Hemolytic Biomarkers Predict Adhesiveness of Sickle Blood Cells in a Clinical Adhesion Bioassay

Abstract

Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that produce severe pain, organ damage, and early death. Lack of reliable biomarkersto objectively define VOEs, hindersthe development of clinically useful interventions to improve the care for these patients.VOEs result from a complex interplay of adhesive eventsmediated by cell surface adhesion molecules elevated in the SCD microenvironment. We developed a clinical adhesion assay that incorporates the adhesive properties of mature erythrocytes, reticulocytes, and leukocytes to measure adhesion from blood samples obtained from SCD subjects. Blood is acquired by venipuncture in sodium citrate tubes and can be stored for up 48hrs at 4°C.Flow conditions (37°C, 1.0 dyne/cm2, 1.67Hz) mimic blood flow in the post-capillary venules where these adhesive interactions are likely to occur. Adherent cells are quantified within a standard viewing area (cells/mm2) to generate an adhesion index(AI).Adhesion was evaluated in SCD subjects in a 6-month, longitudinal, observational study. Hematologic lab values were obtained at each blood draw. An electronic patient reported outcome (ePRO) tool defined steady state and VOE status. We have shown that AIs are elevated in SCD subjects with severe disease phenotypes and during VOEs and decreased in SCD subjects receiving SCD-modifying therapies. The objective of this study was to identify hematologic lab values that contribute to cellular adhesion in our clinical adhesion bioassay. A multi-variantmixed-effects regression model was used to allow for the estimation of different item factor loadings (regression coefficients) for the multiple outcomes. Coefficient of determination for each variable or model was calculated using MuMin package in Rstatistical software.Here, we report that a lower hematocrit (Hct) and higher reticulocyte percent (retic %) predicts (24%; p=0.009) cellular adhesiveness in blood samples collected from SCD subjects at steady state. However, adhesive predictors vary during VOE (Hct or hemoglobin F-HbF). Our findings suggest that cellular adhesiveness in SCD, as assessed by our clinical adhesion assay, may be a surrogate for clinical state and/or identifying impending VOEs. Further studies are needed to validate the utility of the adhesion bioassay in assessing the clinical utility of adhesion as a biomarker in clinical practice. Disclosures White: Functional Fluidics:Current equity holder in private company.Gao:Functional Fluidics:Current equity holder in private company.Tarasev:Functional Fluidics:Current equity holder in private company.Liu:Functional Fluidics:Current equity holder in private company.Callaghan:Octapharma:Honoraria, Membership on an entity's Board of Directors or advisory committees;Spark:Honoraria, Membership on an entity's Board of Directors or advisory committees;Biomarin:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Site Investigator/sub-I Clinical Trial, Speakers Bureau;Bioverativ:Membership on an entity's Board of Directors or advisory committees;Shire:Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau;Grifols:Honoraria, Membership on an entity's Board of Directors or advisory committees;Bayer:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Roche/Genentech:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Site Investigator/sub-I Clinical Trial, Speakers Bureau;Sancillio:Other;Alnylum:Current equity holder in publicly-traded company;NovoNordisk:Other, Speakers Bureau;Global Blood Therapeutics:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Speakers Bureau;Pfizer:Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Site Investigator/sub-I Clinical Trial, Research Funding;Hema Biologics:Honoraria, Membership on an entity's Board of Directors or advisory committees.Hines:Functional Fluidics:Current equity holder in private company.