Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers.

Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.

Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive crises (VOCs). Sickle erythrocytes (SSRBCs) contribute to VOCs by participating in a series of adhesive events with blood cells and the vascular endothelium. Adhesion assays have been used to evaluate the relationship between SSRBC adhesion and SCD severity. We developed a standardized, clinical flow adhesion assay of whole blood to vascular cell adhesion molecule (FA-WB-VCAM). The objective of this study was to assess the variability and clinical predictive value of FA-WB-VCAM in a six-month longitudinal, observational study (ELIPSIS) in SCD subjects during at-home, steady-state and self-reported VOCs, and following VOC resolution. We observed a strong relationship between FA-WB-VCAM and SCD severity. Adhesion indices were significantly lower in SCD subjects on hydroxycarbamide and increased during VOCs; at-home VOCs had significantly higher FA-WB-VCAM than steady-state and contact VOCs. SCD subjects with a high frequency of self-reported VOCs had a pro-adhesive phenotype at steady state and were stratified into a high-adhesive phenotype cohort; two years prospectively we observed a higher frequency of VOCs in the high-adhesion cohort. This study supports stratifying SCD subjects based on steady-state FA-WB-VCAM and suggests that FA-WB-VCAM may be a plausible surrogate end-point for SCD severity.

Flow adhesion of whole blood to P-selectin: a prognostic biomarker for vaso-occlusive crisis in sickle cell disease.

Blood cell adhesion to P-selectin and vascular cell adhesion molecule-1 (VCAM-1) contributes to the pathophysiology of vaso-occlusion crisis (VOC) events in individuals with sickle cell disease (SCD). We evaluated the use of standardized flow adhesion biomarkers in a six-month, 35-subjects longitudinal study (ELIPSIS). Flow adhesion of whole blood on P-selectin (FA-WB-Psel) and VCAM1 (FA-WB-VCAM), and of isolated white blood cells on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) were elevated on VOC days compared with non-VOC days, but only FA-WB-Psel reached statistical significance (P = 0·015). Optimal cut-off values were established with Cox regression models for FA-WB-Psel [46 cells/mm²; hazard ratio (HR): 2·3; 95% confidence interval (CI):1·4-4·0; P = 0·01] and FA-WB-VCAM (408 cells/mm², HR:1·8; 95% CI: 0·9-3·45; P = 0·01). A combined (FA-WB-Psel and FA-WB-VCAM) multimarker risk score was also significantly (P = 0·0006) correlated with VOC risk that was two-fold higher for intermediate and 5·64-fold higher for high score. The concordance (C)-index for the multimarker score was 0·63 in the six-month period (95% CI: 0·56-0·70), indicating a better ability to distinguish patient risk of VOC, compared to individual biomarkers FA-WB-VCAM (C-index: 0·57; 95% CI: 0·49-0·65) or FA-WB-Psel (C-index: 0·58; 95% CI: 0·53-0·62). The presented multimarker score can be used to risk-stratify individuals with SCD during their steady state into low, intermediate, and high-risk strata for self-reported VOCs. Such risk stratification could help focus healthcare resources more efficiently to maintiain health, personalize treatment selection to each patient's individual needs, and potentially reduce healthcare costs.

Cross-sectional analysis of adhesion in individuals with sickle cell disease using a standardized whole blood adhesion bioassay to VCAM-1.

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that lead to severe pain, organ damage, and early death. Lack of reliable biomarkers that objectively define VOEs remains a critical barrier to improving the care for SCD patients. VOEs result from a complex interplay of cell-cell interactions that promote micro-vascular occlusion. Earlier studies demonstrated that sickle erythrocytes are more adherent than non-sickle erythrocytes and established a direct link between adhesion and frequency of VOEs. We developed a standardized, flow-based adhesion bioassay to assess the adhesive properties of SCD blood samples. The current study provides a cross-sectional analysis of steady state adhesion in SCD patients presenting at monthly out-patient hematology visits. Steady state adhesion varied from patient-to-patient. Adhesion positively correlated with reticulocyte percent and WBC count although there was no significant relationship between adhesion and platelets or hemoglobin in this study. Additionally, steady state adhesion indices were significantly lower in SCD subjects receiving hydroxyurea therapy when compared to the untreated group. The well-plate based microfluidic flow adhesion bioassay described in this report may provide a platform to identify SCD subjects with severe disease phenotypes, predict impending VOEs, and monitor response to current and developing therapies.

VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow.

Very Late Antigen-4 (VLA-4, α4ß1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 µg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.

Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

GBT1118, a voxelotor analog, protects red blood cells from damage during severe hypoxia.

A lack of objective metrics in Sickle Cell Disease (SCD) makes it difficult to assess individual patient therapy options or assess the effects of therapy. This is further complicated by mechanisms of action involving multiple interconnected effects, that combine to relieve SCD symptoms. In 2019, based on the increase in hemoglobin concentration observed in the HOPE trial, the Food and Drug Administration approved voxelotor (Oxbryta®, Global Blood Therapeutics) for SCD patients 12 years and older. The main mechanism of action for voxelotor was increased hemoglobin-oxygen affinity, but other mechanisms may apply. In this study, we assessed the effect of GBT1118, an Oxbryta analog, on hypoxia-induced lethal and sub-hemolytic red blood cell (RBC) membrane damage using RBC Mechanical Fragility (MF), a metric of existing membrane damage and prospective hemolysis. RBC MF was measured non-invasively using a proprietary system comprising an electromagnetic bead mill and fiberoptic spectrophotometry detection. Three cycles of severe hypoxia (<5% oxygenated hemoglobin) with follow-up reoxygenation resulted in a significant increase in RBC MF for all SCD (Hb-S >60%) samples. Supplementation with GBT1118 caused no significant changes in pre-hypoxia RBC MF. However, following GBT1118 treatment, cell stability showed significantly less degradation, as evidenced by a significantly smaller RBC MF increase after three cycles of hypoxia-reoxygenation. These findings indicate that GBT1118 prevents hypoxia-induced membrane damage in sickled RBC, in part by alternative mechanisms not associated with induced changes in hemoglobin-oxygen affinity.

Can red blood cell function assays assess response to red cell-modifying therapies?

BACKGROUND: Red blood cell (RBC)-modifying therapies have provided new opportunities for patients with sickle cell disease, although the absence of validated biomarkers of RBC function is a barrier to FDA approval and clinical adoption. Flow Adhesion (FA) and Mechanical Fragility (MF) biomarkers objectively stratify individuals with SCD into pro-adhesive vs pro-hemolytic phenotypes respectively, which may potentially help predict therapeutic responses. OBJECTIVE: A Phase 3 clinical trial to determine the effectiveness of vepoloxamer, an RBC-modifying therapy in sickle cell disease (SCD), failed to meet its primary clinical outcome. The aim of this study was to determine whether standardized flow adhesion and mechanical fragility bioassays could differentiate cellular level "responders" from "non-responders" to vepoloxamer treatment. METHODS: Standardized biomarkers of RBC function (adhesion and mechanical fragility) were utilized in this study to assess the effect of veploxamer on blood samples collected from SCD subjects and to determine whether our assays could differentiate cellular-level "responders" from "non-responders" to vepoloxamer treatment. A Wilcoxon signed-rank test was used to test for differences in adhesion in response to varying vepoloxamer treatments and a Wilcoxon Mann-Whitney test was used to assess differences in mechanical fragility, pre- and post-vepoloxamer treatment. A p-value<0.05 was considered significant. RESULTS: In this study, we report that in vitro treatment with vepoloxamer reduced adhesion by >75%in 54%of patient samples and induced changes in the membranes of sickle erythrocytes (SSRBCs) making sickle cells behave more like normal erythrocytes (AARBCs) in terms of their resistance to hemolysis. CONCLUSION: This study demonstrates that the standardized flow adhesion and mechanical fragility biomarkers described here may be useful tools to predict clinical responders to RBC-modifying therapies.

Central nervous system events in children with sickle cell disease presenting acutely with headache.

OBJECTIVE: To determine the frequency of acute care visits and risk factors for central nervous system (CNS) events in children with homozygous sickle cell disease (SCD-SS) with an acute headache. STUDY DESIGN: This is a retrospective cohort study of acute care visits for headache in children with SCD-SS. The prevalence of headache visits, neuroimaging evaluation, and acute CNS events were calculated and clinical and laboratory variables assessed. RESULTS: Headache was the chief complaint in 102 of 2685 acute care visits (3.8%) by children with SCD-SS. Acute CNS events were detected in 6.9% of these visits. Neuroimaging was performed in 42.2% of visits, and acute CNS events were identified in 16.3% of studies. Factors associated with acute CNS events included older age, history of stroke, transient ischemic attack, or seizure, neurologic symptoms, focal neurologic exam findings, and elevated platelets. CONCLUSIONS: Acute headache is common in pediatric SCD-SS and more frequently associated with acute CNS events than in the general pediatric population. A history of stroke, transient ischemic attack, seizures, neurologic symptoms, focal neurologic exam, or elevated platelet counts at presentation warrant confirmatory imaging studies. Whether a more limited workup is adequate for other children should be confirmed in a larger, prospective study.

Ghosal hematodiaphyseal dysplasia: a rare cause of a myelophthisic anemia.

Ghosal hematodiaphyseal dysplasia syndrome (GHDD) is a rare clinical syndrome characterized by increased bone density and a severe, myelophthisic anemia. Few cases have been reported worldwide and there are no detailed descriptions of the associated hematologic abnormalities and long-term clinical outcomes after treatment. Here, we report two siblings with GHDD who were successfully treated with chronic, low dose, corticosteroid therapy. Although GHDD is uncommon, these cases illustrate the need to consider GHDD in patients with anemia and bone dysplasia and the use of chronic, low-dose steroid therapy.

Pulsed-dosing with oral sodium phenylbutyrate increases hemoglobin F in a patient with sickle cell anemia.

Increasing hemoglobin F (HbF) appears to be beneficial for patients with sickle cell anemia. We previously demonstrated that daily, oral sodium phenylbutyrate (OSPB) induces HbF synthesis in pediatric and adult patients with hemoglobin SS (HbSS). The high doses and need for daily therapy, however, have limited its use. Here, we report a patient treated with pulsed-dosing of OSPB for over 3 years. This patient developed a modest, but sustained elevation in HbF over the course of therapy without side effects. Although larger studies are needed, this case demonstrates that pulsed-dosing with OSPB enhances HbF synthesis.

Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay.

Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60-120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.

A novel role of h2-calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow.

Calponin is an actin filament-associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2-calponin isoform is expressed in myeloid-derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2-calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2-calponin and actin colocalized in both human and wild-type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow-based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2-calponin knockout versus wild-type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2-calponin knockout mice also had prolonged bleeding time and blood loss. H2-calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2-calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild-type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2-calponin may be a novel target for therapeutic platelet inhibition.

Copeptin as a marker of relative arginine vasopressin deficiency after pediatric cardiac surgery.

PURPOSE: Relative arginine vasopressin (AVP) deficiency after pediatric cardiac surgery has recently been described. Copeptin, a more stable and easily measured product of pro-AVP processing, may be a means of identifying these patients. We aimed to determine if copeptin was correlated with AVP in these children and whether it can be a surrogate marker of relative AVP deficiency. METHODS: Patients <6 years of age with basic Aristotle scores ≥7 requiring surgery with cardiopulmonary bypass were prospectively enrolled. Plasma AVP and copeptin concentrations were measured pre-cardiopulmonary bypass and 4 and 24 h post-cardiopulmonary bypass. Relative AVP deficiency was defined a priori based on our previous work as AVP <9.2 pg/ml at 4 h post-cardiopulmonary bypass. RESULTS: Of 41 children enrolled, relative AVP deficiency was present in 13 (32 %). AVP and copeptin concentrations were significantly lower in these 13 children at 4 h post-cardiopulmonary bypass as compared to the other 28 patients. A significant positive association between plasma AVP and copeptin concentrations over time was determined. Based on log-transformed analyses, a 1 % increase in plasma AVP led to a 0.19 % increase in copeptin. Further, copeptin <1.12 ng/ml at 4 h post-cardiopulmonary bypass had a sensitivity of 92 % and a negative predictive value of 95 % for relative AVP deficiency. CONCLUSIONS: Plasma AVP and copeptin are positively associated in children undergoing cardiac surgery. Copeptin may represent a useful means of identifying relative AVP deficiency in these patients.

Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-alphavbeta3 interactions.

The possible role of physiologic stress hormones in enhancing adhesion of sickle erythrocytes (SS RBCs) to endothelial cells (ECs) in sickle cell disease (SCD) has not been previously explored. We have now found that up-regulation of intracellular cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) by epinephrine significantly increased sickle but not normal erythrocyte adhesion to both primary and immortalized ECs. Inhibition of serine/threonine phosphatases also enhanced sickle erythrocyte adhesion at least partially through a PKA-dependent mechanism. Adhesion was mediated through LW (intercellular adhesion molecule-4 [ICAM-4], CD242) blood group glycoprotein, and immunoprecipitation studies showed that LW on sickle but not on normal erythrocytes undergoes increased PKA-dependent serine phosphorylation as a result of activation. The major counter receptor for LW was identified as the alphavbeta3 integrin on ECs. These data suggest that adrenergic hormones such as epinephrine may initiate or exacerbate vaso-occlusion and thus contribute to the association of vaso-occlusive events with physiologic stress.

Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion.

The vasoocclusive crisis is the major clinical feature of sickle cell anemia, which is believed to be initiated or sustained by sickle (SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but not unaffected (AA) RBCs, adhere avidly to multiple components of the vascular wall, including laminin. Here we report a novel role for epinephrine and cyclic adenosine monophosphate (cAMP) in the regulation of human SS RBC adhesiveness via the laminin receptor, basal cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs under basal conditions. Forskolin or the stress mediator epinephrine further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to laminin in a protein kinase A (PKA)-dependent manner, with the low-density population being the most responsive. Epinephrine-stimulated adhesion to laminin, mediated primarily via the beta 2-adrenergic receptor, occurred in SS RBC samples from 46% of patients and was blocked by recombinant, soluble BCAM/Lu, implicating this receptor as a target of cAMP signaling. Thus, these studies demonstrate a novel, rapid regulation of SS RBC adhesion by a cAMP-dependent pathway and suggest that components of this pathway, particularly PKA, the beta 2-adrenergic receptor, and BCAM/Lu, should be further explored as potential therapeutic targets to inhibit SS RBC adhesion.