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Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.

von Willebrand disease (VWD) is the most common inherited bleeding disorder, and type 1 VWD is the most common VWD variant. Despite its frequency, diagnosis of type 1 VWD remains the subject of debate. In order to study the spectrum of type 1 VWD in the United States, the Zimmerman Program enrolled 482 subjects with a previous diagnosis of type 1 VWD without stringent laboratory diagnostic criteria. von Willebrand factor (VWF) laboratory testing and full-length VWF gene sequencing was performed for all index cases and healthy control subjects in a central laboratory. Bleeding phenotype was characterized using the International Society on Thrombosis and Haemostasis bleeding assessment tool. At study entry, 64% of subjects had VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity below the lower limit of normal, whereas 36% had normal VWF levels. VWF sequence variations were most frequent in subjects with VWF:Ag

Mice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with α-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P < .006). Mutant relative to unaffected RBC adhesion to LM was significantly (P < .01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P < .001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in α-spectrin–deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P < .005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.

The etiology of stroke in sickle cell disease is unclear, but may involve abnormal red blood cell (RBC) adhesion to the vascular endothelium and altered vasomotor tone regulation. Therefore, we examined both the adhesion of sickle (SS)-RBCs to cerebral microvessels and the effect of SS-RBCs on cerebral blood flow when the nitric oxide (NO) pathway was inhibited. The effect of SS-RBCs was studied in the rat cerebral microcirculation using either a cranial window for direct visualization of infused RBCs or laser Doppler flowmetry (LDF ) to measure RBC flow. When fluorescently labeled human RBCs were infused into rats, SS-RBCs had increased adhesion to rat cerebral microvessels compared with control AA-RBCs (P = .01). Next, washed SS-RBCs or AA-RBCs were infused into rats prepared with LDF probes after pretreatment (40 mg/kg intravenously) with the NO synthase inhibitor, N-ω-nitro-L-arginine methyl ester (L-NAME), or the control isomer, D-NAME. In 9 rats treated with systemic L-NAME and SS-RBCs, 5 of 9 experienced a significant decrease in LDF and died within 30 minutes after the RBC infusion (P = .0012). In contrast, all control groups completed the experiment with stable LDF and hemodynamics. Four rats received a localized superfusion of L-NAME (1 mmol/L) through the cranial window followed by infusion of SS-RBCs. Total cessation of flow in all observed cerebral microvessels occurred in 3 of 4 rats within 15 minutes after infusion of SS-RBCs. We conclude that the NO pathway is critical in maintaining cerebral blood flow in the presence of SS-RBCs in this rat model. In addition, the enhanced adhesion of SS-RBCs to rat brain microvessels may contribute to cerebral vaso-occlusion either directly, by disrupting blood flow, or indirectly, by disturbing the vascular endothelium.

The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of “VWF activity” by this assay and may not reflect a functional defect or true hemorrhagic risk.

Patients with sickle cell disease (SCD) experience increased pain to thermal (cold, heat) stimuli compared to healthy controls suggesting peripheral and/or central sensitization. However, the underlying etiology of this thermal hypersensitivity is unknown. Substance P (SP), a primary pain neurotransmitter, is a potent mediator of neurogenic inflammation where inflammatory cell activation, vasodilation, and edema occur after the stimulation of sensory nerve fibers. Chronic elevation of SP can sensitize peripheral nociceptors resulting in thermal hypersensitivity and chronic neuropathic pain as shown in mice and humans without SCD and in SCD mice. Little is known about SP levels in patients with SCD in baseline health compared to healthy African American controls. Furthermore, little is known about changes in SP levels during acute SCD pain. The objective of this study was to evaluate plasma SP levels in SCD patients and controls in baseline health and in SCD patients during acute pain. We hypothesized that SCD patients, in baseline health, will have significantly higher SP levels compared to controls suggesting chronic pain sensitization and that SP levels will significantly increase during acute pain. Patients with SCD in baseline health and during admission for acute pain and healthy African American controls were recruited. Our primary outcome was plasma SP level measured in 3 independent cohorts: healthy African American controls, SCD patients in baseline heath, and SCD patients during acute pain. Plasma samples were assayed in duplicate for SP levels using competitive enzyme-linked immunosorbent assay (Cayman Chemical). Independent samples student's t-test was used to compare SP levels between: 1) SCD patients in baseline health and controls and 2) SCD patients in baseline health and those in acute pain. Multivariable linear regression analysis was used to determine the impact of age and gender on SP levels in the 3 independent groups. Thirty-five African American controls, 25 SCD patients in baseline health, and 12 SCD patients during acute pain completed the study. Controls were older than SCD patients in baseline health (mean (SD) age 21.5 (12.5) vs. 11.8 (3.8) yrs, p=0.0004) and there was no difference in the mean age of SCD patients in baseline health compared to those in acute pain (11.8 (3.8) vs. 12.1 (3.5) yrs, p=0.81). Gender did not differ between the same comparison groups. Genotypes of SCD groups were: 1) baseline: 68% SS, 24% SC, 8% Sβ+thal and 2) acute pain: 67% SS, 25% SC, 8% SOArab. The mean (SD) SP level of SCD patients in baseline health was significantly higher than controls (32.4 (11.6) vs. 22.9 (7.6) pg/mL, p=0.0009) (Figure 1). When controls were restricted to patients less than 19 years of age (n=21) to better match SCD patients in baseline health, the significant elevation of SP levels in SCD patients persisted (32.4 (11.5) vs. 21 (7.1) pg/mL, p The elevated SP levels in SCD patients during baseline health compared to controls suggests that SP may be a mediator of or marker for chronic pain sensitization. SP levels further increase during acute pain suggesting that SP may also play a role in mediating or marking an acute pain event. The downstream effects of chronically increased levels of SP on the development and propagation of pain in patients with SCD warrant further investigation. Ultimately, SP could differentiate those at risk for development of chronic pain and could be used as a novel biomarker for acute pain and resolution of a painful event. Eventually, compounds aimed at decreasing SP levels or blocking the SP receptor could be a target for novel treatments for SCD pain. Figure 1. Figure 1. Disclosures Brandow: NIH, ASH: Research Funding. Hillery:NIH: Research Funding. Panepinto:NKT Therapeutics, Inc: Consultancy; HRSA, NIH: Research Funding.

Introduction: Children receiving treatment for cancer experience impaired health status. Health status during treatment can be systematically quantified using patient-reported outcome measures (PROs). PROs are any report of a patient's health status that comes directly from the patient and provide composite scores from a series of questions around a central concept to quantify the level of distress or impairment caused by a patient's symptoms, disease, or treatment. Previous work in pediatric leukemia patients and brain tumor patients measuring PROs over time demonstrated no change in patient-reported health status late in therapy compared to the end of therapy. Changes in patient function over the early, intensive phases of treatment have not been studied. Additionally, the impact of cancer disease type (Leukemia/Lymphoma, CNS Tumor, Solid Tumor) on patient-reported health status during intensive cancer therapy is not known. The objective of this study was to characterize the impact of disease type on patient function related to mobility, fatigue, pain, anxiety, depression, and peer relationships over the initial six months of therapy. We hypothesized that CNS/Solid Tumor patients have worse self-reported health status than Leukemia/Lymphoma patients and that patient function worsened over time for all domains measured. Methods: We conducted a prospective cohort study of children ages 5-21 yearsold with a new diagnosis of cancer. Patients were enrolled in the study for six months or until their treatment completed, whichever time period was shorter. Our primary outcome was patient function as measured using the Patient-Reported Outcome Measurement Information System (PROMIS) for impaired mobility, pain, fatigue, anxiety, depressive symptoms, and peer relationships. PROMIS is a validated PRO that provides separate scores for each domain measured (reported as T score) with a mean score of 50. Self-report participant assessments were obtained twice monthly on electronic tablets, at the beginning of a treatment cycle and approximately 1 week later. Intensity of therapy was determined using the Intensity of Treatment Rating (ITR-3) scale and classified as moderate, very, and most intensive treatments. Patient demographics were analyzed using simple statistics. Analysis of changes in patient function used a generalized linear mixed model (GLMM) using predictors of type of cancer and time from diagnosis. Results: Forty patients completed the study and had a mean age of 11.7 years (SD 4.7). Fifty-eight percent of the patients were male and 60% of patients had Leukemia/Lymphoma. Forty percent of patients received moderate intensity treatment, 47.5% received very intensive, and 12.5% received most intensive treatment. The intensity of treatments was similar in the Leukemia/Lymphoma group and the CNS/Solid Tumor group (p=0.21). Disease Group: CNS/Solid Tumor patients have better self-reported health status for all domains measured compared to Leukemia/Lymphoma patients. CNS/Solid Tumor patients report better mobility (higher T scores) compared to Leukemia/Lymphoma patients by a factor of 1.1 (p Time from diagnosis: Time from diagnosis has a significant effect on patient-report of pain for both Leukemia/Lymphoma and CNS/Solid Tumor patients, with improved pain over time. At 100 days from diagnosis, pain scores decrease by a factor of 0.91 (p=0.008). Time from diagnosis did not have a significant effect on patient function in any other domain measured. Conclusion: Leukemia/Lymphoma patients experience significantly worse patient functioning compared to patients with CNS/Solid Tumors over the first six months of cancer treatment.In addition, as time from diagnosis increases patient function related to pain improves. The significant burden of disease for Leukemia/Lymphoma patients demonstrated in this study is a surprising finding and perhaps reflects an underestimation of the burden of Leukemia/Lymphoma treatment because of the overall favorable prognosis for most of the children with these diagnoses. Disclosures Panepinto: HRSA, NIH: Research Funding; NKT Therapeutics, Inc: Consultancy.

The diagnosis of variant von Willebrand Disease (VWD) currently involves evaluating multiple aspects of von Willebrand Factor (VWF) functions using separate, individual assays. This process can be time consuming and may delay definitive diagnosis of variant VWD. To address this issue, we have previously described a novel, rapid ELISA-based VWF functional screening assay that allows for variant VWD phenotype assignment and can provide same-day results. When considering application of this assay as a screening test, it is important to investigate its correlation with conventional clinical assays of VWF function. Therefore, the objective of our study was to investigate the relationship of our qualitative and semi-quantitative assay with traditional quantitative assays of VWF functions. 161 plasma samples from the Zimmerman PPG were analyzed on a novel ELISA-based platform, which measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:FVIIIB (binding to FVIII), VWF:CBIII (binding to collagen III), and VWFpp (propeptide). Samples were compared to a 30% standard control plasma by a ratio of each unique VWF functional activity over the VWF:Ag. Data were compared to available quantitative data from the BloodCenter of Wisconsin clinical laboratory on the same patient plasma samples. Plasma samples included 21 type 1, 30 type 1C, 23 type 2A, 23 type 2B, 20 type 2M, 17 type 2N, 5 type 2N carriers (heterozygote), and 22 potential hemophilia A (HA) subjects. Data was analyzed by linear regression and power function. In all VWD samples analyzed by power function, VWF:Ag by our assay correlated with the clinical assay with an R2 of 0.794. For VWFpp from 111 VWD subjects, the power function revealed our assay correlated with the clinical assay with an R2 of 0.630, and sub-analysis of 51 type 1 and 1C VWD subjects showed an R2 of 0.673. VWF:CBIII analysis of all type 2A, 2B, and 2M subjects through linear regression revealed an R2 of 0.675. VWF:IbCo analysis was separated into type 2B and non-type 2B VWD subjects due to the significant VWF:IbCo enhancement seen in our assay because of augmented binding of type 2B VWF to glycoprotein Ib. While correlation of our VWF:RCo with the clinical VWF:RCo was poor, the VWF:IbCo was able to discriminate type 2B from non-type 2B. VWF:FVIIIB analysis showed complete discrimination of type 2N and potential HA subjects. In addition, there was discrimination of type 2N VWD subjects from type 2N carriers; however these data were not fit into the statistical model because there were too few subjects in each category. In summary, our study shows that each individual component of the VWF functional screening assay shows correlation with traditional quantitative assays of VWF functions. The best correlation was seen with the VWF:Ag, and there was also discrimination of type 2B, type 2N, type 2N carriers, and HA subjects demonstrated in our analysis. These data indicate the potential use of our assay as a laboratory screening test to expedite diagnosis of variant VWD. Disclosures No relevant conflicts of interest to declare.

Introduction: Children treated for leukemia and lymphoma receive multi-agent chemotherapy and experience toxicities related to their disease and treatment. Commonly described symptoms in cross-sectional studies of symptom prevalence during treatment are nutrition concerns, nausea/vomiting (n/v), pain, fatigue, and emotional distress. The evolution of these symptoms over the course of treatment and the relationship between symptoms and intensity of therapy remain unknown. We hypothesize that symptom prevalence will change over time (nutrition concerns will decrease, n/v will decrease, pain will increase, fatigue will increase, and emotional distress will increase) and patients who receive more intense treatments will experience more symptoms. Methods: As part of a prospective study investigating the burden of cancer treatment, a retrospective chart review of 44 children treated for leukemia or lymphoma at a single institution was performed. Clinician documentation of nutrition concerns, n/v, pain, fatigue and emotional distress was abstracted from all outpatient clinic visits over the first six months of cancer treatment. Each symptom was recorded as present or absent in the documentation at each clinic visit. The Intensity of Treatment Rating Scale was used to determine intensity of treatment. Data were analyzed using a logistic generalized linear mixed model to model the trajectory of each symptom over time, and consider the covariates of diagnosis and intensity of treatment. Results: The median age of the sample population is 9.2 years, 59% are male. Fifty percent of patients were treated for ALL, 14% for AML, and the remainder for lymphoma. Treatment regimens were classified as moderately intensive (43.2%), very intensive (38.6%), and most intensive (18.2%). Eighty percent of patients reported concerns regarding nutrition at some point during the first six months of therapy, 86% reported n/v, 86% reported pain, 71% reported fatigue, and 30% reported emotional distress. Nutrition concerns were reported at 23% of all clinic visits, n/v at 14%, pain at 20%, fatigue at 3.9%, and emotional distress at 1.3%. Univariate analyses reveals that children with leukemia have a higher prevalence of nutrition concerns than children with lymphoma (p=0.0229). When adjusted for time from diagnosis, the prevalence of nutrition concerns decreases with time in lymphoma patients (p=0.0334) where they remain constant in leukemia patients (p=0.0449). When adjusted for intensity of treatment, the prevalence of nutrition concerns decreases over time, with the low intensity therapy group decreasing to zero (p=0.0004). Leukemia patients have a lower prevalence of n/v than lymphoma patients (p=0.0366). Neither time nor intensity of treatment has an effect on prevalence. Prevalence of pain decreases slightly over time but does not reach statistical significance for leukemia or lymphoma patients (p=0.0554). Pain decreases over time for patients who receive less intensive therapy compared to patients who receive high intensity therapy over time (p=0.0375). Fatigue and emotional distress were not modeled due to low prevalence. Conclusion: Children with newly diagnosed leukemia/lymphoma experience symptoms over the first six months of therapy that change over time. Nutrition concerns are more prevalent in children with leukemia and remain constant throughout the first six months of treatment. Patients with either leukemia or lymphoma who receive low intensity therapy have the least difficulty with nutrition. Pain also improves over time for patients who receive less intensive therapy, but remains constant for those with more intense therapy. The low to zero prevalence of fatigue and emotional distress symptoms likely reflect unrecognized symptoms by the clinician. Improved detection and understanding of symptom trajectory during active treatment can both guide appropriate use of supportive care resources and allow for improved anticipatory guidance for families. As part of an ongoing prospective trial, we plan to correlate the findings described here with data on patient function using patient-reported outcome measures, to improve understanding of the burden of cancer therapy and direct supportive care to minimize symptoms during treatment. Disclosures No relevant conflicts of interest to declare.

Background von Willebrand disease (VWD) is a very common inherited bleeding disorder. The current phenotypic classification of VWD variants includes disorders of both quantitative and qualitative defects in von Willebrand Factor (VWF) that determines optimal treatment of patients. Current phenotype determination is multimodal, cumbersome, performed by only a few specialized laboratories, and may delay the definitive diagnosis necessary in proper selection of therapy. We have developed an ELISA-based strip assay that is capable of rapid determination of relative qualitative and quantitative VWF functionality to correctly assign phenotypic variants of VWD. Methods 136 VWD plasma samples from the Zimmerman PPG were analyzed on a new ELISA based platform. In single, individual wells this assay measures relative values of VWF:Ag (antigen), VWF:IbCo (Ib cofactor, no ristocetin), VWF:RCo (ristocetin cofactor), VWF:F8B (binding to FVIII), VWF:CB3 (binding to collagen III), and VWF:pp (propeptide) in comparison to a 30% normal control standard in a single ELISA strip assay. The study included 22 type 1 VWD, 32 type 1C VWD, 18 type 2A VWD, 23 type 2B VWD, 20 type 2M VWD, 7 type 2N VWD, 4 type 3 VWD, and 10 hemophilia A subjects. Each sample was run in single wells for each assay and optical densities (OD) were compared to the OD of a 30% standard control plasma and a 100% VWF:Ag control. The standard ELISA plate read time was 30 minutes and full assay can be accomplished in 3 hours. Two of the coauthors were blinded as to the Zimmerman PPG VWD phenotypes of test samples. Using the ELISA strip results, phenotype assignment was determined and then compared to the unblinded Zimmerman PPG VWD diagnosis. Further statistical analysis of VWF functional profile relationships was performed using the Mann-Whitney test and ROC analysis, and can quantify the ability to identify these phenotypes. Results VWF functional profiles based on visually observed ratio relationships correctly assigned VWD phenotypic variant on first attempt in 122 of 136 subjects (89.7%). Repeat testing of the 14 incorrectly assigned subjects along with 11 random, correctly assigned subjects for a validation check, accurately re-assigned 9 of 14 previously incorrect phenotypes, suggesting initial plate to plate variability since all ELISA plates were made fresh for each run. Previously correctly assigned subjects, 11 of 11, remained correctly assigned. Comparing specific phenotypes revealed VWF:IbCo/VWF:Ag is good at separating type 1C from 2A; ROC area under the curve 0.875, with an optimal ratio threshold 0.649 (sensitivity 0.969, specificity 0.611, p Conclusions The rapid VWF functional screening assay is able to discriminate VWD phenotypic variants with good sensitivity and specificity, and may facilitate the rapid laboratory assignment of VWD phenotypes once a subject with low VWF is identified. Disclosures: No relevant conflicts of interest to declare.

Background Although neuropathic pain is becoming an increasingly recognized component of sickle cell disease (SCD) pain, national data regarding the use of neuropathic pain drugs in SCD patients do not exist. Furthermore, factors associated with the development of neuropathic pain in SCD are not well characterized. In non-SCD pain populations, neuropathic pain is associated with older age and female gender. Older age is also associated with increased thermal pain sensitivity, a marker of neuropathic pain, in both SCD patients and pain populations without SCD. Thus, the objectives of our study were to describe the use of neuropathic pain drugs in children with SCD and determine factors associated with the use of these drugs. We hypothesized older age and female gender would be associated with increased use of neuropathic pain drugs. Methods We used the Pediatric Health Information System (PHIS) Database (2004-09) to investigate the use of neuropathic pain drugs in SCD patients during hospital admission. The PHIS provides comprehensive inpatient data, including detailed drug information, from 43 children’s hospitals in the United States. All visits with a SCD-related (any genotype) ICD-9 discharge diagnosis (i.e., primary, secondary, tertiary) were included. To limit confounding, visits that included any psychiatric, seizure, or headache diagnosis in addition to a SCD diagnosis were excluded since neuropathic pain drugs also treat these disorders. The drugs of interest were based on published neuropathic pain treatment guidelines originally created for patients without SCD and included antiepileptics, tricyclic antidepressants, and selective serotonin reuptake inhibitors. Multivariate logistic regression analysis was used to determine the impact of age and gender on the use of neuropathic pain drugs with the outcome being whether a neuropathic pain drug was or was not prescribed during that admission. Age was classified into 4 groups (0-4, 5-10, 11-14, and 15-18 years) for clinical interpretation. Results A total of 61,325 visits met inclusion criteria. Mean age was 9.8 (±5.7) years. The proportions of visits in each age group were: 0-4 (24%), 5-10 (26%), 11-14 (22%), 15-18 (28%) and 49% were female. The majority had HbSS disease (73.7%). We found 3.6% were prescribed one or more neuropathic pain drugs with the majority (3.3%) receiving one drug. There was no difference in the use of neuropathic pain drugs over time (2004-09). The most commonly used drugs were: amitriptyline (47.4%), gabapentin (27.7%), phenytoin (12.3%), and fluoxetine (11.2%) with pregabalin, venlafaxine, paroxetine, and nortriptyline used infrequently. In multivariate analysis, compared to the reference group of 0-4 years, the odds of receiving a neuropathic pain drug increased significantly and sequentially for each older age group (Table 1 ). Female patients were more likely to be taking a neuropathic pain drug [OR 1.8 (CI 1.6-1.9; p Conclusions Neuropathic pain drugs were infrequently prescribed ( Disclosures: No relevant conflicts of interest to declare.

Abstract 83 Background: Improved survival of patients with sickle cell disease has led to increased attention on the transition of care from pediatric to adult providers. Recent studies have shown increased emergency department (ED) utilization as well as an increased risk of mortality during this transition period for patients with sickle cell disease. Suggested reasons for such changes include loss of a medical home, loss of insurance, and loss of access to outpatient clinics as pediatric sickle cell patients transition to adult services. Emergency Department Reliance (EDR), defined as the number of ED visits divided by the number of ED and outpatient visits, views ED visits in relation to all ambulatory visits. Those with more severe disease, who utilize the ED more, should also have more outpatient visits, while those without adequate outpatient clinic access simply use the ED more without a rise in outpatient clinic visits, thus increasing EDR. EDR, therefore, is used to differentiate increased ED use due to need for care from increased ED use as an access issue. We hypothesized that as patients transition from pediatric to adult centered care, loss of a primary medical home would lead to an increase in the EDR. Methods: We conducted a retrospective cohort study using the Wisconsin State Medicaid database from 2003–2007. This database includes 757 patients of any age with sickle cell disease who are part of the Wisconsin Medicaid system. Consistent with previous literature, having sickle cell disease was defined by having at least one inpatient hospitalization, or two outpatient visits at least one month apart, with a diagnosis of sickle cell disease. All encounters during the study period were extracted from the database and linked by unique anonymous identifiers. Information extracted included age, all diagnosis codes, and classification as ED visit, outpatient visit, or inpatient hospitalization. The study population was divided into three mutually exclusive age groups: 1) children: ≤ 18 years old for the entire study period, 2) the transition group who turned 19 years old during the study period; and 3) Young adults age ≥ 19–45 years at first encounter. Adults age 3 46 at first encounter were eliminated from the analysis as the goal was to compare the transition group to the age groups immediately above and below. The median Emergency Department Reliance (ED visits/[ED + outpatient visits]) was compared for each age group for sickle cell related diagnoses, all diagnoses, and sickle cell crisis diagnoses using non-parametric, multiple-comparisons adjusted tests. Results: The 687 remaining patients had a total of 32,258 ambulatory visits over the five year study period, including 20,418 outpatient visits and 11,840 emergency department visits. There were 345 children, 65 patients in the transition group, and 277 young adults. For sickle cell diagnoses, the transition group had a significantly higher EDR than children (0.50 vs. 0.39, p=0.031) and a higher EDR than young adults, though this increase failed to achieve statistical significance (0.50 vs. 0.41, p=0.360). The difference in EDR between the children and adults for sickle cell diagnoses was not significant. A similar pattern emerged when computing EDR based on all diagnoses, with the EDR for the transition group being significantly higher than for children (0.35 vs. 0.28, p Conclusions: Patients with sickle cell disease who are transitioning from pediatric to adult centered care have an increased reliance on the emergency department as a location of care for sickle cell diagnoses. As adults, emergency department reliance returns to similar levels as childhood, suggesting a stabilization of a medical home following the transition period. The EDR is highest for all groups at times of acute pain crisis related to sickle cell disease, consistent with the need for emergent care in those situations. These findings support the hypothesis that increased ED utilization during the transition period from pediatric to adult providers is potentially due to limitations in access to care. Disclosures: No relevant conflicts of interest to declare.

Abstract 2208 Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p Disclosures: Montgomery: GTI Diagnostics, Inc: Consultancy.

Abstract 3839 Menorrhagia is a common symptom of haemostatic disorders and often triggers an evaluation for a bleeding disorder. Clinical screening tools, to identify women with menorrhagia who have an underlying bleeding disorder, are very desirable. Scoring systems to quantify menstrual blood loss in women, the Pictorial Blood Loss Assessment Chart, (PBAC), and to assess the severity of bleeding symptoms in individuals diagnosed with von Willebrand disease (VWD), European Union Bleeding Score (EU-BS) (MCMDM-1VWD), were evaluated for their ability to predict the presence of low von Willebrand factor (VWF) level in a cohort of 150 women recruited from an outpatient gynecology clinic. One of us (SG or ML) completed a PBAC and an EU Bleeding questionnaire with the participant. Menorrhagia cases were defined by a PBAC ≥185 and controls by a PBAC Disclosures: Gill: CSL Behring: Research Funding.

Abstract 2489 Poster Board II-466 Elevated hospitalization rates for patients with sickle cell disease are largely a result of frequent vaso-occlusive crises. Recent hospital utilization concerns have placed increasing emphasis on rehospitalization as a cost saving and quality of care measure. For sickle cell disease, 30 day rehospitalization has become a benchmark for care quality. Previous reports have shown 30 day rehospitalization rates as high as 30-47% and propose outpatient follow-up as a preventive measure. However, these studies have been limited to single centers and have focused only on specific age groups. Our study sought to examine the efficacy of outpatient follow-up on 30 day rehospitalization prevention across all ages in a statewide Medicaid program. We hypothesized that a post-discharge outpatient visit is associated with lower rehospitalization rates for patients with sickle cell disease. The study is a retrospective cohort using Wisconsin Medicaid claims data for hospitalized children and adults with sickle cell disease from January 1, 2003 to December 31, 2007. Patients at risk for rehospitalization were identified using sickle cell disease-related ICD-9 diagnosis codes (28241, 28242, 28260-28269) at inpatient discharge. The first hospitalization with a sickle cell diagnosis for each individual was extracted and then only those hospitalizations with a diagnosis of sickle cell crisis (28242, 28262, 28264, 28269) were included. Each patient participated in the study only once. The main outcome measure was a rehospitalization within 30 days of discharge from the previous hospitalization. Individuals were considered to have had an outpatient follow-up visit based on a claim for an outpatient visit within 30 days of discharge or prior to a rehospitalization if the rehospitalization occurred in fewer than 30 days. Outpatient visits that occurred on the same day as the rehospitalization were not included. Outpatient visits were calculated as rates to avoid the bias of those not being rehospitalized having more time for an outpatient visit. Multiple logistic regression was performed to evaluate the association between having an outpatient visit, disease severity, and asthma on the outcome of rehospitalization. Severe cases were defined as individuals with 3 or more hospitalizations within 1 year beginning 2 months after the index hospitalization. Patients were excluded from our study if the index hospitalization resulted in death, as they were not at risk for rehospitalization. Four hundred and eight patients with a hospitalization for sickle cell crisis were included in the analysis. Of these 408 patients, 70 (17%) were rehospitalized within 30 days following discharge from the index hospitalization. Of the 70 re-hospitalized patients, 35 (50%) were re-hospitalized within 11 days, and 54 (77%) were rehospitalized within 22 days from discharge. Multiple regression revealed that severe disease was associated with rehospitalization (4.693, 95% CI (2.674, 8.236)), but neither the outpatient visit rate (OR 0.970, 95% CI (0.367, 2.560)) nor a diagnosis of asthma (OR 0.862, 95% CI (0.467, 1.592)) were associated with rehospitalization. Of note, when the unadjusted analysis using the simple existence of an outpatient visit prior to rehospitalization was used, outpatient visits did show an association with decreased rehospitalizations; however this association was no longer significant when adjusted for the increased time for an outpatient visit in those not rehospitalized within 30 days. Contrary to our hypothesis, outpatient follow-up was not associated with fewer rehospitalizations in our study, when the outpatient follow-up rate was used to correct for the increased time for outpatient visits in those not rehospitalized. Disease severity was found to be the only statistically significant predictor of rehospitalization for patients with sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 1365 Poster Board I-387 Background Although sickle cell disease is a genetic disease diagnosed by state newborn screening programs, it is not a reportable condition. Therefore it is difficult to ascertain the actual number of affected individuals living with sickle cell disease in the United States. One NIH estimate puts the number between 50,000 and 75,000 while the Sickle Cell Disease Association of America estimates the number to be “over 70,000”. Without accurate estimates, clinicians, health services researchers, and policy makers are all working with incomplete information when determining the extent of the cost and health consequences of sickle cell disease. Our objective was to estimate the number of people with sickle cell disease for the United States as a whole and for each individual State, adjusting for the increased mortality of sickle cell disease. Methods Census estimates by age and race/ethnicity were obtained for both the United States as a whole and for each individual State from the US Census website. The prevalence of sickle cell disease for blacks was uniformly applied to the U.S. and individual states using a rate of 289 black children with sickle cell disease per 100,000 live births. Based on previously published prevalence rates for Hispanics, prevalence rates of 89.8 Hispanic children of non-Mexican ancestry with sickle cell disease per 100,000 live births and 3.14 Hispanic children of Mexican ancestry with sickle cell disease per 100,000 live births were calculated. We did not include sickle cell disease for whites or Asians in our estimate. Year 2005 was used as the baseline year for all calculations. Consistent with previous literature, at age 0, 60% of children with sickle cell disease were classified as having HgbSS/SB0 and 40% of children were classified as having HgbSC/SB+. These proportions were altered towards higher proportions of HbSC/SB+ with increasing age based on the increased mortality of the more severe forms of sickle cell disease. To adjust for mortality, we analyzed the data based on age and sickle cell type, and used published mortality data for different sickle cell genotypes to calculate survival of the population to a given age. A population multiplier was then used to adjust population estimates for the difference in population across age groups and differences in population patterns by race/ethnicity. Results Analysis revealed an estimate of 89,079 (95% CI: 88,494 – 89,664) people with sickle cell disease in the United States, of which 80,151 are black and 8,928 Hispanic. The South, with a sickle cell population of 47,354 people, comprised more than 53% of all people with sickle cell disease in the United States. The five states with the highest estimated number of people with sickle cell disease were New York with 8,308; Florida with 7,539; Texas with 6,765; California with 6,474; and Georgia with 5,890. These five states comprised more than 43% of the total sickle cell population for the nation. Finally, the increased mortality for HgbSS/SB0 leads to an alteration in the relative percentages of sickle cell genotypes, with HgbSS/SB0 comprising 60% of people with sickle cell disease at birth, half of the sickle cell population at slightly over 30 years old, and only 25% of the sickle cell population by 60 years old. Conclusion This study reveals that the sickle cell population in the United States is higher than previously reported, with almost 90,000 people with sickle cell disease. In addition, differential mortality increases the percentage of people with HbSC/SB+, such that after 40 years of age they represent the majority of the sickle cell population. The population estimates for the country as well as the individual states provide important information with regard to allocation of resources for this chronic disease which primarily affects lower income, underserved individuals. Disclosures No relevant conflicts of interest to declare.

Previous studies have shown that poverty is associated with worse health-related quality of life (HRQL). Children with sickle cell disease have been shown to have poor HRQL. The impact that poverty and sickle cell disease have on the HRQL of children has not been well delineated. The objective of this study was to determine the impact of poverty and sickle cell disease on the HRQL of children with and without sickle cell disease. A cross-sectional study was conducted. Parents of children with sickle cell disease and parents of children without sickle cell disease (controls) who presented to the clinic (ages 2 to 18 years) were asked to complete the PedsQL questionnaire. Detailed demographic information on total family income and family size was collected to classify family income based on the federal poverty threshold. The primary outcome measured was HRQL. An impaired HRQL, a score similar to that of children with chronic disease, is defined as any score that is less than the population mean -1 standard deviation (Varni, et al. 2003). Three HRQL summary scores were assessed: total, physical and psychosocial summary scores. Poverty level and sickle cell disease were the independent variables of interest. Descriptive statistics and non-parametric tests were used to look for significant differences. An ordinal multinomial regression model was used to look at the independent effects of income and disease on the HRQL of children. A total of 104 children with sickle cell disease and 74 control patients participated in the study. Approximately half of the study population was female, 98% of the cases and 78% of the controls were African-American. Approximately half (58%) of the controls and 42% of the cases with sickle cell disease had a family income at poor or near poor (100% or 100–124% of the federal poverty threshold). There were 15% of the controls and 25% of the cases that had a low family income (125–200% of the federal poverty threshold). The remainder in each group were considered as middle/high income (greater than 200% of the federal poverty threshold). The median HRQL was significantly worse in the children with sickle cell disease compared to the control group in all summary scores, p < 0.05 adjusted for multiple comparisons (total score 67.4 versus 80.7, physical summary score 68.8 versus 87.5, psychosocial summary score 68.1 versus 75.0). When both disease and family income were examined in the regression model, children who were poor and who had sickle cell disease were more likely to have lower physical and psychosocial HRQL. Children with sickle cell disease were more than twice as likely to have an impaired physical HRQL compared to controls at each family income level. (Table 1). In conclusion, children with sickle cell disease have an increased probability of having impaired physical HRQL compared to children without sickle cell disease across each family income level. Predicted Probabilities of Poor Physical Health Score (PHS) (based on multinomial regression model) Group Income Level Pr (PHS ≥ 63.28)* 95% Confidence Interval *Denotes probability that PHS is≤ 1 standard deviation below the population mean reported by Varni et al. 2003 Control Poor/Near Poor 0.23 0.14–0.36 Low 0.24 0.12–0.41 Middle/High 0.10 0.04–0.22 Sickle Cell Disease Poor/Near Poor 0.49 0.37–0.61 Low 0.49 0.34–0.64 Middle/High 0.26 0.14–0.42

We performed a prospective randomized trial of antithoracic duct lymphocyte globulin (ATDLG), HLA-haploidentical marrow, and androgen (regimen ABA) versus androgen alone (concurrent STANDARD care controls) in 42 newly diagnosed individuals with severe aplastic anemia. ABA patients also were matched with patients from our preceding study (historical STANDARD care controls). Supportive care and pretreatment patient characteristics were the same in all groups. By life table analysis, 76% of patients receiving ABA are alive at 2 yr compared to 31% of the concurrent control group (p less than 0.002 versus ABA) and 19% of the historical controls (p less than 0.0001 versus ABA) given STANDARD care. ABA patients had greater hematologic improvement than either control group (p less than 0.001). However, improvement with ABA was often incomplete. Toxicity of ATDLG was considerable but manageable. Further studies to determine the mechanism of action and active component(s) of ABA are indicated.