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Blood utilization review programs educate clinicians on guidelines for appropriate transfusion, review local transfusion practice, and provide feedback on transfusion trends. To gather data on transfusion practice, modern blood utilization programs leverage electronic medical records and computerized physician order entry with automated decision support. Data may be collected and feedback may be given in real-time for individual transfusions or retrospectively with aggregated data. Important elements for a successful program include a multidisciplinary group that can champion the effort, adequate documentation and data capture for transfusions, and regular discussions about trends with ordering clinicians. Blood utilization programs are popular because they can lower transfusion risk, improve quality outcomes, and lower costs.

Iron overload is an inevitable consequence of chronic red cell transfusions without erythrocytapheresis or chelation therapy. The effectiveness of partial manual exchange, a technique used to slow iron loading, has not been evaluated. We evaluated all children with sickle cell disease (SCD) receiving chronic transfusion to identify chelation naïve subjects who had quantitative liver iron concentration (LIC) studies. Seventeen chelation naïve children with SCD received a median of 29 transfusions prior to first LIC determination. Serum ferritin concentrations were assessed prior to each transfusion. The mean volume of blood phlebotomized prior to each transfusion was 5.1±1.8 cc/kg, which cumulatively resulted in a calculated median 35.0 mg/kg removal of iron. Using linear regression, pretransfusion phlebotomy resulted in a statistically significant reduction in ferritin (-8.8 ng/mL of ferritin for each mg/kg of iron phlebotomized, P = 0.02). A reduction in LIC from pretransfusion phlebotomy could not be established (P =0.4). Partial manual exchanges appear to be an effective strategy for slowing the pace of iron loading in the setting of chronic transfusion for SCD.

To determine if glial fibrillary acidic protein (GFAP) is associated with brain injury in children with sickle cell disease (SCD), we measured plasma GFAP among cross-sectional groups of unselected children with SCD, subsets of children with SCD and normal brain MRI or MRI evidence of cerebral infarct, healthy pediatric controls, and adults with brain injury. Children with SCD had higher plasma GFAP than healthy pediatric controls (mean concentrations 0.14 ± 0.37 vs. 0.07 ± 0.08 ng/mL; P 5 0.003); also, 16.0% (16/100) of children with SCD and cerebral infarct had GFAP elevations above the 95th percentile of healthy pediatric controls (P 5 0.04). Although not statistically significant, children with SCD and cerebral infarct had more elevated GFAP levels than with SCD and no infarct (16/100, 16.0% vs. 14/168, 8.3%; P 5 0.07). Children with SCD and acute brain ischemia had a higher proportion of elevated GFAP than SCD children with normal MRI (3/6, 50% vs.8.3%; P 5 0.01). GFAP was associated with elevated systolic blood pressure in the preceding year and correlated positively with white blood cell count and negatively with age and performance IQ. Plasma GFAP is elevated among children with SCD and may be associated with subclinical brain injury.

Recent randomized controlled trials have shown no benefit for transfusion to a hemoglobin10 g/dL compared with lower hemoglobin thresholds in the perioperative period, even among older adults. Nevertheless, physicians may choose to transfuse older adults more liberally than younger adults. It is unclear whether older patients have higher odds than younger patients of being transfused in the perioperative period. Our objective in this study was to determine whether the odds of transfusion are higher in older patients than in younger patients in the perioperative period.We conducted this retrospective observational cohort study at a tertiary care academic medical center. We included adults who had undergone a surgical procedure as an inpatient at our institution from January 2010 to February 2012. The primary analysis compared the odds of transfusion for patients65 years old with the odds of transfusion in younger patients based on multilevel multivariable logistic regression analyses including adjustment for comorbidities, surgical service, lowest in-hospital hemoglobin value, gender, and estimated surgical blood loss and accounted for clustering by the surgeon and procedure.We included 20,930 patients in this analysis. In multilevel models adjusted for comorbidities, surgical service, estimated surgical blood loss, and lowest in-hospital hemoglobin value, with surgeon and procedure as random effects, patients65 years old had 62% greater odds (odds ratio, 1.62; 95% confidence interval, 1.40-1.88; P0.0001) of being transfused than did younger patients. When patients were stratified by lowest in-hospital hemoglobin (7.00-7.99, 8.00-8.99, 9.00-9.99, and ≥10.00 g/dL), the odds of transfusion generally increased with each additional decade of age in every stratum, except for that containing patients in whom the lowest in-hospital hemoglobin did not decrease below 10 g/dL. When the odds of transfusion were compared between younger and older patients, significant differences were observed among surgical services (P = 0.02) but not among anesthesia specialty divisions (P = 0.9).Older adults have greater odds of receiving red blood cell transfusion in the perioperative period than do younger patients, despite the lack of evidence supporting higher hemoglobin triggers in elderly patients. Further research is needed to determine whether transfusion practice in the elderly is an opportunity for education to improve blood management.

BACKGROUND: The time that red cell units are stored before transfusion may be associated with postoperative complications, although the evidence is conflicting. However, the association between the length of red cell unit storage and postoperative delirium has not been explored. We hypothesized that the length of storage of transfused red cell units would be associated with delirium after cardiac surgery. METHODS: We conducted a case-control study in which patients undergoing coronary artery bypass, valve, or ascending aorta surgery with cardiopulmonary bypass at Johns Hopkins from 2005 to 2011 were eligible for inclusion. Patients were excluded if they did not receive red cell units, received >4 red cell units during hospitalization, received any transfusion after the first postoperative day, or received red cell units that were not exclusively stored for ≤14 days or >14 days. Eighty-seven patients met transfusion-related inclusion criteria and developed postoperative delirium. Controls who did not develop delirium were selected from the same source population of eligible patients and were matched 1:1 based on age (± 5 years), 2- to 2.5-year band of date of surgery, and surgical procedure. For each patient, we calculated the average storage duration of all transfused red cell units. The primary outcome was odds of delirium in patients who were transfused red cell units with exclusive storage duration >14 days compared with that of ≤14 days. Secondary outcomes were odds of delirium with each increasing day of average red cell unit storage duration. We used conditional multivariable regression to test our hypotheses. RESULTS: In conditional multivariable analysis of 87 case-control pairs, there was no difference in the odds of patients developing delirium if they were transfused red cell units with an exclusive storage age >14 days compared with that ≤14 days (odds ratio [OR] 1.83; 95% confidence interval, 0.73–4.58, P = 0.20). Each additional day of average red cell unit storage beyond 14 days was associated with a 1.01- to 1.13-fold increase in the odds of postoperative delirium (OR, 1.07; P = 0.03). Each additional day of average storage beyond 21 days was associated with a 1.02- to 1.23-fold increase in the odds of postoperative delirium (OR, 1.12; P = 0.02). CONCLUSIONS: Transfusion of red cell units that have been stored for >14 days is not associated with increased odds of delirium. However, each additional day of storage >14 or 21 days may be associated with increased odds of postoperative delirium in patients undergoing cardiac surgery. More research is needed to further characterize the association between delirium and storage duration of transfused red cell units.

Transfusion-related characteristics have been hypothesized to cause allergic transfusion reactions (ATRs) but they have not been thoroughly studied. The primary objective of this study is to evaluate the associations of infusion rate, infusion volume, ABO mismatching, component age, and pretransfusion medication with the incidence and severity of ATRs. A secondary objective is to compare the risk of these attributes relative to the previously reported risk factor for aeroallergen sensitization in transfusion recipients, as measured by an aeroallergen-specific immunoglobulin (Ig)E antibody screen.Clinical and transfusion-related data were collected on subjects with reported ATRs and uneventful (control) apheresis platelet (PLT) transfusions over a combined 21-month period at two academic medical centers. Control transfusions were selected as the next uneventful transfusion after an ATR was reported. Logistic regression, Mann-Whitney, and t tests were used to assess associations with ATRs. Previously reported aeroallergen-specific IgE screening data were incorporated into a multivariable logistic regression.A total of 143 ATRs and 61 control transfusions were evaluated among 168 subjects, ages 2 to 86 years. Infusion rate, infusion volume, ABO mismatching, component age, and pretransfusion medication showed no significant association with ATRs (p 0.05). Neither infusion rate nor infusion volume increased the risk of anaphylaxis versus mucocutaneous-only ATRs. Aeroallergen sensitization has previously been associated with ATRs. After transfusion-related covariates were controlled for, aeroallergen sensitization remained significantly associated with ATRs (odds ratio, 2.68; 95% confidence interval, 1.26-5.69).Transfusion- and component-specific attributes are not associated with ATRs. An allergic predisposition in transfusion recipients is associated most strongly with ATR risk.

The effect of mixed chimerism on the pace of post-transplant immune reconstitution is unknown. Using flow cytometry, recall and neo-antigen vaccine responses, and T cell receptor recombination excision circle (TREC) quantification, we evaluated phenotypic and functional characteristics of T and B cells in nine patients following non-myeloablative, HLA-identical peripheral blood stem cell transplantation for chronic granulomatous disease. Engraftment of T cell, B cell, and myeloid lineages proceeded at similar paces within each patient, but engraftment kinetics segregated patients into two groups: adults, who became full donor T cell chimeras before 6 months (rapid engrafters) and children, who became full donor T cell chimeras after 6 months or not at all (slow engrafters). Quantitative B cell recovery was achieved by 6 weeks after transplantation in children, but was delayed until 1 year in adults. Early quantitative B cell recovery was not accompanied by an early humoral immune response to tetanus toxoid (TT). Emergence of TT-specific T cell responses coincided with naive T cell reconstitution, as measured by CD4/CD45RA T cell recovery and TREC quantification. These data suggest that immune reconstitution occurs faster in pediatric patients who have prolonged mixed hematopoietic chimerism compared to adults, who have rapid donor stem cell engraftment.

Importance Sickle cell disease (SCD) is a life-threatening genetic disorder affecting nearly 100 000 individuals in the United States and is associated with many acute and chronic complications requiring immediate medical attention. Two disease-modifying therapies, hydroxyurea and long-term blood transfusions, are available but underused. Objective To support and expand the number of health professionals able and willing to provide care for persons with SCD. Evidence Review Databases of MEDLINE (including in-process and other nonindexed citations), EMBASE, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, CINAHL, TOXLINE, and Scopus were searched using prespecified search terms and keywords to identify randomized clinical trials, nonrandomized intervention studies, and observational studies. Literature searches of English-language publications from 1980 with updates through April 1, 2014, addressed key questions developed by the expert panel members and methodologists. Findings Strong recommendations for preventive services include daily oral prophylactic penicillin up to the age of 5 years, annual transcranial Doppler examinations from the ages of 2 to 16 years in those with sickle cell anemia, and long-term transfusion therapy to prevent stroke in those children with abnormal transcranial Doppler velocity (≥200 cm/s). Strong recommendations addressing acute complications include rapid initiation of opioids for treatment of severe pain associated with a vasoocclusive crisis, and use of incentive spirometry in patients hospitalized for a vasoocclusive crisis. Strong recommendations for chronic complications include use of analgesics and physical therapy for treatment of avascular necrosis, and use of angiotensin-converting enzyme inhibitor therapy for microalbuminuria in adults with SCD. Strong recommendations for children and adults with proliferative sickle cell retinopathy include referral to expert specialists for consideration of laser photocoagulation and for echocardiography to evaluate signs of pulmonary hypertension. Hydroxyurea therapy is strongly recommended for adults with 3 or more severe vasoocclusive crises during any 12-month period, with SCD pain or chronic anemia interfering with daily activities, or with severe or recurrent episodes of acute chest syndrome. A recommendation of moderate strength suggests offering treatment with hydroxyurea without regard to the presence of symptoms for infants, children, and adolescents. In persons with sickle cell anemia, preoperative transfusion therapy to increase hemoglobin levels to 10 g/dL is strongly recommended with a moderate strength recommendation to maintain sickle hemoglobin levels of less than 30% prior to the next transfusion during long-term transfusion therapy. A strong recommendation to assess iron overload is accompanied by a moderate strength recommendation to begin iron chelation therapy when indicated. Conclusions and Relevance Hydroxyurea and transfusion therapy are strongly recommended for many individuals with SCD. Many other recommendations are based on quality of evidence that is less than high due to the paucity of clinical trials regarding screening, management, and monitoring for individuals with SCD.

Background: A general understanding of allergic transfusion reaction (ATR) mechanisms remains elusive. Various hypotheses invoke proteins, small molecules, mitochondria, or microparticles that may be plasma or platelet derived and suggest antibody dependent or independent mechanisms. There has been no systematic comparison of these proposed mechanisms. The aim of this study is to characterize the mechanistic determinants of ATRs. Methods: Basophil enriched cell suspensions were collected from healthy donors (n=8). Basophil histamine release was measured in response to platelet-derived components: platelet component supernatant (plasma), platelet lysate, and manipulated platelet lysates were examined to characterize the unknown allergic stimuli. Lysate manipulations were: 1) dialysis against a 3,500 molecular weight cutoff membrane, 2) butanol/DIPE delipidation, 3) trypsinization with immobilized trypsin beads, 4) mild heat denaturation at 47˚C for 20 minutes, and 5) ultracentrifugation at 200,000 gfor 1hr. Immunoglobulin-dependent mechanisms were investigated through lactic acid immunoglobulin depletion from the basophil cell surface and signaling inhibition with ibrutinib. Histamine release from magnetic bead isolated platelet mitochondria was compared to platelet lysate with or without DNase treatment. Platelet lysate histamine content represented an average of Results: Robust, dose-responsive histamine release to platelet lysate was observed in two of eight healthy donors. No histamine release was observed with plasma. Reactivity did not correlate with the clinical allergic phenotype, and reactive donors were nulliparous and had no prior transfusion. Trypsin treatment of platelet lysate reduced histamine release by 39% (p=0.008). Delipidation of platelet lysate decreased histamine release by 20% (p=0.051). Dialysis, ultracentrifugation, and mild heat denaturation of platelet lysate did not significantly affect histamine release, compared to unmanipulated platelet lysate. To investigate the immunoglobulin dependence of basophil activation, in separate experiments we 1) depleted immunoglobulins, including IgE, from the basophil cell surface, and 2) inhibited immunoglobulin-mediated intracellular signaling with ibrutinib. Histamine release in response to platelet lysate significantly decreased in both cases. Finally, isolated platelet mitochondria induced minimal basophil histamine release, and DNase treatment did not inhibit activation of basophils by platelet lysate. Conclusion: Type I immediate hypersensitivity to platelet, not plasma, proteins is a primary mechanism for ATRs. Small molecules, microparticles, and mitochondria are not significant contributors to ATRs in this human basophil model. Prior sensitization to human proteins is not required for basophil responses to platelet proteins. Donor variability and storage conditions that promote accumulation of soluble platelet-derived proteins may contribute to ATRs. Disclosures No relevant conflicts of interest to declare.

Introduction: Non-stem cell therapies including cellular vaccines and chimeric antigen receptor (CAR) T cells are experiencing explosive growth in research development and clinical applications. Demand is growing, with several CAR products heading for imminent FDA approval. The variety of products poses a challenge to even experienced cancer centers. Our goal was to create clinical and operational infrastructure to deliver these therapies efficiently and safely. We leveraged existing workflows and care models spanning inpatient and outpatient settings and disease groups previously unfamiliar with cell therapies. Methods: We conducted over 30 internal stakeholder interviews across research, cell processing, nursing, physician, pharmacy, and operational areas and queried experts at other cell-engineering sites. Key areas identified were 1) cell distribution, 2) trial initiation, 3) communication, training, and clinical care, 4) resource utilization, and 5) safety and outcome monitoring. A steering committee formulated guiding principles: make safety paramount, systematically coordinate care, recognize each cell therapy and sponsor's unique requirements, and minimize unnecessary infrastructure. Aspects of the care spectrum evaluated spanned from patient identification, financial clearance, outpatient workup, cell collection, admission/administration, to post-infusion follow-up. Results: 1) Cell distribution: despite product manufacturing at central contracted sites, novel internal workflows were required to ensure chain of identity through multiple hand-offs. A standard questionnaire was devised to determine unique trial hurdles and a consistent approach to labeling, shipping and communication with sponsors and clinicians. Biweekly meetings between apheresis, cell processing, nursing, and pharmacy staff to discuss logistics and impediments were established. (Figure) 2) Trial initiation: A "start up kit" was developed to explain key entities and workflows (e.g. apheresis, pharmacy for tocilizumab, biosafety review) to help investigators unfamiliar with cell therapies navigate submission. 3) Communication, training and clinical care: guidance in budget design, tools to flag high-risk patients in electronic medical records (EMR), and education on nomenclature were developed. Over 20 general education sessions were conducted across clinical and operational areas, in addition to trial-specific training, for financial coordinators, patient safety groups, MDs, residents, PAs and nurses in all disease areas both inpatient and outpatient, even statisticians. We created email distribution lists, EMR flags, a case management system, and toxicity management order sets to ensure awareness of admissions, availability of crucial medications, recognition of cytokine release or neurotoxicity requiring unique intervention, and priming of ICU resources. A weekly forum for MDs and clinical stakeholders to share clinical events and needs was instituted. 4) Resource utilization: shortfalls were identified in cell-collection capacity and scheduling, product distribution, and clinical expertise. Dedicated clinician positions were created, and scheduling was consolidated to optimize capacity within and outside existing BMT infrastructure. 5) Safety and outcome monitoring: a format and forum was developed for quarterly review of major outcomes allowing institutional oversight over the spectrum of investigational cell therapies and standard of care administrations. Discussion: With the explosion in outpatient cancer vaccines and CAR T cells, the need for additional physical resources, new medications, experienced clinicians, and communication tools became evident. We describe our approach to creating novel workflows to ensure patients receive the latest cell therapies safely, timely distribution of cells, educated ED and ICU involvement, and rapid expert management including anti-cytokine therapy. The investment of individual centers to bring cellular therapies safely into clinical care should not be under-appreciated, particularly given the high-risk nature of these treatments and the unique processes and high-coordinated activities these new modalities require. Regulatory agencies, such as FACT, are likewise requesting that such safety measures, standard procedures and oversight be addressed. Figure Figure. Disclosures Soiffer: Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Consultancy.

Allergic transfusion reactions (ATRs) are a spectrum of hypersensitivity reactions that are the most common adverse reaction to platelets and plasma, occurring in up to 2% of transfusions. Despite the ubiquity of these reactions, little is known about their mechanism. In a small subset of severe reactions, specific antibody has been implicated as causal, although this mechanism does not explain all ATRs. Evidence suggests that donor, product, and recipient factors are involved, and it is possible that many ATRs are multi-factorial. Further understanding of the mechanisms of ATRs is necessary so that rationally designed and cost-effective prevention measures can be developed.

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.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 pretransfusion medication 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.Under base-case variables, using APs stored in PAS for all patients as an initial transfusion protocol is expected to avert ATRs and associated costs, compared to current practice. Using PAS for all patients along with pretransfusion medication would be cost-saving only when the additional cost of PAS is below $9.14. If PAS storage could eliminate pretransfusion medication 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 one 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.Using PAS storage for all AP transfusions to prevent ATRs may be financially and clinically beneficial, compared to current practice.

Background Recent literature suggests that a restrictive approach to red blood cell transfusions is associated with improved outcomes in cardiac surgery patients. Even in the absence of bleeding, intravascular fluid shifts cause hemoglobin levels to drift postoperatively, possibly confounding the decision to transfuse. The purpose of this study was to define the natural progression of hemoglobin levels in postoperative cardiac surgery patients. Methods All cardiac surgery patients from October 2010 through March 2011 who did not receive a postoperative transfusion were included. Primary stratification was by intraoperative transfusion status. Change in hemoglobin was evaluated relative to the initial postoperative hemoglobin. Maximal drift was defined as the maximum minus the minimum hemoglobin for a given hospitalization. Final drift was defined as the difference between initial and discharge hemoglobin. Results The final cohort included 199 patients: 71 (36%) received an intraoperative transfusion, whereas 128 (64%) did not. The average initial and final hemoglobin levels for all patients were 11.0 ± 1.4 g/dL and 9.9 ± 1.3 g/dL, respectively, giving a final drift of 1.1 ± 1.4 g/dL. The maximal drift was 1.8 ± 1.1 g/dL and was similar regardless of intraoperative transfusion status ( p = 0.9). Although all patients' hemoglobin initially dropped, 79% of patients reached a nadir and experienced a mean recovery of 0.7 ± 0.7 g/dL by discharge. On multivariable analysis, increasing cardiopulmonary bypass time was significantly associated with total hemoglobin drift (coefficient/hour, 0.3 [0.1–0.5] g/dL; p = 0.02). Conclusions In this report of hemoglobin drift after cardiac surgery, although all postoperative patients experienced downward hemoglobin drift, 79% of patients exhibited hemoglobin recovery before discharge. Physicians should consider the eventual upward hemoglobin drift before administering red blood cell transfusions.

Concentrating and washing apheresis platelets (APs) substantially reduce the number of allergic transfusion reactions likely due to removal of plasma. However, these processes may damage platelets (PLTs). This study evaluated whether concentrating or washing APs decrease the corrected count increment (CCI).This retrospective study evaluated individuals who initially received unmanipulated APs and subsequently received concentrated and/or washed APs at a large university hospital between 1998 and 2009. Concentrated units were prepared by reducing the plasma volume of APs by a goal of more than 67%. Washed units were prepared by washing the APs with 1 L of normal saline. The CCI (PLTs [×10(6)] × m(2)/L) for all transfusions was calculated. Hypothesis testing was performed with t tests for continuous variables and chi-square tests for dichotomous variables.We evaluated 121 individuals: 46 patients who received unmanipulated, concentrated, and then washed APs; 59 patients who received unmanipulated and then concentrated APs; and 16 patients who received unmanipulated and then washed APs. Patient demographics were similar among the three groups. The mean CCI for unmanipulated AP transfusions at 0 to 2 hours posttransfusion was significantly higher than concentrated and washed PLT transfusions (p0.001). When accounting for PLT loss due to manipulation, concentrating APs did not impact the CCI. However, the CCI remained significantly lower for washed products at all time points after transfusion (40.7% mean reduction at 20-24 hr, p0.001).Washing APs significantly reduces PLT count recovery and survival, as demonstrated by a significantly reduced CCI.

The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet (PLT) products associated with ATRs and controls.Using bead-based and standard enzyme-linked immunosorbent assays, we tested supernatants from 20 consecutive apheresis PLT transfusions associated with ATRs and 30 control products for concentrations of mediators in three categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth and/or priming factors of basophils and mast cells.Median concentrations of the direct allergic agonists C5a, brain-derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6, 41.8, and 13.9% higher, respectively, in the supernatant of apheresis PLT products that were most strongly associated with ATRs (p0.05 for each mediator). Other direct agonists (macrophage inflammatory protein-1α, monocyte chemotactic protein-1, eotaxin-1, interleukin-8) were similar between groups. Concentrations of acute inflammatory mediators and basophil growth and/or priming factors were also similar between groups (p0.2 for all associations).The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis PLT products. Acute inflammatory proteins and basophil and/or mast cell growth and priming factors do not appear to be associated with apheresis PLT products that cause ATRs.

Objective Glial fibrillary acidic protein (GFAP) is specific to astrocytes in the central nervous system. We hypothesized that serum GFAP would be increased in neonates with hypoxic-ischemic encephalopathy (HIE) treated with whole-body cooling. Study Design We measured GFAP at birth and daily for up to 7 days for neonates in the intensive care unit. We compared neonates with HIE treated with whole-body cooling to gestational age–matched controls without neurological injury and neonates with HIE by brain abnormalities on magnetic resonance imaging (MRI). Results Neonates with HIE had increased GFAP levels compared with controls. Neonates with HIE and abnormal brain imaging had elevated GFAP levels compared with neonates with HIE and normal imaging. Conclusion Serum GFAP levels during the first week of life were increased in neonates with HIE and were predictive of brain injury on MRI. Biomarkers such as GFAP could help triage neonates with HIE to treatment, measure treatment efficacy, and provide prognostic information.

The biologic mechanisms of allergic transfusion reactions (ATRs) are largely unknown. We sought to compare the atopic predisposition of platelet (PLT) recipients who experienced an ATR to nonreactive control recipients.We identified 37 consecutive apheresis PLT recipients who experienced an ATR and 26 matched controls. Total immunoglobulin (Ig)E and aero- and food allergen-specific IgE were quantified in plasma by a blood test for allergies (ImmunoCAP: Phadiatop and Fx5). IgE testing of apheresis PLT supernatants was also performed.Pruritus and urticaria were manifest in 91.9 and 83.8% of all ATRs, with more severe respiratory symptoms and angioedema occurring in less than 15% of cases. No subject had anaphylaxis. Sex, age, and primary diagnosis were balanced between the two groups. Total and aeroallergen-specific IgE was higher among subjects experiencing an ATR in comparison to control subjects (median total IgE, 55.5 kU/L vs. 8.3 kU/L, p = 0.002; and median aeroallergen-specific IgE, 0.57 kUa/L vs. 0.36 kUa/L, p = 0.046). IgE antibody levels in apheresis products associated with ATRs were similar to control products (p0.1 for all IgE tests).Recipient atopic predisposition, as defined by IgE sensitization, is a risk factor associated with ATRs.

A 12-year-old boy with HbSS sickle cell disease (SCD) was admitted with an acute febrile illness and developed overt stroke 3 days later. Plasma glial fibrillary acidic protein levels were elevated, as compared to pediatric controls, 32 h prior to the clinical diagnosis of stroke, peaked immediately prior to the exchange transfusion, and remained elevated 1 year later despite chronic transfusion therapy. Stroke in SCD can occur in the setting of acute illness, and a biomarker that could predict the onset and triage ill children to therapeutic intervention more quickly would be useful.

Objective Demonstrate that high-dose cyclophosphamide (CY) is effective therapy for hepatitis-associated aplastic anemia (HAA). Background HAA is a sequence of seronegative hepatitis followed by aplastic anemia. Optimal treatment is matched-sibling allogeneic bone marrow transplantation (BMT). The combination of antithymocyte globulin (ATG) and cyclosporine (CSA) has also been studied, but there are scarce data regarding treatment of HAA. Procedure Five patients (median age 14 years; range 6–17 years) with HAA and without an HLA-matched sibling were treated with high-dose CY (50 mg/kg/day IV × 4 days) followed by granulocyte-colony stimulation factor (G-CSF). Results After at least 1 year of follow-up, four of five patients are in remission without further immune suppression beyond high-dose CY. Of the 4 responders, median time to absolute neutrophil count (ANC) >500 µl−1 was 51 days (range 44–369). Median time to transfusion independence for erythrocytes and platelets was 109 (range 57–679) and 160 (range 48–679) days, respectively. The fifth patient did not respond and proceeded to an unrelated donor transplant. One patient met criteria for autoimmune hepatitis (AIH) in addition to HAA. In this case, high-dose CY successfully induced remission of both diseases. Conclusions High-dose CY induces durable remissions in HAA and may be an effective treatment for AIH. Pediatr Blood Cancer 2007;49:947–951. © 2007 Wiley-Liss, Inc.

In this study, we have identified an altered B cell compartment in patients with chronic granulomatous disease (CGD), a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH activity. This is characterized by an expansion of CD5-expressing B cells, and profound reduction in B cells expressing the memory B cell marker, CD27. Both findings were independent of the age, genotype, and clinical status of the patients, and were not accompanied by altered CD5 and CD27 expression on T cells. Focusing on CD27-positive B cells, considered to be memory cells based on somatically mutated Ig genes, we found that the reduction was not caused by CD27 shedding or abnormal retention of CD27 protein inside the cell. Rather, it was determined that CD27-negative B cells were, appropriately, CD27 mRNA negative, consistent with a naive phenotype, whereas CD27-positive B cells contained abundant CD27 mRNA and displayed somatic mutations, consistent with a memory B cell phenotype. Thus, it appears that CGD is associated with a significant reduction in the peripheral blood memory B cell compartment, but that the basic processes of somatic mutation and expression of CD27 are intact. X-linked carriers of CGD revealed a significant correlation between the percentage of CD27-positive B cells and the percentage of neutrophils with normal NADPH activity, reflective of the degree of X chromosome lyonization. These results suggest a role for NADPH in the process of memory B cell formation, inviting further exploration of secondary Ab responses in CGD patients.

Acquired inhibitors of coagulation factors, particularly to factor V (FV) and thrombin, after topical bovine thrombin exposure may result in clinically important coagulopathies. While bovine thrombin is commonly used in pediatric patients for surgical hemostasis, the reported cases of acquired inhibitors in children are few. We report two cases of children who developed factor inhibitors after bovine thrombin exposure. One child developed a FV inhibitor at 3 months of age after exposure to bovine thrombin during cardiac surgery. The inhibitor resolved with intravenous immunoglobulin (IVIG) and steroids. The other child developed concomitant FV and bovine thrombin inhibitors after cardiac surgery at age 11 years. The presence of these inhibitors complicated post-operative anti-coagulation management, but the inhibitors were transient. In addition to these two cases, we identified all the pediatric patients with bovine thrombin-induced inhibitors who were reported in the world's literature, and reviewed their clinical characteristics. These cases underscore the fact that bovine thrombin can be antigenic in infants and children and can result in significant coagulopathies.

A 7-year-old Caribbean-American boy with a 5-month history of hemophagocytic lymphohistocytosis and previous ceftriaxone exposure presented with fever. He was empirically given amikacin and ceftriaxone after 1:00 PM on Day 1 and acutely developed back and abdominal pain, vomiting, hematuria, and hypotension. Both plasma (see figure, top panel) and urine showed hemolysis. The peripheral blood smear showed diffuse agglutination of RBCs without schistocytes (see figure, lower left). Serum LDH was elevated (1787 U/L); urine was negative for myoglobin. The patient's Hb dropped from 11.7 to 4.4 g/dL in 32 hours. The symptoms, hemolysis, and agglutination resolved (see figure, lower right) with discontinuation of ceftriaxone and two double-volume whole blood exchange transfusions. Immunohematology studies demonstrated a positive DAT due to C3 only, and serologic drug studies were positive with ceftriaxone, but not with amikacin. The RBC agglutination seen on peripheral smear was most likely due to drug/antidrug immune complexes, a theory supported by the finding of IgM ceftriaxone-dependent antibodies (titer = 32) at presentation. The titer decreased to 8 on Day 2 and to 0 by Day 5, suggesting that the antibody was consumed by the RBCs and/or removed by treatment. Drug-induced immune hemolytic anemia (DIHA) due to ceftriaxone-dependent antibodies is uncommon, but well described. The majority of documented cases have occurred in children with a history of exposure to ceftriaxone. Autoagglutination may be a feature of DIHA due to ceftriaxone. It is important to recognize and treat DIHA early, since it can be fatal, but is reversible with treatment.

Abstract 3426 Background: Allergic transfusion reactions (ATRs) occur in ∼2–3% of platelet transfusions, but their etiology remains largely unclear. Both donor and recipient factors have been shown to contribute to ATR risk. An in vitro model of ATRs would enable mechanistic studies. Objective: To develop a basophil activation test as a model of ATRs to apheresis platelets (AP) by measuring histamine release (HR) and leukotriene C4 (LTC4) production after exposing basophils to AP supernatants. Methods: Basophil-enriched suspensions (∼5–15% basophils) from healthy donors were prepared using double-Percoll density centrifugation. After washing and counting basophil numbers using Alcian blue staining, cells were resuspended in buffer containing 2 ng/mL IL-3 and 5mM Ca++/Mg++ such that 20–30×104 basophils were added in 0.025mL aliquots. IL-3 is a potent basophil priming agent and was used to model the increased recipient susceptibility to allergic reactions that has been observed clinically. Reaction tubes (1.5 mL) were set-up containing 0.025mL of 2× (twice the final conc.) of ATR/control supernatant (final titer 1:2–1:8), negative (buffer alone), and positive control reagents (100 ng/mL anti-IgE antibody, 50 ng/mL C5a, 1 μM FMLP). Cells and reaction tubes were pre-warmed separately to 37°C for 15' before aliquoting 0.025mL cell suspension to reaction tubes for a final volume of 0.050mL. After 30′ incubation at 37°C, 0.950mL of cold buffer (without IL-3 or Ca++/Mg++) was added to each reaction tube. Tubes were centrifuged 10" at ∼300g (4°C) before removing 0.900mL for histamine detection by automated fluorimetry and LTC4 detection by ELISA. AP supernatants that were (n=10) or were not (n=10) associated with ATRs were tested. HR values were determined as a percentage of the total histamine content by lysis with perchloric acid (1.6%). Results used in analysis are net HR or LTC4 production (AP sample minus IL-3 primed negative control) normalized to HR or LTC4 production observed with positive control. Results: IL-3 priming of basophils was necessary in order to evoke a response after exposure to AP supernatant. HR in response to AP incubation with unprimed basophils was HR and LTC4 production within individual reactions were correlated (Spearman rho=0.83, P No statistically significant differences were observed in HR or LTC4 production when ATR and control groups were compared (P>0.1). ATRs were stratified into clinical categories of mild (pruritus/urticaria, n=3), moderate (angioedema/dyspnea, n=5), and severe (any symptom with hypotension, n=2). There were no differences or trends in HR and LTC4 production among the categories (P>0.2). There was no correlation between pre/post transfusion tryptase elevations in recipient plasma (n=4) and HR or LTC4 production. HR and LTC4 production was similar among blood types of AP products (A: n=12, O: n=5, AB: n=3), (P>0.7). We previously reported that concentrations of C5a are higher in AP products associated with ATRs than controls. C5a content in AP supernatant modestly correlated with basophil HR release (rho: 0.47, P=0.04), but less with LTC4 production (rho: 0.27, P=0.3). Summary: An in vitro basophil activation test that measures HR and LTC4 production was developed to study ATRs. We find that 1) IL-3 priming of basophils was necessary to elicit responses to AP supernatants; 2) LTC4 and HR are correlated within each reaction; 3) there are no differences in HR and LTC4 production between ATR and control AP supernatants were observed, regardless of clinical ATR severity; and 4) HR is associated with C5a content in the AP supernatant. Conclusions: The observations of 1) an overall requirement for basophil priming and 2) the lack of correlation between the clinical severity of ATRs and basophil HR and LTC4 release implicate recipient atopic priming as a risk factor for ATRs. However, the association of AP C5a content with HR suggests a donor/product role in ATRs, as well. Studies focusing on the nature of recipient susceptibility may further elucidate mechanisms of ATRs. Disclosures: No relevant conflicts of interest to declare.

94 Background: Patients with malignant glioma often develop increased enhancement in the tumor bed after chemoradiation (CRT). Current imaging techniques cannot distinguish if these changes are due to recurrent disease or treatment-related brain injury (TRBI). Serum biomarkers could help noninvasively make this distinction. Brain-specific proteins have been used as markers of the severity of other brain injuries including stroke, hypoxia, and traumatic brain injury. We hypothesized that similar changes could be observed following brain CRT and could provide preliminary data supporting biomarker development for TRBI. Methods: Patients receiving concurrent radiation and temozolomide (TMZ) for malignant glioma were eligible. The study was approved by the institutional review board. Informed consent was obtained from all patients. Serum is sampled before CRT, in the last week of CRT, and at 1 month, 6 mos, and 1 year after CRT. Neurologic exams, cognitive testing, and imaging are performed at the same intervals. Serum samples were tested for glial fibrillary acidic protein (GFAP), neurogranin, ICAM-5, B-syn-nuclein, and brain-derived neurotrophic factor (BDNF). Samples were measured in duplicate using an electrochemiluminescent immunoassay. Results: Data are available for the first 7 patients enrolled in this study. All of the tested markers except GFAP were measurable at baseline and all other time points. None of the markers were consistently elevated during the last week of CRT or at any other time point during follow-up. Three patients had resection of suspected recurrent disease. Two had recurrent tumor and 1 had no evidence of disease. No correlation was apparent with the findings at surgery and serum levels of GFAP, neurogranin, ICAM-5, B-synuclein, or BDNF. Conclusions: This study describes a mechanism for identifying a serum protein marker of TRBI. Preliminary results did not identify elevations in any of the selected markers immediately after CRT when signs of TRBI should be apparent or at other points in the clinical course. Testing of additional serum markers is needed to identify a biomarker of TRBI which would provide valuable information for managing high-grade glioma patients.

21 Background: Current imaging techniques are unable to accurately measure tumor burden in gliomas, because they rely on blood-brain barrier (BBB) permeability that is altered following surgery, radiation, steroids, and antiangiogenic therapies. This often leads to misinterpretation of disease status and incorrect clinical decisions. The development of tumor-specific blood-based markers for gliomas is therefore critically important. This study evaluates the detectability of five novel protein biomarker candidates in the plasma of patients with gliomas: neurogranin, ICAM-5, beta-synuclein, brain-derived neurotrophic factor (BDNF), and glial fibrillary acidic protein (GFAP). These biomarker candidates were chosen because of their specificity for brain tissue. Methods: Pre- and postoperative plasma samples were obtained from patients with newly diagnosed gliomas with IRB approval. Plasma concentrations were measured in duplicates using an electrochemiluminescent immunoassay with a lower limit of quantification of >0.04ng/mL (GFAP) and >0.02 ng/mL (other markers). Results: Plasma levels were detectable for all 5 tested markers at baseline and postoperatively. Baseline levels were highly variable and postoperative GFAP levels were much higher than preoperative levels likely related to brain injury (see table). Conclusions: Five potentially useful protein markers have been identified in the plasma of patients with gliomas. Further studies are underway to assess the specificity of each marker and its ability to detect dynamic changes in tumor burden. [Table: see text]

identification of periventricular white matter injury in preterm neonates with intraventricular hemorrhage at birth Christopher P. Wayock, Elisabeth Nigrini, William J. Savage, Jacky M. Jennings, Frances J. Northington, Allen D. Everett, Ernest Graham Johns Hopkins University School of Medicine, Department of Gynecology & Obstetrics, Division of Maternal-Fetal Medicine, Baltimore, MD, Johns Hopkins University School of Medicine, Department of PathologyTransfusion Medicine, Baltimore, MD, Johns Hopkins University School of Medicine, Departments of Pediatrics, Division of General Pediatrics and Adolescent Medicine; Department of Epidemiology, Bloomberg School of Public Health, Baltimore, MD, Johns Hopkins University School of Medicine, Department of Pediatrics, Division of Neonatology, Baltimore, MD, Johns Hopkins University School of Medicine, Department of Pediatrics, Division of Cardiology, Baltimore, MD, Johns Hopkins University School of Medicine, Gynecology and Obstetrics, Division of Maternal-Fetal Medicine, Baltimore, MD OBJECTIVE: Premature neonates are at increased risk for both intraventricular hemorrhage (IVH) and periventricular white matter injury (PWMI). It is unclear which neonates with IVH will develop PWMI, which is associated with the subsequent development of cerebral palsy. Our objective is to determine if a serum biomarker can identify which neonates with IVH will develop PWMI. STUDY DESIGN: From 4/09-4/11 there were 49 neonates born between 23-34 weeks gestation diagnosed with IVH by head ultrasound performed on day of life 3 or 7. PWMI was diagnosed by head ultrasound performed at 2 months of life. A serum biomarker glial fibrillary acidic protein (GFAP) was measured in cord blood at the time of birth, in neonatal serum upon admission to the intensive care unit, and daily for the first 4 days of life. Neonates with IVH and PWMI (n 20) were compared to those with IVH alone(n 29) using t-tests, Chi-square and Fisher’s exact where appropriate. Logistic regression was used to measure the ability of GFAP to identify PWMI. RESULTS: Neonates with IVH & PWMI did not differ from those with IVH only in gestational age (26.6 / 2.7, 27.1 / 2.8 wks), birth weight (960 / 444, 952 / 366g) or incidence of cesarean delivery (60%, 48.3%). Umbilical arterial pH (7.22 / 0.2, 7.27 / 0.08) and base deficit (4.6 / 6.9 mM, 3.8 / 2.8 mM) did not differ, but those with IVH & PWMI had a significantly higher incidence of seizures (45%, 6.9%, p 0.004) and severe IVH (85%, 17.2%, p 0.0001). GFAP was significantly increased in those with IVH & PWMI on day of life 2-4 (p 0.04, 0.02, 0.02). Logistic regression showed that GFAP on day of life 4 predicted PWMI with sensitivity 41.2%, specificity 95.6%, PPV 87.5%, and NPV 68.6%. CONCLUSION: Among preterm neonates diagnosed with IVH by head ultrasound after birth, serum GFAP may identify neonates at risk for developing PWMI. Identifying these neonates during the first week of life, rather than on a head ultrasound performed at 2 months, could provide early prognostic information, assist in the triaging of these patients to investigational therapies, and help monitor the efficacy of therapy. 166 Does penile ring block anesthesia make a difference in pain response to Neonatal circumcision? Thomas Toussaint, Clones Lans, Douglas Sherlock, Joshua Fogel, Chaur-Dong Hsu Nassau University Medical Center, OBGYN, East Meadow, NY OBJECTIVE: To compare the use of oral sucrose (SUC) versus oral sucrose plus lidocaine ring block (SUC-RB) in the management of pain during neonatal circumcision. STUDY DESIGN: This is a retrospective review of 826 charts of newborns circumcised from January 1st 2008 to December 31st 2010 at our single medical center. Due to insufficient documentation, 35 charts were excluded. The charts were divided in two groups: SUC group where sucrose only was used for pain management during neonatal circumcision and SUC-RB group where 1% lidocaine ring block was added to the sucrose for pain management. Pain levels were recorded using the Neonatal Infant Pain Scale (NIPS) before the procedure, at 1 minute after starting the procedure, at 5 minutes after the procedure, and 30 minutes after the procedure. ANOVA, Pearson Chisquare test, and Fischers exact test were used for statistical analyses .Within groups, analyses were performed for each of the pain timepoints using the McNemar test. RESULTS: With regard to pain, the SUC group had a significantly greater percentage of pain than the SUC-RB group at both 1 minute (77.7% vs.69.4%; p 0.01) and 5 minutes (65.7% vs. 55.7%; p 0.004). There was no significant pain difference at 30 minutes. In the multivariate logistic regression analyses, those in the SUC group had significantly greater odds for pain at 1 minute than those in the SUC-RB group (OR: 2.02 vs. 1.45: p 0.03). No significant difference was noted at 5 minutes. There was a continuous significant decrease in pain percentage at 5 minutes and 30 minutes (p 0.001). CONCLUSION: Those in the SUC-RB group had significantly lower levels of pain at 1 minute. These findings suggest the addition of lidocaine ring block to oral sucrose is helpful to prevent pain in neonates undergoing circumcision.

Abstract 1052 Background: Chronic red cell transfusions are commonly used for the treatment and prevention of complications in sickle cell disease (SCD). Liver injury from transfusional iron overload is a recognized morbidity of chronic transfusion therapy, but little is known about the progression of liver injury over time in SCD. Methods: We conducted a retrospective cohort study of all chronically transfused people with SCD who had 2 or more serial liver biopsies at a single academic hospital. Subjects with viral hepatitis were excluded. Serum ferritin, serum ALT, chelation status, and transfusion volumes were extracted from the electronic record and validated against paper records in all subjects. Quantitative liver iron concentration (LIC) was determined at the time of biopsy by inductively coupled plasma-mass spectrometry. Core liver biopsy slides stained for iron and fibrosis were retrieved and scored in a blinded fashion by a hepatopathologist (RA) for total iron score (TIS, Deugnier, 2007) and fibrosis score (Ishak, 1995). Analyses evaluated how liver fibrosis changed over the first 2 biopsies and how changes in biomarkers correlated with changes in fibrosis. Cross sectional analyses assessed the relationship of biomarkers to the presence or absence of fibrosis. Ferritin was analyzed as an average of the 3 closest values ± 6 weeks of liver biopsy. Area under receiver operator characteristic curve (AUC) analysis, likelihood ratios for positive tests (LR+), and summary statistics were calculated using Stata v11.2. Results: 26 people had at least 2 serial core liver biopsies for evaluation (n=70 biopsies total, range 2–7 biopsies per subject). Fibrosis was Ishak grade 0 or 1 in all biopsies. Median age at first biopsy was 13.3 years and median total transfusion duration was 9.4 years. The first 2 biopsies were obtained a median of 2.3 years apart. Evaluation of the first 2 biopsies showed that fibrosis was present in 7/26 initial biopsies and 3/26 second biopsies: fibrosis regressed in 6 subjects, developed in 2 subjects and persisted in 1 subject. Among non-chelated subjects at the time of first biopsy, 2/11 had fibrosis, as compared to 5/15 subjects who had received a mean of 3.7 years of chelation at the time of first biopsy (18% vs 33%, P=0.6). Eleven subjects had 3 or more serial biopsies performed during a median of 9.2 years of chronic transfusion. There was no consistent pattern of fibrosis development, nor was there an apparent association of fibrosis with LIC over time (Figure, asterisks indicate presence of fibrosis). On a cross-sectional basis, ALT performed better than ferritin in classifying fibrosis, with ALT having an AUC of 0.80 (95% CI 0.66–0.94) and ferritin having an AUC of 0.63 (95% CI 0.38–0.87). LIC performed poorly at discriminating fibrosis from no fibrosis (AUC 0.30; 95%CI 0.0–0.82). The highest positive likelihood ratios for fibrosis were for a ferritin cutoff of 5000 ng/mL (LR+ 5.7) and an ALT cutoff of 65 U/L (LR+ 5.2). Longitudinal analysis did not reveal any statistically significant relationship between changes in fibrosis status and changes in ferritin, ALT, LIC, TIS, and cumulative red cell transfusion volume. Summary: Among chronically transfused people with SCD, liver fibrosis most often does not persist or progress, as detected by serial core liver biopsies. On a cross-sectional basis, serum ALT >65 U/L and serum ferritin >5000 ng/mL are cutoffs associated with the highest likelihood of liver fibrosis. Cross-sectional or longitudinal measurements of LIC are not associated with fibrosis. Conclusion: The progression of liver fibrosis is minimal among people with SCD who receive chronic red cell transfusions for up to 17 years. Serum biomarkers may be used to inform when investigations for fibrosis are warranted. Disclosures: No relevant conflicts of interest to declare.

2095 Background: Magnetic resonance imaging (MRI) of the brain primarily assesses blood brain barrier (BBB) dysfunction which provides indirect information regarding tumor size. The limitations of MRI have become evident with the use of temozolomide (pseudo-progression) and VEGF based therapies (pseudo-response). As a result, a blood-based marker to assess tumor burden is increasingly important. Previous studies suggested that plasma glial fibrillary acidic protein (GFAP) levels are sensitive and specific for glioblastoma (GBM). This pilot study examines the utility of GFAP as a biomarker in pre- and post-operative gliomas. Methods: Plasma samples were collected in patients pre-operatively (N=34) and 24-48 hours post-operatively (N=24) for what was post-operatively confirmed to be newly diagnosed glioma. Plasma GFAP was detected using an electrochemiluminescent immunoassay with a detection threshold of >0.04ng/ml. Presence or absence of contrast enhancement (CE) on the pre-op MRI was compared with the pre...

Abstract 4428 Introduction: Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are closely related bone marrow failure disorders. Most AA results from an autoimmune attack directed against hematopoietic stem/progenitor cells. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a PIG-A mutation. The PIG-A gene mutation leads to glycosylphosphatidylinositol-anchor protein (GPI-AP) biosynthesis deficiency and subsequent hemolysis secondary to the absence of complement regulatory proteins (CD55 and CD59). Both PNH and AA can be cured by allogeneic bone marrow transplantation (alloBMT), but only a minority of patients is offered this approach due to the potential morbidity and mortality. AA can be treated with immunosuppressive therapy (IST) and PNH can be controlled by eculizumab. It has been estimated that more than 50% of AA patients harbor small, but expandable PNH populations at diagnosis. The natural history of PNH clones in AA patients following non-transplant therapy is not well studied. The purpose of this study is to determine the fate and clinical relevance of these PNH clones in patients with AA who did not receive an alloBMT. Patients and Method: Twenty-seven patients with AA and a detectable PNH clone were monitored for a median of 5.3 years (range,1.5 to 11.5 years). The PNH granulocyte clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE-conjugated anti-CD15 and fluoresceinated aerolysin variant (FLAER) staining were used to define granulocytes and GPI-AP deficient cells respectively. Serum lactate dehydrogenase (LDH) value was used as a surrogate for monitoring hemolysis and 1.5× the upper limit of normal LDH value (330mg/dL) as a cut-off point to define clinically apparent hemolysis. A PNH size change Result: We found a linear relationship between PNH granulocyte clone size and LDH values (Pearson correlation coefficient=0.73; P15%) initial PNH granulocytes. Of 9 patients who initially were treated with traditional IST (ATG, CsA, and prednisone), 7 did not respond to treatment and eventually received high-dose cyclophosphamide (HiCy) salvage therapy, 2 achieved a remission and did not require further treatment though one demonstrated PNH clone size expansion to 50% after 37 months. After HiCy salvage, all 7 patients became transfusion independent and 4 of them had no further PNH clone expansion. PNH clone expansion was observed in 7 of 9 patients at a median time of 3 (range: 2 to 87) months after treatment. Of 15 patients who received HiCy as initial therapy, 14 achieved remissions. Later expansion of PNH size was observed in 7 patients, of which 5 eventually required intermittent blood transfusion but only 1 developed symptomatic hemolysis necessitating eculizumab therapy. The median time to PNH granulocyte clone expansion after HiCy was 52 (range: 18 to 106) months. In 5 patients who received HiCy and then relapsed, their PNH clone size only increased (1±0.7)% in (71±31) months observation during post treatment remission; however, their PNH clone size increase accelerated to (38±14)% in (34±21) months after AA relapse (P=0.04). Two nSAA patients with an initial PNH clone size ≤15% spontaneously recovered hematopoiesis at 84 and 56 months respectively, neither had PNH clone size expansion. In this study, 25.9% patients kept a stable PNH size, 48.1% patients increased the size, and 26% patients decreased the size. The group with small initial PNH clone sizes (≤15%) was the most stable over time. Conclusion: The risk of developing clinically significant PNH over 10 years appears to be low in AA patients with PNH clones, especially for those with small initial PNH granulocyte clones (≤15%) and for those who maintain remission following therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 259 Objectives: Silent cerebral infarction (SCI) occurs in approximately 27% of children with sickle cell disease (SCD) by age 6 years, and is associated with decreased neurocognitive function and a 14-fold increased risk of progression to overt stroke. While several clinical parameters, such as increased white blood cell (WBC) and platelet counts and decreased hemoglobin (Hb) or hematocrit, have been reported in the literature to be associated with SCI, to date no validated biomarkers exist to predict SCI in patients with SCD. Furthermore, recent unpublished data from the Silent Infarct Transfusion (SIT) Trial has identified systolic blood pressure and total hemoglobin as risk factors. The aim of this study was to identify candidate biomarker plasma proteins that correlate with SCI in patients with SCD. Methods: We used a proteomic discovery approach involving three sequential separation steps to compare the plasma proteomes of 15 children with SCD (7 with SCI and 8 without SCI), aged 5–15 years. Baseline steady-state plasma samples were obtained from the SIT Trial Biologic Repository and matched for age, Hb and WBC. Plasma samples were Hb depleted in the first dimension, separated using immunoaffinity depletion and reverse phase liquid chromatography fractionation, and then trypsin-digested for characterization using label-free quantification on a LTQ-Orbitrap (Thermo) mass spectrometer. The resulting MS/MS data were analyzed using PASS (Integrated Analysis, Bethesda, MD) with X! Tandem searches (www.thegpm.org; version 2008.12.01) of the International Protein Index peptide database (human, 3.19). We measured candidate proteins in a validation cohort of 116 children with SCD (n=65 SCI, 51 non-SCI) and 24 age-matched, healthy African American control subjects using enzyme-linked immunosorbent assays (thrombospondin 1 [TSP1], L-selectin, RandD Systems, Minneapolis, MN) and immunoassays (E- and P-selectin, Mesoscale Discovery, Maryland). All samples were run in duplicate according to the manufacturers' protocols. Statistical differences in biomarker plasma concentrations between groups were compared by the Mann-Whitney U test. Results: TSP1 (5 peptides) and L-selectin (3 peptides) were among 335 proteins that showed differential detection between the SCI and non-SCI groups based on the spectral counts. TSP1 is an extracellular matrix glycoprotein that is involved with platelet aggregation, inhibition of neovascularization and tumorigenesis and has been shown to promote the adherence of sickle erythrocytes to the vascular endothelium. L-selectin is an adhesion molecule that mediates leukocyte interaction with the vascular endothelium. In a validation cohort of 116 children with SCD (n=65 SCI, 51 non-SCI) and 24 age matched, healthy African American control subjects, TSP1 and L-selectin were both significantly increased in SCI vs. non-SCI groups (median 8.5 vs. 6.2 μ g/ml for TSP1, P =0.03; 1.5 vs. 1.4 μ g/ml for L-selectin, P =0.03). As expected, neither TSP nor L-selectin were elevated in the age-matched normal controls (median=4.6 μ g/ml for TSP1, P =0.10, 1.2 μ g/ml for L-selectin, P =0.10). The specificity of the L-selectin results was verified by demonstrating that E-selectin and P-selectin were not increased in the SCI group. TSP1 was correlated with baseline oxygen saturation in both the SCI and non-SCI groups (r=-0.51, and r=-0.35, P Conclusions: TSP1 and L-selectin may represent the first two plasma biomarkers of SCI in children with SCD. Although further studies are needed, these and other potential biomarkers may provide insight into the pathophysiology of SCI, and may fill an important clinical need in identifying children with SCD who are at risk for SCI. Disclosures: No relevant conflicts of interest to declare.

Abstract 2579 Poster Board II-556 Silent cerebral infarction (SCI) is part of a spectrum of cerebrovascular disease, occurs in approximately 22% of children with sickle cell disease (SCD) and is associated with decreased cognitive function. While several plasma biomarkers have been shown to be elevated in acute stroke, to our knowledge none have been evaluated in SCD or SCI. The aim of this study was to develop a reliable pipeline to identify low abundance plasma proteins that correlate with SCI in patients with SCD. We used a proteomic discovery approach involving three sequential separation steps in order to compare the plasma proteomes in a discovery set of 15 children with SCD (7 children with SCI and 8 children without SCI). Baseline steady state plasma samples obtained from the Silent Infarct Transfusion (SIT) Trial Biologic Repository were matched for age and WBC. In the first dimension, hemoglobin was depleted with nickel-nitrilotriacetic acid (Ni-NTA). Subsequently, second dimension separation and enrichment was achieved by immunoaffinity depletion of the 12 most abundant proteins (ProteomeLab IgY-12 LC10) followed by third dimension separation by reverse phase liquid chromatography fractionation (RPLC) using a C18 column and a linear acetonitrile gradient. Collected fractions were subjected to tryptic digestion and analyzed using label-free quantification on a LTQ-Orbitrap (Thermo) mass spectrometer. The MS/MS data were analyzed using the Proteomics Analyzer Software System (PASS) version 4.0.10 (Integrated Analysis Inc, Bethesda, MD) with X! Tandem searches (www.thegpm.org; version 2008.12.01) using the human IPI database. Identified proteins were compared to databases of brain specific and brain enriched proteins to identify candidate biomarker proteins for SCI. After hemoglobin depletion, 71% of total protein was removed. On average, protein recovery after the LC-12 column was 4% of the total Ni-NTA depleted protein. Of the 9800 proteins that were identified in the plasma proteome of children with SCD, 23 were brain specific proteins. Evaluation of the relative abundance by spectral counts (SC) revealed 3 brain-related proteins that were over-represented in patients with SCI: microtubule-associated protein tau (a neurofibrillary tangle protein implicated in Alzheimer disease, frontotemporal dementia and parkinsonism), neuroligin-3 (a neuronal cell surface protein proposed to be involved in cell-cell-interactions via binding to beta-neurexins and implicated in autism and Asperger syndrome), and nucleosome-remodeling factor subunit BPTF (a histone-binding component of nucleosome-remodeling factor that is abundantly expressed in the fetal brain and re-expressed in neurodegenerative diseases). Reticulon-4, a potent neurite growth inhibitor involved in the restriction of axonal regeneration after injury, was under-represented in patients with SCI. After depletion of hemoglobin and other high abundant proteins, we were able to develop a database of plasma proteins in children with SCD and to identify brain specific proteins as potential surrogate markers of brain injury. These markers may be implicated in the pathophysiology of SCI. Although validation studies are necessary to determine the relevance of these candidate biomarkers in SCI and SCD, our methodology appears to be a practical approach to proteomic discovery in patients with hemolytic anemia. Disclosures: Casella: Boehringer Ingelheim: Honoraria, Research Funding, Travel funding.

Abstract 1512 Poster Board I-535 Introduction In children with sickle cell disease (SCD), silent cerebral infarct (SCI) is an independent risk factor for lower IQ, poorer school performance, and overt stroke. MRI is the only method to identify patients with SCI. A blood biomarker of SCI in patients with sickle cell disease (SCD) would fill a clinical void because blood is easy to obtain and measure, a biomarker may determine risk of or progression of neurologic injury to overt stroke, and a biomarker could benchmark current and new therapies for SCI and other subclinical forms of neurologic injury in SCD. We hypothesized that an unbiased proteomics screen of plasma from sickle cell patients would yield biomarkers of brain injury. Methods A cross-sectional sample of children 5-14 years old with sickle cell disease (HbSS and HbSB0) who were screened for the Silent Infarct Transfusion (SIT) Trial (ClinicalTrials.gov NCT00072761) were studied (n=258) along with 60 age-matched healthy controls and 28 adults with overt brain injury. For biomarker discovery, plasma underwent depletion of hemoglobin with nickel- nitrilotriacetic acid and immunoaffinity depletion of the 12 most abundant plasma proteins. After fractionation by reverse phase liquid chromatography over a C18 column and acetonitrile gradient and trypsin digestion, peptide spectra from each fraction were obtained by LC/MS/MS (LTQ-Orbitrap) and were searched using X!Tandem and human IPI database version 3.5. Post search analysis was performed using Proteomics Analysis Software System (Integrated Analysis Inc., Bethesda, MD). An electrochemiluminescent immunoassay was developed for one of the candidate proteins identified, glial fibrillary acidic protein (GFAP), for protein validation (MesoScale Discovery). Results GFAP, a brain-specific intermediate filament known to be a marker of acute stroke and head trauma in adults, was identified in discovery sample plasma. Four percent of sickle cell subjects in steady-state had plasma GFAP concentrations similar to stroke and brain surgery controls (>0.45 ng/mL). Among sickle cell subjects 5-14 years old screened for the SIT Trial, 9.3% had GFAP concentrations above the 95th percentile of age-matched controls (95th percentile cutoff: 0.227 ng/mL; p=0.08). Sickle cell subjects with silent cerebral infarct (SCI) had more elevations above the normal 95th percentile (10/69; 14.5%) than those without SCI (9/131; 6.8%), although this difference was not statistically significant (p=0.08). The sensitivity of GFAP as a marker of brain injury was demonstrated in a case of an 11 year-old child with HbSS and acute stroke in which plasma GFAP was 1.52 ng/mL (6 times the normal control 95th percentile) before the stroke was clinically evident and peaked at 2.83 ng/mL. We discovered GFAP as a circulating brain protein in plasma of children with SCD. Plasma GFAP is a marker of acute stroke in sickle cell disease but discriminates only minimally between SCI and non-SCI status in the SIT Trial using baseline cross-sectional samples. Four percent of children with sickle disease 5-14 years old have plasma GFAP levels similar to controls with severe brain injury without clinical evidence of overt stroke. Conclusions Elevations in circulating brain proteins such as GFAP show promise as indicators of subacute brain injury in children with SCI, but will require longitudinal studies of plasma GFAP in children with SCI to clarify the utility of GFAP as a plasma biomarker of SCI and a predictor of neurologic risk. Disclosures No relevant conflicts of interest to declare.

Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p0.05) and allogeneic (28% vs 58%; p0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.