stressors.
Conclusion
These studies suggest that RBC eNOS does not modulate susceptibility to hemolysis in response to selected stress conditions or prolonged hypothermic storage. Other strategies to increase NO bioactivity following prolonged storage utilizing NOS-independent pathways such as the nitrate-nitrite-NO pathway may prove a more promising approach.
doi:10.1111/j.1537-2995.2012.03850.x
PMCID: PMC4313879  PMID: 22897637
Hemolysis; red blood cell storage lesion; RBC endothelial nitric oxide synthase (eNOS)
4.  [No title available] 
PMCID: PMC3760974  PMID: 23676138
5.  [No title available] 
PMCID: PMC3766489  PMID: 23711284
6.  [No title available] 
PMCID: PMC3929224  PMID: 24517134
7.  Strain-Specific RBC Storage, Metabolism, and Eicosanoid Generation in a Mouse Model 
Transfusion  2013;54(1):137-148.
Background
RBC transfusion is a life-saving therapy, the logistical implementation of which requires RBC storage. However, stored RBCs exhibit substantial donor variability in multiple characteristics, including hemolysis in vitro and RBC recovery in vivo. The basis of donor variability is poorly understood.
Study Design and Methods
We applied a murine model of RBC storage and transfusion to test the hypothesis that genetically distinct inbred strains of mice would demonstrate strain-specific differences in RBC storage. In vivo recoveries were determined by monitoring transfused RBCs over 24 hours. Timed aliquots of stored RBCs were subjected to tandem chromatography/mass spectrometry analysis to elucidate metabolic changes in the RBCs during storage.
Results
Using independent inbred mouse strains as donors, we found substantial strain-specific differences in post-transfusion RBC recovery in vivo following standardized refrigerated storage in vitro. Poor post-transfusion RBC recovery correlated with reproducible metabolic variations in the stored RBC units, including increased lipid peroxidation, decreased levels of multiple natural antioxidants, and accumulation of cytidine. Strain-dependent differences were also observed in eicosanoid generation (i.e. prostaglandins and leukotrienes).
Conclusion
These findings provide the first evidence of strain-specific metabolomic differences following refrigerated storage of murine RBCs. They also provide the first definitive biochemical evidence for strain specific variation of eicosanoid generation during RBC storage. The molecules described that correlate with RBC storage quality, and their associated biochemical pathways, suggest multiple causal hypotheses that can be tested regarding predicting the quality of RBC units prior to transfusion and developing methods of improved RBC storage.
doi:10.1111/trf.12264
PMCID: PMC4284097  PMID: 23721209
8.  CTLA4-Ig Prevents Alloantibody Production and BMT Rejection in Response to Platelet Transfusions in Mice 
Transfusion  2012;52(10):2209-2219.
Background
Platelet transfusions can induce humoral and cellular alloimmunity. Anti-HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to platelet transfusions other than leukoreduction. Targeted blockade of T cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of platelet transfusion.
Study Design and Methods
We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused platelets. BALB/c (H-2d) mice and C57BL/6 (H-2b) mice were used as platelet donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c platelets and splenocytes as targets. Bone marrow transplants were carried out under reduced intensity conditioning using BALB/b (H-2b) donors and C57BL/6 (H-2b) recipients to model HLA identical transplants. Experimental groups were given CTLA4-Ig (before or after platelet transfusion) with control groups receiving isotype matched antibody.
Results
CTLA4-Ig abrogated both humoral alloimmunization (anti-H-2d antibodies) and transfusion induced bone marrow transplant rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent platelet transfusions, administration of CTLA4-Ig after initial platelet transfusion was ineffective. Delaying treatment until after platelet transfusion failed to prevent bone marrow transplant rejection.
Conclusions
These findings demonstrate a novel strategy using an FDA approved drug that has the potential to prevent the clinical sequela of alloimmunization to platelet transfusions.
doi:10.1111/j.1537-2995.2011.03550.x
PMCID: PMC4284104  PMID: 22321003
9.  Transfusion of murine RBCs expressing the human KEL glycoprotein induces clinically significant alloantibodies 
Transfusion  2013;54(1):179-189.
Background
Red blood cell (RBC) alloantibodies to non-self antigens may develop following transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans.
Study Design and Methods
We recently generated transgenic mice with RBC specific expression of the human KEL glycoprotein, with the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system.
Results
Transfusion of RBCs from KEL2 donors into wild type recipients (lacking the human KEL protein but expressing the murine KEL orthologue) resulted in dose dependent anti-KEL glycoprotein IgM and IgG antibody responses, enhanced by recipient inflammation with poly (I:C). Boostable responses were evident upon repeat transfusion, with morbid appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic following transfusion into wild type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses.
Conclusions
This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long term translational goals of these studies include improving transfusion safety for at risk patients.
doi:10.1111/trf.12217
PMCID: PMC3732531  PMID: 23621760
10.  Cost-effectiveness of Prospective Red Cell Antigen-Matching to Prevent Alloimmunization among Sickle Cell Patients 
Transfusion  2013;54(1):86-97.
Background
Sickle cell disease is associated with extensive healthcare utilization; estimated lifetime costs exceed $460,000 per patient. Approximately 30% of chronically transfused sickle cell patients become alloimmunized to red cell antigens, but these patients cannot be identified a priori. Prospective antigen-matching can prevent alloimmunization, but is costly and may not benefit most patients.
Study Design and Methods
A Markov-based model was constructed to compare the health and financial implications of four alternative antigen-matching strategies for chronically transfused sickle cell patients. The strategies varied by the selection method of patients receiving matched blood (contingent on prior alloimmunization or prospectively for all patients) and the extent of antigen-matching (limited or extensive). Direct medical costs and alloimmunization events were assessed over 10 and 20-year periods, for a hypothetical cohort of initially transfusion-naïve patients and for a dynamic population.
Results
Within a hypothetical cohort of initially transfusion-naïve patients, implementing prophylactic limited matching for chronically transfused patients instead of history-based limited matching is expected to cost an additional $766 million over 10 years, but result in 2,072 fewer alloimmunization events. Within the same cohort, implementing prospective extensive matching is expected to cost $1.86 billion more than history-based extensive matching, but result in 2,424 fewer alloimmunization events. Averting a single alloimmunization event using prospective matching would cost $369,482–769,284. Among a dynamic population over 10 years, prospective limited matching is expected to cost $358 million more than history-based limited matching.
Conclusions
While prospective matching for all transfused patients would reduce alloimmunization, this benefit requires considerable expenditure.
doi:10.1111/trf.12250
PMCID: PMC3758770  PMID: 23692415
cost-effectiveness; delayed hemolytic transfusion reaction (DHTR); red blood cells; transfusion; alloimmunization; phenotype matching; sickle cell disease; Markov model; decision-tree
11.  Evaluation of Preoperative and Intraoperative RBC Transfusion Practices at Maputo Central Hospital, Mozambique 
Transfusion  2013;54(1):42-48.
BACKGROUND
The purpose of this study was to evaluate preoperative and intraoperative blood transfusion practices in Hospital Central (Maputo, Mozambique) and estimate the number of potentially avoidable transfusions.
STUDY DESIGN AND METHODS
A retrospective cohort study was performed. Age, comorbidities, hemoglobin, the potential for blood loss, and units of packed red blood cell (RBC) transfusions were recorded. Preoperative transfusions were evaluated to determine whether they met criteria established by the Mozambican Ministry of Health as well as proposed guidelines based on more restrictive protocols. Avoidable blood transfusions were defined as those preoperative transfusions that were not indicated based on these guidelines. Multivariate logistic regression was used to identify factors that predicted transfusion.
RESULTS
Two-hundred and five patients (age range: 0.1 - 86 years) underwent surgery in the main operating room during the two-week study period. Overall, thirty-five (17%) patients received sixty-eight transfusions. Of these, thirty-six transfusions were given preoperatively and thirty-two were given intraoperatively. Thirty-six percent of preoperative transfusions were avoidable according to national guidelines. Ninety-two percent were avoidable using more restrictive guidelines. The primary predictors of preoperative blood transfusion were lower hemoglobin (odd's ratio 0.390 / 1 g/dl; p<0.0001) and the potential for blood loss (odd's ratio 3.73; p=0.0410).
CONCLUSIONS
Adherence to existing hemoglobin thresholds recommended by national blood transfusion guidelines could significantly reduce the number of transfusions and the association risk of transfusion-transmissible infections. Adoption of more restrictive guidelines is recommended to further improve blood transfusion utilization and further reduce the transmission risk of HIV and hepatitis.
doi:10.1111/trf.12252
PMCID: PMC3751985  PMID: 23692441
preoperative; intraoperative; blood transfusion; HIV; transfusion-transmissible infections
12.  In vivo reduction of cell-free methemoglobin to oxyhemoglobin results in vasoconstriction in canines 
Transfusion  2013;53(12):10.1111/trf.12162.
BACKGROUND
Cell-free hemoglobin (Hb) in the vasculature leads to vasoconstriction and injury. Proposed mechanisms have been based on nitric oxide (NO) scavenging by oxyhemoglobin (oxyHb) or processes mediated by oxidative reactions of methemoglobin (metHb). To clarify this, we tested the vascular effect and fate of oxyHb or metHb infusions.
STUDY DESIGN AND METHODS
Twenty beagles were challenged with 1 h similar infusions of (200uM) metHb (n=5), oxyHb (n=5), albumin (n=5), or saline (n=5). Measurements were taken over 3 h.
RESULTS
Infusions of the two pure Hb species resulted in increases in mean arterial blood pressure (MAP), systemic vascular resistance index, and NO consumption capacity of plasma (all p<0.05) with the effects of oxyHb being greater than that from metHb (MAP; increase 0 to 3h; 27±6 % vs.7±2 %, respectively) (all p<0.05). The significant vasoconstrictive response of metHb (vs. albumin and saline controls) was related to in vivo auto-reduction of metHb to oxyHb, and the vasoactive Hb species that significantly correlated with MAP was always oxyHb, either from direct infusion or after in vivo reduction from metHb. Clearance of total Hb from plasma was faster after metHb than oxyHb infusion (p<0.0001).
CONCLUSION
These findings indicate that greater NO consumption capacity makes oxyHb more vasoactive than metHb. Additionally, metHb is reduced to oxyHb post-infusion and cleared faster or is less stable than oxyHb. Although we found no direct evidence that metHb itself is involved in acute vascular effects, in aggregate, these studies suggest that metHb is not inert and its mechanism of vasoconstriction is due to its delayed conversion to oxyHb by plasma-reducing agents.
doi:10.1111/trf.12162
PMCID: PMC3686899  PMID: 23488474
methemoglobin; cell-free hemoglobin; nitric oxide; vasoconstriction; hemoglobin
13.  Neutrophils release extracellular DNA traps during storage of red blood cell units 
Transfusion  2013;53(12):10.1111/trf.12203.
BACKGROUND
Blood transfusion is associated with an increased risk of organ damage, infection and alloimmunity. Neutrophil extracellular traps (NETs) are extracellular chromatin fibers decorated with neutrophil granular proteins that have been linked to cytotoxicity, thrombosis and autoimmunity. We questioned whether neutrophils in blood products release NETs during storage and thus could contribute to adverse reactions from blood transfusions.
STUDY DESIGN AND METHODS
We analyzed supernatants and blood smears of human red blood cell (RBC) units that either were or were not leukoreduced before storage for markers of NETs.
RESULTS
We identified extracellular DNA, which was associated with histones and myeloperoxidase, a marker of neutrophil granules, in supernatants and blood smears of non-leukoreduced RBC units. These markers of NETs were absent in leukoreduced RBC units. Importantly, NETs passed through blood transfusion filters and could therefore potentially be infused into patients.
CONCLUSIONS
Our studies indicate that NETs are liberated during storage of non-leukoreduced RBC units. Future studies should address whether NETs in RBC units could potentially contribute to transfusion-associated complications.
doi:10.1111/trf.12203
PMCID: PMC3728163  PMID: 23560771
Neutrophil extracellular traps; NETs; DNA; histones; red blood cell units; blood transfusion; storage
14.  Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay 
Transfusion  2014;54(9):2237-2244.
Background
Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA).
Study Design and Methods
A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA.
Results
Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR.
Conclusion
The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti.
doi:10.1111/trf.12763
PMCID: PMC4163072  PMID: 24995863
15.  Risk-adjusted clinical outcomes in patients enrolled in a bloodless program 
Transfusion  2014;54(10 0 2):2668-2677.
BACKGROUND
Although clinical outcomes have been reported for patients who do not accept allogeneic blood transfusion (ABT), many previous studies lack a control group, fail to use risk adjustment, and focus exclusively on cardiac surgery.
STUDY DESIGN AND METHODS
We report a risk-adjusted, propensity score–matched, retrospective case-control study of clinical outcomes for inpatients who did not accept ABT (bloodless, n = 294) and those who did accept ABT (control, n = 1157). Multidisciplinary specialized care was rendered to the bloodless patients to conserve blood and optimize clinical outcomes. Differences in hemoglobin (Hb), mortality, five morbid outcomes, and hospital charges and costs were compared. Subgroups of medical and surgical patients were analyzed, and independent predictors of outcome were determined by multivariate analysis.
RESULTS
Overall, mortality was lower in the bloodless group (0.7%) than in the control group (2.7%; p = 0.046), primarily attributed to the surgical subgroup. After risk adjustment, bloodless care was not an independent predictor of the composite adverse outcome (death or any morbid event; p = 0.91; odds ratio, 1.02; 95% confidence interval, 0.68–1.53). Discharge Hb concentrations were similar in the bloodless (10.8 ± 2.7 g/dL) and control (10.9 ± 2.3 g/dL) groups (p = 0.42). Total and direct hospital costs were 12% (p = 0.02) and 18% (p = 0.02) less, respectively, in the bloodless patients, a difference attributed to the surgical subgroup.
CONCLUSIONS
Using appropriate blood conservation measures for patients who do not accept ABT results in similar or better outcomes and is associated with equivalent or lower costs. This specialized care may be beneficial even for those patients who accept ABT.
doi:10.1111/trf.12752
PMCID: PMC4234090  PMID: 24942198
16.  Restless Legs Syndrome, pica, and iron status in blood donors 
Transfusion  2013;53(8):1645-1652.
BACKGROUND
The association of blood donation related iron deficiency with pica or Restless Leg Syndrome (RLS) remains poorly elucidated. This study evaluated the prevalence of RLS and pica in blood donors completing the REDS-II Iron Status Evaluation (RISE) Study.
STUDY DESIGN AND METHODS
RISE enrolled 2425 blood donors in a prospective cohort study; 1334 donors provided blood samples to characterize iron status and answered a questionnaire inquiring into symptoms of RLS and pica at a final visit after 15–24 months of follow-up. Associations between both conditions and iron status were evaluated.
RESULTS
There were 9% and 20% of donors reporting symptoms of Probable or Probable/Possible RLS, respectively. Iron depletion and donation intensity were not predictive of RLS. Pica was reported by 65 donors (5.5%), half of whom reported daily cravings. Prevalence of pica increased with degree of iron depletion in women (2% in iron replete females, 13% in those with ferritin < 12ng/mL), but not in men. Probable RLS and pica co-expressed in 8 individuals, but no more frequently than expected by chance.
CONCLUSION
RLS and pica have been associated with iron deficiency in non-donor populations. This study indicates a potentially high prevalence of RLS in frequent blood donors but shows no association with iron status or donation intensity. Low iron stores were associated with higher prevalence of pica, but only in females. Furthermore, the results are incompatible with RLS and pica sharing a common pathophysiology.
doi:10.1111/trf.12260
PMCID: PMC4226336  PMID: 23763445
blood donor; iron depletion; donation interval; pica; Restless Legs Syndrome
17.  The Cost-effectiveness of Platelet Additive Solution to Prevent Allergic Transfusion Reactions 
Transfusion  2013;53(11):2609-2618.
Background
Allergic transfusion reactions (ATRs) are among the most common complications of transfusion. Storage in platelet additive solution (PAS) has been shown to reduce ATRs from apheresis platelets (APs). This study evaluated the cost-effectiveness of using PAS storage as an alternative method to reduce ATRs.
Study Design and Methods
A Markov-based decision tree was constructed to compare ATR rates and associated costs expected from current practice and from alternative strategies of using APs stored in PAS. The potential use of premedication was also incorporated. Using a hospital perspective and including direct medical expenses only (US$2012), Monte Carlo microsimulations were run to evaluate outcomes under a base-case analysis. One-way and probabilistic sensitivity analyses were used to assess outcome uncertainty.
Results
Under base-case parameters, using APs stored in PAS for all patients as an initial transfusion protocol is expected to avert ATRs and associated costs, as compared to current practice. Using PAS for all patients along with premedication would be cost-saving only when the additional cost of PAS is below $9.14. If PAS storage could eliminate premedication use, it is expected to result in cost savings when the additional unit cost of PAS is under $11.90. At a PAS cost of $15, averting 1 ATR would cost $701.95. Using PAS storage only in response to recurring mild ATRs is associated with cost savings under all costs of PAS evaluated.
Conclusions
Using PAS storage for all AP transfusions to prevent ATRs may be financially and clinically beneficial, as compared to current practice.
doi:10.1111/trf.12095
PMCID: PMC3650119  PMID: 23363552
allergic transfusion reaction (ATR); platelet additive solution (PAS); platelet; plasma; urticaria; hives; anaphylaxis; hypersensitivity; Markov model; decision-tree
18.  D category IV: a group of clinically relevant and phylogenetically diverse partial D 
Transfusion  2013;53(11 0 2):10.1111/trf.12145.
Background
The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing.
Study design and methods
RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels.
Results
DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV type 1.0 harboring 350H and the dispersed amino acids 62F, 137V and 152T. The DIVb phenotype is expressed by DIV type 3 to type 5 representing RHD-CE-D hybrids. 4 of the 6 postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and 7 likely immunized by pregnancy. 2 DIV related alleles are newly described: DWN which differs from DIV type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density.
Conclusion
DIV alleles arose from at least 2 independent evolutionary events. DIV type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV type 2 to type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted.
doi:10.1111/trf.12145
PMCID: PMC3681876  PMID: 23461862
19.  Red Blood Cell Transfusion-Related Necrotizing Enterocolitis in Very Low Birth Weight Infants: A Near-Infrared Spectroscopy Investigation 
Transfusion  2013;53(11):2650-2658.
Background
Recent evidence suggests that antecedent packed red blood cell (PRBC) transfusions increase the risk for necrotizing enterocolitis (NEC), the most common gastrointestinal emergency encountered by very low birth weight (VLBW) infants. The underlying mechanism for this association is unknown. Altered oxygenation of the mesenteric vasculature during PRBC transfusion has been hypothesized to contribute to NEC development and was investigated in this study.
Study design and methods
Oxygenation patterns among four VLBW infants who developed transfusion-related NEC (TR-NEC) were compared to four VLBW infants with similar gestational age who were transfused but did not develop NEC (non-NEC). Cerebral and mesenteric patterns were recorded before, during and 48 hours subsequent to PRBC transfusion using near-infrared spectroscopy technology (NIRS). Percentage change from mean baseline regional saturation (rSO2) values and cerebro- splanchnic oxygenation ratio (CSOR) were analyzed.
Results
All TR-NEC infants (24–29 weeks gestation; 705–1080 grams) demonstrated greater variation in mesenteric oxygenation patterns surrounding transfusions than non-NEC infants (27.6–30 weeks gestation; 980–1210 grams). TR-NEC infants received larger mean volumes of total blood (27.75 ml/kg ± 8.77) than non-NEC infants (15.25ml/kg ± 0.5).
Conclusion
Wide fluctuation and decreases in mesenteric oxygenation patterns are more pronounced in TR-NEC infants, especially prior to TR-NEC onset, as compared to non-NEC infants. Greater total volume of infused blood was associated with TR-NEC in preterm infants. Using NIRS, larger prospective studies are needed to further evaluate potential risk factors for NEC in this high risk population.
doi:10.1111/trf.12158
PMCID: PMC3686850  PMID: 23480548
Transfusion-related NEC; necrotizing enterocolitis; near-infrared spectroscopy
20.  Undisclosed Human Immunodeficiency Virus Risk Factors Identified through a Computer-based Questionnaire Program among Blood Donors in Brazil 
Transfusion  2013;53(11):2734-2743.
Background
HIV risk factor screening among blood donors remains a cornerstone for the safety of blood supply and is dependent on prospective donor self-disclosure and an attentive predonation interview. Residual risk of HIV transmission through blood transfusion is higher in Brazil than in many other countries. Audio computer-assisted structured-interview (ACASI) has been shown to increase self-reporting of risk behaviors.
Study design and methods
This cross-sectional study was conducted between January 2009 and March 2011 at four Brazilian blood centers to identify the population of HIV-negative eligible blood donors that answered face-to-face interviews without disclosing risks, but subsequently disclosed deferrable risk factors by ACASI. Compared to the donor interview, the ACASI contained expanded content on demographics, sexual behavior and other HIV risk factors questions.
Results
901 HIV-negative blood donors were interviewed. On the ACASI, 13% of donors (N=120) declared a risk factor that would have resulted in deferral that was not disclosed during the face-to-face assessment. The main risk factors identified were recent unprotected sex with an unknown or irregular partner (49 donors), sex with a person with exposure to blood/ fluids (26 donors), multiple sexual partners (19 donors), and male-male sexual behavior (10 donors). Independent factors associated with the disclosure of any risk factor for HIV were age (≥40 years vs. 18–25 years, AOR=0.45; 95% CI 0.23–0.88) and blood center (Hemope vs. Hemominas, AOR=2.51; 95% CI 1.42–4.44).
Conclusion
ACASI elicited increased disclosure of HIV risk factors among blood donors. ACASI may be a valuable modality of interview to be introduced in Brazilian blood banks.
doi:10.1111/trf.12166
PMCID: PMC3708980  PMID: 23521083
HIV; transfusion; blood donors; ACASI; Brazil; risk factors
21.  Molecular basis of two novel and related high-prevalence antigens in the Kell blood group system, KUCI and KANT, and their serological and spatial association with K11 and KETI 
Transfusion  2013;53(11 0 2):10.1111/trf.12200.
Background
The numerous antigens in the Kell blood group system result from missense nucleotide changes in KEL. Antibodies to antigens in this system can be clinically important. We describe six probands whose plasma contained antibodies to high-prevalence Kell antigens, and discuss their relationship.
Study design and methods
PCR amplification, direct sequencing, RFLP assays, hemagglutination, flow cytometry, and protein modeling were performed by standard methods.
Results
Proband 1 (KUCI), and her serologically-compatible sister, were heterozygous for a nucleotide change in exon 11 (KEL*1271C/T; Ala424Val). Proband 2 (KANT) was heterozygous for KEL*1283G/T (Arg428Leu) and KEL*1216C/T (Arg406Stop) in exon 11. RBCs from Proband 1 and her sister were not agglutinated by plasma from Proband 2; however, RBCs from Proband 2 were agglutinated by plasma from Proband 1. Probands 3, 4, 5, and 6 had the KEL*1391C>T change associated with the previously reported KETI− phenotype. Proband 5 was also homozygous for KEL*905T>C encoding the K11−K17+ phenotype. Hemagglutination studies revealed an association between KUCI, KANT, KETI and K11. Protein modeling indicated that whereas Ala424 and Arg428 are clustered, Val302 and Thr464 are not.
Conclusion
Ala424 in the Kell glycoprotein is associated with the high-prevalence Kell antigen, KUCI (ISBT 006032), which is detected by the antibody of Proband 1. Arg428 is associated with the high-prevalence Kell antigen, KANT (ISBT 006033). The association between KUCI, KANT, KETI, and K11, and the results of protein modeling are discussed.
doi:10.1111/trf.12200
PMCID: PMC3722303  PMID: 23560718
22.  RHCE*ceMO is frequently in cis to RHD*DAU0 and encodes a hrS–, hrB–, RH:–61 phenotype in Blacks; Clinical Significance 
Transfusion  2013;53(11 0 2):10.1111/trf.12271.
Background
RHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has nt1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the RBC antigen expression, antibody specificity, RHD association, and the prevalence in African-Americans.
Methods
Serologic testing was performed by standard methods. Genomic DNA was used for PCR-RFLP and RH- exon sequencing, and for some, Rh-cDNA was sequenced. Seventy-seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African-Americans were screened for allele prevalence.
Results
RBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weakly or non-reactive with some anti-e , and were non-reactive with polyclonal anti-hrS and anti-hrB. Twenty-three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had allo anti-e, anti-f, anti-hrS/hrB, or an antibody to a high prevalence Rh antigen. Three patients with allo-anti-c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African-Americans tested and in 69 of 77 (90%) of samples with RHCE*ceMO.
Conclusions
RHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hrS– and hrB– phenotype, and the absence of a high prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in 1 in 50 African-Americans with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion.
doi:10.1111/trf.12271
PMCID: PMC3784631  PMID: 23772606
RH genotype matching; Rh blood group; blood transfusion; high prevalence antigen; partial antigens; Rh clinical significance; RHCE alleles; RH alleles; RHD alleles
23.  External quality assessment (EQA) in molecular immunohematology: the INSTAND proficiency test program 
Transfusion  2013;53(11 0 2):10.1111/trf.12414.
Background
Genotyping for red blood cell (RBC), platelet and granulocyte antigens is a new tool for clinical pathology, transfusion medicine services and blood banks. Proficiency in laboratory tests can be established by external quality assessments (EQAs), which are required for clinical application in many health care systems. There are few EQAs for molecular immunohematology.
Methods
We analyzed the participation and pass rates in an EQA for RBC, platelet and granulocyte antigens. This EQA was distributed by INSTAND, a large non-profit provider of proficiency tests, twice per year since fall 2006 as EQA no. 235 Immunohematology A (molecular diagnostic). The coordinators defined at the outset which alleles are mandatory for detection.
Results
The number of participants steadily increased from 51 to 73 per proficiency by fall 2012. More than 60 institutions utilized this EQA at least once a year. Approximately 80% of them participated in RBC, 68% in platelet and 22% in granulocyte systems. With the exceptions of RHD (82%) and granulocytes (85%), pass rates exceeded 93%. While the pass rate increased for granulocyte and decreased for the ABO system, the pass rates for the other systems changed little over 6 ½ years.
Conclusions
The INSTAND proficiency test program was regularly used for EQA by many institutions, particularly in Central Europe. While the technical standards and pass rates in the participating laboratories were high, there has been little improvement in pass rates since 2006.
doi:10.1111/trf.12414
PMCID: PMC3830650  PMID: 24111785
24.  Laboratory variables for assessing iron deficiency in REDS-II Iron Status Evaluation (RISE) blood donors 
Transfusion  2013;53(11):2766-2775.
BACKGROUND
Iron deficiency is common in regular blood donors. We evaluated the diagnostic sensitivity and specificity of red blood cell (RBC) hematology analyzer indices to assess iron status as a part of donor management.
STUDY DESIGN AND METHODS
A total of 1659 male and female donors from the Retrovirus Epidemiology Donor Study-II (REDS-II) Donor Iron Status Evaluation (RISE) study who were either first-time/reactivated (FT/ RA; no donations for 2 years) or frequent donors were recruited into a longitudinal study of regular donation of RBCs. Of these, 1002 donors returned 15 to 24 months later for a final assessment. Absent iron stores (AIS) was defined as plasma ferritin level of less than 12 µ.g/L. Logarithm of the ratio of soluble transferrin receptor to ferritin of at least 2.07 (≥97.5% in FT/RA males) was used to define iron-deficient erythropoiesis (IDE). Receiver operating characteristics analysis was performed to assess selected RBC indices (e.g., percentage of hypochromic mature RBCs, proportion of hypochromic mature RBCs [HYPOm], and hemoglobin [Hb] content of reticulocytes [CHr]) in identifying AIS and IDE.
RESULTS
HYPOm and CHr detected IDE with comparable sensitivity, 72% versus 69%, but differed in specificity: HYPOm 68% and CHr 53%. For detecting AIS, sensitivity was improved to 85% for HYPOm and 81% for CHr but specificity was reduced for both. Venous Hb had high specificity but poor sensitivity for IDE and AIS. A plasma ferritin level of less than 26.7 u.g/L was a good surrogate for assessing IDE.
CONCLUSION
RBC indices correlate with AIS and IDE and are more informative than Hb measurement, but lack sufficient sensitivity and specificity to be used as diagnostic tools in blood donors at risk for iron deficiency.
doi:10.1111/trf.12209
PMCID: PMC3895107  PMID: 23617531
25.  Blood utilization in patients with burn injury and association with clinical outcomes 
Transfusion  2012;53(10):2212-2221.
BACKGROUND
Uncontrolled bleeding is an important cause of increased transfusion in burn victims; however, description of blood utilization patterns in the burn population is lacking.
STUDY DESIGN AND METHODS
We conducted a single-institution, retrospective cohort study to measure blood utilization in 89 consecutive burn patients with 15–65% total body surface area (TBSA) burn within 60 days of injury. We also evaluated the relationship of blood product utilization with clinical variables including anticoagulant usage and mortality.
RESULTS
We determined that: (a) the predictors for increased packed red blood cells (PRBC) and plasma transfusions were high TBSA burn and the use of argatroban anticoagulation (for suspected heparin-induced thrombocytopenia); (b) TBSA burn and patient age were independent predictors of mortality, but not PRBC or plasma transfusion; and (c) the incidence of symptomatic venous thromboembolic events is not uncommon (11.2%), although heparin-induced thrombocytopenia is rare (1.1%).
CONCLUSION
Despite concerns about adverse correlation between increased number of transfusions and mortality in other clinical settings, we did not find this association in our study. However, we demonstrated that the type and intensity of anticoagulation carries substantial risk for increased PRBC as well as plasma usage.
doi:10.1111/trf.12057
PMCID: PMC3620962  PMID: 23278449
burn injury; blood transfusion; blood utilization; mortality; anticoagulation; heparin; argatroban; venous thromboembolic events; heparin-induced thrombocytopenia

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