ALBERT

Description: Background: Hydroxyurea (HU) is highly clinically beneficial in patients with sickle cell disease (SCD). While HU reduces leukocytosis and inflammation, in general, patients with high levels of fetal hemoglobin (HbF) induction experience the greatest clinical benefit from HU. Despite over 25 years of use in patients with SCD, and intense study, the mechanism by which HU induces HbF has not been elucidated, nor the reasons for the considerable inter-individual variability in the amount of HU-mediated HbF induction. Proposed mechanisms of HbF induction by HU include erythropoietic stress response, production of nitric oxide, or reduction of BCL11A, KLF-1, and MYB, negative regulators of HbF. Various signaling pathways have been implicated in HU-mediated HbF induction, such as the activation of the ERK-1/ERK-2 by erythropoietin; Giα/JNK/Jun; p38/MAPK/CREB1; histone deacetylase (HDAC) and DNA methyl-transferase (DNMT) inhibitor-mediated epigenetic modification of γ-globin expression. To identify new genes and pathways involved in HU activity, we studied the in vivo effects of HU on the gene expression profile of paired samples obtained from the reticulocytes of children with SCD before and after HU treatment. Methods : 25 paired RNA samples were collected before HU treatment (pre-HU) and at the stable maximum tolerated dose (MTD) from peripheral blood CD71+ reticulocytes of pediatric patients from Texas Children's Hospital. Subjects were 1.2 to 19.1 years of age at the time of pre-HU sample collection (23 HbSS, 2 HbSβ0) and 48% were female. Hematologic indices were extracted from patient's medical record. RNA sequencing was performed through Illumina platform, paired-end 150 bps, with at least 20 million read in-depth. Quality control of raw sequence data was done with FastQC software. We used Kallisto to determine the compatibility of reads with targets and quantify abundances of transcripts. Then, we used Sleuth to implement statistical algorithms for differential analysis. After validation of results through relative quantity assessment of selected genes by RT-qPCR, the Enrichr web server was used for gene set enrichment (GSE) and pathway analysis. We chose two candidate genes, KLF4 and STAT5A, to analyze functionally based on differential expression, inclusion in enriched pathway analysis, and reported involvement in relevant biological processes, including erythroid maturation or HbF induction. siRNA knockdown of KLF4, and STAT5A in K562 cells was performed by chemical transfection (DharmaFECT) and was verified by RT-qPCR. K562 cells were treated with HU at 50 µM for 24 hours; RNA was extracted and analyzed for γ-globin expression levels by RT-qPCR. Results and conclusions: We compared our pre-HU and at MTD paired samples using a two-fold change expression cut-off and 0.1 FDR multiple comparison correction threshold. Using this threshold, 139 genes had statistically significant differential levels of expression.135 were upregulated and 4 downregulated in response to HU treatment. Pathway analysis primarily grouped the upregulated genes more into the EPO, IL-2, and IL-3 signaling pathways (adjusted p-value

Description: Background: In sickle cell anemia (SCA), hemoglobin S (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These deoxygenated RBCs have strongly reduced deformability, which contributes to the etiology of vaso-occlusive crises and chronic hemolytic anemia. There are no widely available clinical laboratory tests to directly monitor effects of disease modifying therapies (i.e. hydroxyurea) on RBC deformability. RBC deformability can be measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, the Netherlands), which measures RBC deformability over a range of osmolalities. Recently, a new module was added which consists of a method to measure RBC deformability, expressed as Elongation Index (EI), during controlled deoxygenation. This test, termed oxygenscan, has 3 key read out parameters: 1) EImax, which represents RBC deformability at normoxia; 2) EImin represents deformability upon deoxygenation; and 3) the point of sickling (PoS), the point at which a 〉5% decrease in EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1). In this study, we correlated laboratory parameters associated with SCA disease severity with oxygenscan parameters to establish the clinical utility of this test. Methods: The discovery cohort consisted of 15 SCA patients (median age 22.0 years, 33.3% on hydroxyurea (HU)) enrolled at University Medical Center Utrecht (UMCU). The validation cohort consisted of 21 patients with SCA (median age 12.5 years, 76.2% on HU) from Texas Children's Hematology Center (TCHC). Oxygenscans were carried out in duplicates at both sites. Percentage dense RBC (%DRBC) were measured using an ADVIA hematology analyzer (Siemens) at TCHC only. In this study, we used Pearson's correlation to test for linear correlations between oxygenscan parameters EImax, EImin and PoS and clinically relevant laboratory parameters: total hemoglobin (Hb), absolute reticulocyte count (ARC), %HbS and %HbF, and %DRBC. Results: In both cohorts PoS significantly positively correlated with ARC (Figure 2A-B). In the UMCU cohort, total Hb levels also significantly positively correlated with EImax (Figure 2C), which was validated in the TCHC cohort (Figure 2D). HbF positively correlated with the EImin in both cohorts (Figure 2E-F). EImin also significantly negatively correlated with HbS (r=-0.828 p=

Description: Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect 1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity 〉98% and specificity 〉99%, even in the absence of paternal DNA. Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with 〉20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is

Description: Background: Many patients with sickle cell disease (SCD) require a surgical splenectomy for repeat splenic sequestration or hypersplenism, resulting in worsening anemia and/or thrombocytopenia, or abdominal discomfort. Higher rates of thrombosis, pain crises and acute chest syndrome (ACS) have been reported following surgical splenectomy, although the reasons for this are not known. We hypothesize that this clinical worsening post-splenectomy is due to hemorheological changes; studies of the effects of surgical splenectomy on hemorheology in non-SCD animal models found significant reductions in red cell deformability and increase in whole blood viscosity, or blood thickness, following splenectomy. Understanding the impact of surgical splenectomy on blood rheology is especially relevant for patients with SCD, who have many clinical complications as a result of their high whole blood viscosity for their given hemoglobin levels, and low hematocrit-to-viscosity ratio (HVR), a measure of oxygen carrying capacity. Another important measure of SCD rheology is percent dense red blood cells (%DRBC), red cells with a density〉1.11 mg/mL; they are typically the result of cellular dehydration, and are less deformable and more likely to sickle. We therefore sought to use our existing longitudinal rheology data, including measures of viscosity and %DRBC, to evaluate the impact of surgical splenectomy on our pediatric patients with SCD. Methods: We identified seven pediatric patients with multiple measurements of whole blood viscosity and %DRBC, collected before and after surgical splenectomy between November 2013 and April 2018 from SCD patients at Texas Children's Hospital on an IRB approved protocol. The cohort included 4 female and 3 male patients, ages 3-12 years old. Whole blood viscosity was measured using a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 37 degrees Celsius within 4 hours of sample collection in an EDTA vacutainer tube. CBC data including %DRBC was measured on an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Samples collected 1 month before or after an emergency department visit or within 3 months of a packed red blood cell transfusion were omitted from analysis. Results: We found a significant rise in %DRBC following splenectomy (p=0.01). There was a significant increase in whole blood viscosity at 45 s-1 and 225 s-1 (p=0.006 and p=0.004, respectively) and a decline in hematocrit-to-viscosity ratio (HVR) at 45 s-1 and 225 s-1 (p=0.03 and p=0.03, respectively) (Table 1). Hemoglobin and hematocrit did not significantly change after splenectomy (p=0.6 and p=0.5, respectively), suggesting that the rise in viscosity was due to intrinsic changes in red cell rheology. Platelets increased markedly (p

Description: Background. Avascular necrosis (AVN) is a serious complication of sickle cell disease (SCD) that can lead to significant morbidity including chronic pain and physical impairment with treatment depending on the patient's pain severity and functional limitation. Patient age, hemoglobin levels, and abnormal rheology, particularly high blood viscosity, or thickness, and percentage of dense red blood cells, defined as cells 〉1.11 mg/mL have been identified as risk factors for the development of AVN, but their contributions to the subsequent clinical course has not been described. The natural history of AVN has been described as chronic and progressive without significant surgical or physical therapy based intervention. However, this description was established before widespread MRI use in diagnosis, or hydroxyurea (HU). A new evaluation of the natural history of AVN is needed. Objective: Review the natural history of AVN at our institution in patients for whom repeat imaging is available, and determine if patient demographics, treatment type, and laboratory data, including rheological values, predict of clinical course. Methods. We identified all patients with a diagnosis of AVN with diagnostic and follow-up imaging studies (pelvic X-rays or MRI of the hip), receiving care at Texas Children's Hospital Hematology Center 2006 to 2018, and collected demographic, treatment, rheological and clinical laboratory data. Patient radiographic images at diagnosis and follow up were reviewed and scored by a board certified pediatric radiologist using the Steinberg criteria. Laboratory values for each patient were collected as available from time of diagnosis through to the date of follow up imaging. Longitudinal patient blood samples were collected for rheological measurements under an IRB approved protocol. Whole blood viscosity was measured on a cone and plate viscometer (DV3T Rheometer, AMETEK Brookfield, Middleboro, MA, USA) at 45 and 225 s-1 at 37oC within 4 hours of sample collection in an EDTA vacutainer tube. Percent dense red blood cells (%DRBCs) were measured by an ADVIA 120 Hematology System (Siemens Medical Solutions USA, Inc., Malvern, PA, USA). Statistical analysis comparing groups with and without radiographic improvement was performed using a two-tailed Student's t-test. Results. 16 patients had repeat imaging, allowing for assessment of radiographic improvement (Table1). All were diagnosed via X ray or MRI of the pelvis. Five of 16 patients had resolution or significant radiographic improvement of AVN without orthopedic intervention or prolonged physical therapy, while 11 patients had stable or worsening disease. In the group with radiographic improvement, the mean age was 9.9 years (SD=2.5) and 14.6 years (SD=2.2) in the group without radiographic improvement (p= 0.002). Five patients underwent core decompression with bone marrow aspirate concentrate injection; one progressed to complete femoral head collapse while the rest had stable or worsening disease. Mean hemoglobin (Hb), fetal hemoglobin (HbF), percent DRBCs, whole blood viscosity, and extent of AVN at diagnosis by Steinberg criteria were not statistically different between the patients that showed improvement and those that did not. Conclusions. Our results challenge the paradigm of untreated AVN in SCD as an inexorably progressive disease. Here we present 5 cases of significant radiographic improvement of AVN without surgical or significant physical therapy interventions. Conversely, 5 AVN cases treated with core decompression remained stable or worsened. Given our small cohort, we were unable to establish laboratory or rheological predictors of spontaneous resolution. However, patients in the radiographic improvement group were younger than patients with progressive disease. HU has shown promise in improving vascular complications in SCD patients however more research is needed to delineate its role as a modifier of AVN natural history as it was not correlated with the improved group in our study (all our study patients were on HU either prior to diagnosis or started during the follow up period). Given the findings that significant radiographic improvement is possible with minimal or no intervention, providers should closely weigh the risks and benefits of observation versus surgical intervention, particularly in young patients with SCD. Disclosures No relevant conflicts of interest to declare.

Description: Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a range of oxygen concentrations using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, Zwaag, the Netherlands) with Oxygenscan module. The Oxygenscan measures 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in normoxic EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that Oxygenscan parameters correlate with measures of SCD disease severity and hemolysis (absolute reticulocyte count, %fetal hemoglobin, %HbS, total Hb, %dense RBC, (Rab et al. Blood 2018). In this study, we investigated the relationship between oxygenscan parameters and incidence of vaso-occlusive crisis (VOC), and we tested the ability of the Oxygenscan parameters to assess response to hydroxyurea (HU) and chronic transfusion (CTF) in patients with SCD. Methods: We analyzed 2 cohorts: a European cohort (EUC) of 62 SCD patients (all HbSS or HbS/β-thalassemia), enrolled at either University Medical Center Utrecht (UMCU, n= 42) or Hospital Lyon (LIBM, n=20), and a US cohort (USC) of 97 SCD patients enrolled at Texas Children's Hospital (TCH). Differences in Oxygenscan parameters in SCD patients without/with VOC (requiring a doctor's evaluation) in the past 2 years were measured in 46 adult EUC SCD patients and 80 pediatric USC SCD patients. EUC patients without VOC (n=18, median age 40.6 years (y); 11 female (F), 44% on HU, 6% on CTF, 28% on both treatments), were compared to patients with a positive history of VOC (n=28, median age 23.9y, 13F, 39% on HU, 11% on CTF and 11% on both). Patients without VOC in the USC (n= 34, median age 8.0y, 15F, 53% on HU, 15% on CTF and 21% on both treatments) were compared to patients with VOC (n=46, median age 11.8y, 20F, 74% on HU, 13% on CTF and 11% on both treatments). To establish treatment related Oxygenscan parameter changes, we analyzed RBCs from 9 SCD patients from UMCU (median age 19y, 5F), before and during HU treatment (measurements performed at baseline, and 1, 3 and 6 months after starting HU), 7 SCD patients from UMCU (median age 26.7y; 6F) before and after transfusion and 17 SCD patients from TCH (median age 10.8y; 6F) on HU before and after transfusion. Results: In the EUC, PoS differed significantly between patients without VOC in the last 2 years (median 41.6mmHg) and patients with VOC in the last 2 years (median 53.7 mmHg, p

Description: Background: Patients with sickle cell disease (SCD) often experience severe chronic pain. In chronic pain, microglia are readily activated, stimulating neurons to send a pain signal. Human microglia are difficult to obtain; we have cultured peripheral blood derived microglia-like cells (PBMLC) from human peripheral blood to develop a model system to investigate chronic pain in sickle cell disease. We have shown that our PB-MLC resemble microglia morphologically, have the same surface markers, and can be activated by lipopolysaccharide (LPS). Our PB-MLC are more easily activated and secrete more inflammatory cytokines if they are derived from a SCD patient with chronic pain compared to a HbAA individual without pain or a SCD patient with acute pain only. Now, we seek to assess the suitability of our model system to screen for compounds to treat chronic pain by using of a panel of compounds to reduce microglia and PB-MLC activation and cytokine release in response to LPS. Finally, we propose to validate our model system by comparing the morphology, surface markers, and cytokine release between PB-MLC and true in situ brain derived microglia (BDM), both from Sprague Dawley (SD) rats. Methods: Human peripheral blood mononuclear cells (HPB-MCs) were obtained from three individuals with SCD with chronic pain and three normal donors (WT). HPB-MCs were cultured with human GM-CSF (10 ng/ml) and human IL-34 (100 ng/ml) to induce peripheral blood derived microglia (HPB-MLC). TNF-alpha and IL-1beta secretion by HPB-MLCs in response to LPS with or without a panel of drugs were measured with ELISA. Rat BDM were isolated from fresh rat brain tissues using anti‐rat CD11b/c microbeads and cultured with murine IL-34(100 ng/ml) and murine GM-CSF (10 ng/ml). Rat PB-MLCs were developed from rat PB-MCs by culturing with murine IL-34 (100 ng/ml) and murine GM-CSF (10 ng/ml). On day 7 of culture, rat BDM and PB-MLCs were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry and indirect immunofluorescence with anti-CX3CR1, TMEM119, CD68, Iba1 antibodies. Microglia morphology was evaluated by quantitative analysis of cell body roundness and branch length. TNF-alpha, IL-1beta, and IL-6 secretion by rat BDM and PB-MLCs in response to LPS with or without piceatannol was measured with ELISA. Results: To evaluate the possibility of using our PB-MLC model system to screen compounds to reduce abnormal microglia activation and treat chronic pain, we tested HPB-MLC cells from patients with SCD and chronic pain with the following drugs: gabapentin, metformin, piceatannol, and resveratrol, chosen based on published reports of their effect on microglia activation. All suppressed the release of proinflammatory cytokine from LPS-induced HPB-MLC in a dose-dependent manner (Figure 1), and reversed the deramification, or rounding of activated PB-MLC upon LPS stimulation by quantitative analysis of cell body roundness and branch length with immunostaining of Iba1. Gabapentin exhibited the smallest effect on reduction of HPB-MLC activation. Rat BDM and PBMLC both exhibited characteristic microglia branched morphology in culture, and rat PBMLC from SD rats expressed microglia specific marker TMEM119. Both PB-MLC and BDM from rats became amoeboid with LPS treatment, and showed increased expression of CD68, and Iba1 (Figure 2). In both BDM and PBMLC from rats, piceatannol reduced LPS activation and TNF-alpha, IL-1beta, and IL-6 secretion (Figure 3). Conclusions: We have established the microglia-like nature of PBMLC from patients with SCD and normal blood donors, and the preservation of the patient pain phenotype in culture. Here, we show that compounds reported to reduce microglia activation reduce the inflammatory cytokine release from HPB-MLCs from patients with SCD. BDM and PB-MLCs from SD rats have similar morphology, both quiescent and activated, and secrete inflammatory cytokines in the same manner in response to LPS. Piceatannol reduces activation and cytokine release in both. This comparison between peripheral blood derived and brain derived microglia supports our assertion that PB-MLC capture significant aspects of true in situ brain microglia biology, particularly that of activation and drug response. We therefore propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Description: Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Mixed donor chimerism (MDC) occurs in nearly 35% of patients with sickle cell disease (SCD) post allogeneic hematopoietic cell transplant (alloHCT). Donor myeloid engraftment of 〉20% is considered necessary to support disease resolution. However, in a disease known for its clinical variability, we hypothesize that patients (pts) with a severe pre-HCT phenotype may have abnormal RBC rheology at a level of chimerism and %HbS believed to be consistent with a cure. RBC rheology is markedly abnormal in SCD; these abnormalities are associated with SCD related clinical complications. Even fully oxygenated, sickle RBC are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. Sickle RBC morphology reflects the tendency to polymerize, with characteristic sickle forms. Sickle RBC are more adherent to the endothelial cell lining of the vasculature than HbAA or HbAS RBCs. The goal of any cell-based therapy of curative intent should be normalize the red cell rheology. To determine if the rheology of blood from SCD pts post-HCT with varying degrees of donor chimerism fall into the HbSS range of values, we propose to functionally assess the peripheral blood from a series of 6 post alloHCT SCD patients using a battery of rheological tests measuring deformability, sickling, adherence, percent dense cells (%DRBC), and RBC morphology. Methods: Peripheral blood samples (EDTA) from 6 SCD pts post alloHCT with a matched sibling donor were collected and immediately analyzed using oxygenscan (Lorrca), artificial microvascular network (AMVN), microfluidic image acquisition, and an ADVIA hematology analyzer. The Lorrca with oxygenscan measures RBC deformability (elongation index EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which RBC deformability rapidly declines. RBC adhesiveness is measured by the difference in rate of perfusion of RBC through the AVMN coated with adhesive proteins (adherent AVMN) and a non-adherent, uncoated AVMN. %DRBC was measured by an ADVIA hematology analyzer. Microscopic RBC images were acquired and classified to determine the fraction of sickled RBCs in well-oxygenated samples. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA. Results: In Figure 1, we show the measurements obtained by the Lorrca with oxygenscan on 5 pts post-alloHCT with a range of donor chimerism, as well as typical values for HbAA, HbAS, and HbSS patients. Patient 1 and 3 exhibit normal rheology; both were transplanted with a HbAA donor, with high chimerism and no detectable HbS. Patient 2 has 40% whole blood chimerism, well above the 20% threshold described with myeloid chimerism, and a HbS less than 50%, yet their plot resembles that of an untransplanted HbSS pt; the donor to Patient 2 exhibits the expected HbAA deformability. Patients 5 and 6 exhibit rheology in the HbAS range; they have intermediate chimerism from a HbAS donor. Clinical details and biomarker panel values for all 6 pts analyzed are in Table 1, as well as ranges of values for HbSS and HbAS subjects generated in the Sheehan and Shevkoplyas labs. Patient 2 is the only subject with values in the HbSS range, and the only subject with SCD symptoms. Conclusions: Our rheological tests identified a pt with values consistent with a HbSS pt of moderate severity or treated with hydroxyurea despite variable donor chimerism above the 20% threshold and HbS

Description: Background: Cerebrovascular disease, particularly overt stroke, is one of the most critical clinical complications of sickle cell disease (SCD). Individual stroke risk for patients with SCD is assessed by measuring trans-cranial Doppler ultrasound (TCD) velocity. While TCD screening and resulting use of chronic blood transfusion therapy in patients with abnormal TCDs has reduced stroke incidence in patients with SCD from 11% to 1%, the test has significant limitations. It can only be performed on children old enough to remain still for the assessment, or young enough to have open bony windows; typically 2-16 years of age. Additionally the test has poor positive predictive value, causing patients to be placed on chronic transfusion therapy who would not have gone on to have a stroke. Hydroxyurea (HU) is replacing chronic transfusion therapy in many institutions for primary stroke prevention, but many sites initiate a "cooling off" period of transfusions prior to initiating HU, and transfusion therapy may be indicated for abnormal TCD velocities in patients already on HU. The pathophysiology of stroke in SCD patients is not completely understood, but vascular remodeling, abnormal cerebral blood flow, and vaso-occlusion all play a role. Plasma levels of Brain derived neurotrophic factor (BDNF), myeloperoxidase (MPO), vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be associated with high trans-cranial Doppler ultrasound (TCD) velocity in children with SCD; but there is limited data on their potential association with stroke in this population. BDNF is a neurotropin that is responsive to hypoxia and promotes neurogenesis as well as playing a key role in the regulation of the cell survival pathway; myeloperoxidase (MPO) promotes vascular injury by oxygen radical generation, leukocyte/neutrophil recruitment, and oxidative stress; vascular cell adhesion molecule 1 (VCAM-1) is and endothelial adhesion molecule involved in the pathophysiology of vaso-occlusion in SCD through promoting RBC adhesion; and intercellular adhesion molecule 1 (ICAM-1) is expressed on endothelial and immune system cells and is thought to be a ligand for leukocyte adhesion. We hypothesized that BDNF, MPO, VCAM-1 and ICAM-1 may be involved in stroke in SCD, and may be potential biomarkers to be used to assess an individual with SCD's stroke risk in addition to TCD. Methods: We collected plasma samples from the peripheral blood of fifteen SCD patients (HbSS) at the time of either silent cerebral infarct (SCI) or stroke. Stroke (n=8) or SI was confirmed by MRI using diffusion imaging and T2 FLAIR. The cohort included 6 females and 9 males between the ages of 3 and 20 years. Plasma from fifteen age and gender matched control patients with HbSS but had normal MRIs and TCD velocities less than 170 m/s. We measured plasma levels of BDNF, MPO, VCAM-1 and ICAM-1 using antibody immobilized fluorescent beads (Millipore, Billerica, MA) and Luminex xMAP technology (Bio-Rad, Hercules, CA). Student's t-test was used to analyze the difference in plasma levels between the stroke and control groups. Results and Conclusions: BDNF levels were significantly higher in the stroke group than in the control group; 2978.2 ± 960.3 pg/ml compared to 2200.8 ± 758.4 pg/ml, p=0.005. Difference in MPO levels between the two groups approached significance; 36617.2 ± 14828.9 pg/ml compared to 29521.9 ± 7889.6 pg/ml, p=0.07. Difference in VCAM-1 levels also approached significance; 143997.0 ± 9963.8 pg/ml compared to 138126.9 ± 11902.0 pg/ml, p=0.08, but there was no significant difference in ICAM-1 levels. Only BDNF plasma levels positively correlated with the area and volume of the infarct, p=0.009. These results further support the assertion that BDNF is involved in the pathophysiology of cerebrovascular disease in SCD. It is possible that MPO and VCAM-1 may also play a role, and that a significant difference was not found due to sample size limitations. Plasma BDNF levels, and possibly MPO and VCAM-1 plasma levels, have potential as biomarkers for stroke risk to improve the positive predictive value of TCD velocities in patients with SCD, or to assess risk in patients unable to receive TCDs. Disclosures No relevant conflicts of interest to declare.