GBT1118, A Voxelotor Analog, Protects Red Blood Cells From Damage During Severe Hypoxia
Michael Tarasev, Marta Ferranti, Andrew Herppich, Patrick Hines
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.
Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease
White, J., Callaghan, M.U., Gao, X., Liu, K., Zaidi, A., Tarasev, M. and Hines, P.C. (2021), Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease. Br J Haematol.
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
Flow Adhesion of Whole Blood to P-selectin: A Prognostic Biomarker for Vaso-Occlusive Crisis in Sickle Cell Disease
Hines, P.C., Callaghan, M.U., Zaidi, A.U., Gao, X., Liu, K., White, J. and Tarasev, M. (2021), Flow adhesion of whole blood to P-selectin: a prognostic biomarker for vaso-occlusive crisis in sickle cell disease. Br J Haematol, 194: 1074-1082. https://doi.org/10.1111/bjh.17643
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 theuse of standardized ﬂow adhesion biomarkers in a six-month, 35-subjectslongitudinal study (ELIPSIS). Flow adhesion of whole blood on P-selectin(FA-WB-Psel) and VCAM1 (FA-WB-V CAM), and of isolated white bloodcells on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) wereelevated on VOC days compared with non-VOC days, but only FA-WB-Pselreached statistical signiﬁcance (P = 0015). Optimal cut-off values wereestablished with Cox regression models for FA-WB-Psel [46 cells/mm; haz-ard ratio (HR): 23; 95% conﬁdence inte rval (CI):14–40; P = 001] and FA-WB-VCAM (408 cells/mm, HR:18; 95% CI: 09–345; P = 001). A com-bined (FA-WB-Psel and FA-WB-VCAM) mu ltimarker risk score was also sig-niﬁcantly (P = 00006) correlated with VOC risk that was two-fold higherfor intermediate and 564-fold higher for high score. The concordance (C)-index for the multimarker score was 063 in the six-month period (95% CI:056–070), indicating a better ability to distinguish patient risk of VOC,compared to individual biomarkers FA-WB-VCAM (C-index: 0 57; 95% CI:049–065) or FA-WB-Psel (C-index: 058; 95% CI: 053–062). The presentedmultimarker score can be used to risk-stratify individuals with SCD duringtheir steady state into low, intermediate, and high-risk strata for self-reportedVOCs. Such risk stratiﬁcation could help focus healthcare resources moreefﬁciently to maintiain health, personalize treatment selection to eachpatient’s individual needs, and potentially reduce healthcare costs.
Keywords: ﬂow adhesion, P-selectin, vascular cell adhesion molecule-1,biomarker, sickle cell disease
Cross-Sectional Analysis of Adhesion in Individuals with Sickle Cell Disease Using a Standardized Whole Blood Adhesion Bioassay to VCAM-1
White J, Lancelot M, Gao X, McGraw BJ, Tabb C 2nd, Hines P. Cross-sectional analysis of adhesion in individuals with sickle cell disease using a standardized whole blood adhesion bioassay to VCAM-1. Blood Cells Mol Dis. 2020 Mar;81:102397. doi: 10.1016/j.bcmd.2019.102397. Epub 2019 Dec 6. PMID: 31864103.
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.
Can Red Blood Cell Function Assays Assess Response to Red Cell-Modifying Therapies?
White J, Moira L, Gao X, Tarasev M, Chakraborty S, Emanuele M, Hines PC. Can red blood cell function assays assess response to red cell-modifying therapies? Clin Hemorheol Microcirc. 2021 Jan 7. doi: 10.3233/CH-200944. Epub ahead of print. PMID: 33459699.
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.
Keywords: Sickle cell disease; adhesion; blood function assays; mechanical fragility; vepoloxamer.
Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by Electronic Patient-Reported Outcomes, Actigraphy, and Biomarkers
Pittman DD, Hines PC, Beidler D, Rybin D, Frelinger AL, Michelson AD, Liu K, Gao X, White J, Zaidi AU, Charnigo RJ, Callaghan MU. Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers. Blood. 2021 Apr 15;137(15):2010-2020. doi: 10.1182/blood.2020006020. PMID: 33067606;
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.
Red Blood Cell Mechanical Fragility as Potential Metric for Assessing Blood Damage Caused by Implantable Durable Ventricular Assist Devices: Comparison of Two Types of Centrifugal Flow Left Ventricular Assist Devices.
Tarasev M, Chakraborty S, Light L, Alfano K, Pagani F.D. Red blood cell mechanical fragility as potential metric for assessing blood damage caused by implantable durable ventricular assist devices: Comparison of two types of centrifugal flow left ventricular assist devices, Progress in Pediatric Cardiology, Volume 56, 2020,101198, ISSN 1058-9813
Implantable Ventricular Assist Devices (VADs) have become a treatment of choice for patients with end-stage heart failure or cardiogenic shock, significantly increasing both survival rates and the quality of life of patients. Moreover, VAD use is growing as destination therapy for patients who require permanent mechanical cardiac circulatory support. This heightens the need to ensure VAD reliability and safety, even amidst challenges in optimization of pump design for minimal blood damage. Advanced design LVADs like the HVAD (Medtronic, Inc., Minneapolis, MN) and the Heartmate 3 (HM3; Abbott Labs, Chicago, IL) are centrifugal systems that had been favorably compared to the continuous flow axial pumps, such as the HeartMate II (HMII; Abbott Labs, Chicago, IL). While the HVAD and HMII are increasingly utilized in older pediatric populations in addition to the Berlin Heart Excor (Berlin Heart GmbH, Berlin Germany), implementation of VAD support for pediatric patients still lags behind adults. Recently, the HM3 has demonstrated superior performance to the HMII with respect to pump thrombosis in clinical trials and may also find utility in pediatric applications, particularly for adolescents. In this present work, performance of HVAD and HM3 were compared using basic laboratory tests and Red Blood Cell (RBC) Mechanical Fragility (MF), an assay that provides assessment of sub-hemolytic RBC damage which can be caused by VAD operation. RBC MF was assessed using electromagnetically driven bead milling with cylindrical beads in multiple different regimes for different sample stressing configurations. Induced hemolysis in the sample was measured non-invasively at different (increasing) cumulative stress duration intervals, to obtain a MF profile for each stressing regime. In a cohort of 13 HVAD and 7 HM3 patients, blood samples were obtained before surgery and at 1 h, 24 h, 1 week and 4 weeks after surgery. No significant differences were observed between the two VAD devices in conventional hemolysis markers including free hemoglobin, bilirubin, total LDH and haptoglobin, as well as in changes patient hemoglobin. HM3 demonstrated elevated, compared to HVAD, levels of LDH-1 at 24 h (p < 0.05) and 1 week (0.05 < p < 0.1) after surgery, with LDH-1 reverting to about pre-surgery levels at 4 weeks. These differences between pumps may have been attributed to confounders such as duration of cardiopulmonary bypass at time of LVAD implant. By one metric, RBC MF was similarly elevated for HM3 at both 24 h and 1 week after surgery, and also reverted to about pre-surgery levels at 4 weeks. Such changes in RBC MF results were observed for only one of the employed stressing regimes, highlighting the potential importance of matching the applied in vitro stress parameters to the particular nature of in vivo blood damage involved. Forthcoming work is planned for further analysis and reporting on additional aspects of this study.
Impact of the Oscillating Bead Size and Shape on Induced Mechanical Stress on Red Blood Cells and Associated Hemolysis in Bead Milling
Tarasev M, Muchnik M, Chakraborty S (2019) Impact of the Oscillating Bead Size and Shape on Induced Mechanical Stress on Red Blood Cells and Associated Hemolysis in Bead Milling. Int J Blood Res Disord 6:041. doi.org/10.23937/2469-5696/1410041
While in circulation, red blood cells (RBC) need to elastically undergo large deformations without lysing, an ability that may be compromised by cell membrane damage. Such can be tested in vitro by subjecting an RBC sample to external mechanical stress, e.g. through bead milling or oscillation of an object in a sample. In addition to controlling frequency and duration of oscillations, this approach can be further tailored by bead selection/design.
This work studies effects of different beads in creating qualitatively as well as quantitatively different shear stresses when oscillated in a sample containing RBC.
Identical, diluted RBC samples were stressed via bead milling using different beads, with hemolysis profiles developed in each case.
Bead dimensions significantly impacted induced stress, both in magnitude as well as type, as reflected by hemolysis under respective conditions. Specifically, cell protection (from lysis) afforded by albumin (when present in the medium) showed a complex dependence on bead length and diameter, with the effect seemingly indicative of multiple combined stresses induced by the bead oscillation. The effect is described as an interplay of stresses generated in bead wake in combination with those generated in the annulus.
Bead oscillation based mechanical fragility (MF) profiling of RBC presents a useful tool for exploring effects of different shear stress types for various potential applications for assessment of blood damage, and particularly of sub-hemolytic red cell damage.
Sevuparin Blocks Sickle Blood Cell Adhesion and Sickle-Leucocyte Rolling on Immobilized L-selectin in a Dose Dependent Manner
White J, Lindgren M, Liu K, Gao X, Jendeberg L, Hines P. Sevuparin blocks sickle blood cell adhesion and sickle-leucocyte rolling on immobilized L-selectin in a dose dependent manner. Br J Haematol. 2019 Mar;184(5):873-876. doi: 10.1111/bjh.15188. Epub 2018 May 16. PMID: 29767405.
Adhesion of sickle red blood cells (SSRBC) to the vascular endothelium may initiate and propagate vascular obstruction in sickle cell disease (SCD) (Hoover et al, 1979; Hebbel et al, 1980). Hebbel et al (1980) were the first to report a correlation between erythrocyte adherence and disease severity. Subsequent studies demonstrated that the pathological adhesion of SSRBCs involves red cell receptors, adhesive bridging proteins and endothelial receptors (Joneckis et al, 1993; Swerlick et al, 1993; Udani et al, 1998; Hillery et al, 2000). These complex and multimodal mechanisms of SSRBC adhesion may require a multi‐targeted approach to achieve the best clinical outcome.
Individual Variability in Response to a Single Sickling Event for Normal, Sickle Cell, and Sickle Trait Erythrocytes
Tarasev M, Muchnik M, Light L, Alfano K, Chakraborty S. Individual variability in response to a single sickling event for normal, sickle cell, and sickle trait erythrocytes. Transl Res. 2017 Mar;181:96-107. doi: 10.1016/j.trsl.2016.09.005. Epub 2016 Sep 23. PMID: 27728824.
Hemoglobin S (Hb-S) polymerization is the primary event in sickle cell disease causing irreversible damage to red blood cell (RBC) membranes over repeated polymerization cycles. A single polymerization triggered by a hypoxic environment was reported to result in reversibly (upon reoxygenation) decreased RBC deformability and increased mechanical fragility (MF). Individualized responses have not been reported, although RBC fragility can vary significantly even among healthy individuals. This study evaluates individual variability in response to a single hypoxia-induced sickling event, through changes in RBC MF. Blood was drawn from 10 normal (AA), 11 sickle cell (SS), and 7 sickle trait (AS) subjects-with Hb-S fraction, osmotic fragility, and medical history also collected. Mechanical stress was applied using a bead mill at 50-Hz oscillation for 0.5-30 minutes. MF profiles here give percent hemolysis upon successive durations of stressing. MF was measured for AA, SS, and AS cells-each equilibrated (1) with air, (2) with nitrogen in an anaerobic chamber, and (3) with air after the hypoxic event. While AA subjects exhibited significantly different changes in fragility upon hypoxia, in all cases there was recovery to close to the initial MF values on reoxygenation. For AS subjects, recovery at reoxygenation was observed only in about half of the cases. Fragility of SS cells increased in hypoxia and decreased with reoxygenation, with significantly variable magnitude of recovery. The variability of response for individual AS and SS subjects indicates that some are potentially at higher risk of irreversible hypoxia-induced membrane damage.
Copyright © 2016 Elsevier Inc. All rights reserved.
Differences in Bead-Milling-Induced Hemolysis of Red Blood Cells Due to Shape and Size of Oscillating Bead
Alfano KM, Chakraborty S, Tarasev M. Differences in bead-milling-induced hemolysis of red blood cells due to shape and size of oscillating bead. Biomed Mater Eng. 2016 Sep 28;27(4):405-412. doi: 10.3233/BME-161594. PMID: 27689573.
Background: Red blood cell (RBC) susceptibility to hemolysis - or fragility - can be profiled by subjecting a sample to progressive durations of mechanical stress and measuring hemolysis upon each. The ability to control stress application with multiple variable parameters can be useful in various areas of research. Bead milling, by oscillating an object in a blood sample, can offer control of parameters including oscillation force and frequency.
Objective: This work addresses the role of bead shape and size, for a given container, in potentially creating qualitatively as well as quantitatively different fluidic stresses in the sample.
Methods: Identical, diluted RBC samples were stressed via bead milling using different beads, with other parameters the same. Resulting hemolysis was plotted for several time increments in each case.
Results: For a cylindrical bead oscillating at a given frequency and force, bead length was a determinant of albumin's protective effect on RBC, as reflected by mechanical fragility. Compared to a sphere of same diameter, the protective effect was absent with shorter cylinders, whereas for longer ones it appeared enhanced.
Conclusions: Bead milling based RBC fragility testing could present a useful tool for creating, and studying effects of different shear stress types in inducing hemolysis.
Keywords: RBC; bead mill; flow; fragility; hemolysis; shear.
Impact of Environment on Red Blood Cell ability to Withstand Mechanical Stress.
Tarasev M, Chakraborty S, Light L, Davenport R. Impact of environment on Red Blood Cell ability to withstand mechanical stress. Clin Hemorheol Microcirc. 2016 Nov 4;64(1):21-33. doi: 10.3233/CH-152037. PMID: 26890109.
Susceptibility of red blood cells (RBC) to hemolysis under mechanical stress is represented by RBC mechanical fragility (MF), with different types or intensities of stress potentially emphasizing different perturbations of RBC membranes. RBC membrane mechanics were shown to depend on cell environment, with many details not yet understood. Here, stress was applied to RBC using a bead mill with oscillation up to 50 Hz, over durations up to 50 minutes. MF profiles plot percent lysis upon stresses of progressive durations. Supplementing media with polyethylene glycol (PEG) which interacts with the cell membrane, but not Dextran which does not, resulted in higher resistance to hemolysis. Albumin, and to a lesser extent fibrinogen and globulins (at physiological concentrations), significantly increased cell ability to withstand mechanical stress versus with un-supplemented buffer solution and with PEG. This is partly due to changes in rheology, per tests done including (PEG) and Dextran, but is mostly due to cell-protein interaction, noting the effect of pH on RBC MF with albumin but not with buffer. Presence of lipids reduced RBC resistance to potentially hemolytic stress with lypemic plasma effecting lower "protection" from induced hemolysis than essentially fatty-acid free plasma. This effect was less dependent on incubation than on fatty-acid presence during stressing. The reduced propensity for hemolysis afforded by plasma proteins also depended markedly on the speed of the bead, potentially reflecting changes from a predominantly Von Karman trail at lower frequencies to an increasingly disorganized turbulent wake at higher frequencies.
Keywords: Red Blood Cells; albumin; blood storage; fatty acid; fibrinogen; gamma-globulin; hemolysis; mechanical fragility; plasma.
Low Molecular Weight Heparin Inhibits Sickle Erythrocyte Adhesion to VCAM-1 through VLA-4 Blockade in a Standardized Microfluidic Flow Adhesion Assay
Lancelot M, White J, Sarnaik S, Hines P. Low molecular weight heparin inhibits sickle erythrocyte adhesion to VCAM-1 through VLA-4 blockade in a standardized microfluidic flow adhesion assay. Br J Haematol. 2017 Aug;178(3):479-481. doi: 10.1111/bjh.14137. Epub 2016 Jun 24. PMID: 27341635.
The vaso-occlusive events in sickle cell disease (SCD) begin in early childhood, warranting the need for more preventative and therapeutic interventions for those affected. Vaso-occlusion is partly caused by adhesion of sickle erythrocytes (SSRBCs) to components of the vascular wall and to circulating leucocytes (WBCs). SSRBCs have greater adhesion to the vascular endothelium and sub-endothelial matrix (SEM) compared to RBCs from unaffected individuals.
VLA-4 Blockade by Natalizumab Inhibits Sickle Reticulocyte and Leukocyte Adhesion During Simulated Blood Flow.
White J, Krishnamoorthy S, Gupta D, Lancelot M, Moore N, Sarnaik S, Hobbs WE 2nd, Light DR, Hines P. VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow. Br J Haematol. 2016 Sep;174(6):970-82. doi: 10.1111/bjh.14158. Epub 2016 Jun 12. PMID: 27291690.
Very Late Antigen-4 (VLA-4, α4β1-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.
Keywords: adhesion; microfluidics; natalizumab; sickle cell; very late antigen-4.
An Approach to Measuring RBC Haemolysis and Profiling RBC Mechanical Fragility
Alfano KM, Tarasev M, Meines S, Parunak G. An approach to measuring RBC haemolysis and profiling RBC mechanical fragility. J Med Eng Technol. 2016;40(4):162-71. doi: 10.3109/03091902.2016.1153741. Epub 2016 Mar 23. PMID: 27004768.
Red blood cells (RBC) can be damaged by medical products, from storage or from disease. Haemolysis (cell rupture and haemoglobin release) is often a key indicator, with mechanical fragility (MF) offering the potential to assess sub-haemolytic damage as well. This article reports on a unique approach to measuring haemolysis, without the need for centrifugation or other sample separation. It also reports on employing that in measuring blood fragility (susceptibility to haemolysis) under shear stress, utilising an electromagnet to cause a bead to oscillate within a cartridge that contains the sample. Cycling between stressing and optical measurement of induced haemolysis at progressively increasing durations of stress provides a fragility profile. Sub-system-level testing shows high accuracy for the haemolysis measurements and fair consistency for MF profiling. Improving accuracy and precision of profiling is a current focus and a fully integrated and automated version of this system is under development.
Increased Erythrocyte Adhesion to VCAM-1 during Pulsatile Flow: Application of a Microfluidic Flow Adhesion Bioassay.
White J, Lancelot M, Sarnaik S, Hines P. Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay. Clin Hemorheol Microcirc. 2015;60(2):201-13. doi: 10.3233/CH-141847. PMID: 24898561;
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.
Keywords: Pulsatile flow; VCAM-1; VLA-4; adhesion; erythrocyte; microfluidics; sickle cell disease; variable shear.
RBC Mechanical Fragility as a Direct Blood Quality Metric to Supplement Storage Time
Tarasev M, Chakraborty S, Alfano K. RBC mechanical fragility as a direct blood quality metric to supplement storage time. Mil Med. 2015 Mar;180(3 Suppl):150-7. doi: 10.7205/MILMED-D-14-00404. PMID: 25747646.
Introduction: Lengthy storage times and associated storage lesion can result in reduced red blood cell (RBC) efficacy, particularly dangerous for massively transfused patients. Today's inventory management makes storage times the de-facto metric of blood quality. However, RBC units' quality may vary because of time-independent factors. Mechanical fragility (MF) of RBC, reflecting sub-lethal cell damage, can potentially provide a more physiologically relevant predictor of cell's performance "in vivo."
Methods: Mechanical stress was applied using a bead mill (50 Hz) over durations varying from 0.5 to 60 minutes, or using ultrasound (40 W) with durations from 0.1 to 120 seconds. MF profiles were described in terms of percentage hemolysis following stresses of specified durations.
Results: RBC MF declined significantly in the presence of albumin, with albumin protecting membrane against damage from elevated temperature or from methyl-β-cyclodextrin or diamide. MF profiles allowed detection of sub-lethal membrane damage caused by elevated temperature, to a greater extent than was reflected by autohemolysis. Different types of profiles for RBC damage were associated with MF changes at different stress intensities and potentially stress types.
Conclusions: These findings indicate that MF profiles can provide a powerful and versatile tool for investigation of RBC, as well as a potential metric of RBC quality.
Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.
Similar Donors – Similar Blood?
Tarasev M, Alfano K, Chakraborty S, Light L, Doeden K, Gorlin JB. Similar donors-similar blood? Transfusion. 2014 Mar;54(3 Pt 2):933-41. doi: 10.1111/trf.12457. Epub 2013 Oct 28. PMID: 24660765.
Background: Red blood cell (RBC) storage lesions have been suggested as contributing factors to suboptimal clinical outcomes. While undesirable effects of storage are well documented, their clinical relevance is still debated. Focus on storage time as the sole determinant of RBC quality ignores the variability in cell properties that may depend on factors other than age. Mechanical fragility (MF) aggregately reflects many storage-related functional and structural changes. This study evaluates interdonor versus intradonor variability, throughout storage, of both MF and autohemolysis (AH).
Study design and methods: Thirteen uniformly manufactured RBC units were collected initially as whole blood from nonsmoking, group A+, male Caucasian research donors. Mechanical stress was applied using a bead mill with oscillation at 50 Hz over durations varying from 0.5 to 60 minutes. MF profiles were described in terms of percent hemolysis after stresses of specified durations. Two months later, 11 of the 13 donors returned and assays were performed using the same protocol to allow comparison of intradonor versus interdonor variation.
Results: At 5 days postcollection, RBC MF profiles exhibited marked interdonor variability (up to twofold) overall. Both autolysis and MF across all units increased during storage-with rates of these increases varying by up to 10-fold for certain MF variables. Especially high AH and MF were observed for an outlier donor (with p < 0.05), for whom follow-up revealed previously undisclosed hereditary hypertriglyceridemia (levels exceeding approx. 1000 mg/dL).
Conclusions: RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.
© 2013 American Association of Blood Banks.
Mechanical Fragility as a Potential Time-Independent Measure of Membrane Integrity Among Stored RBC Units
Tarasev M, Alfano K, Chakraborty S, Bertholf M, Zubair A (2013)
Mechanical Fragility as a Potential Time-Independent Measure of Membrane
Integrity among Stored RBC Units. J Blood Disorders Transf 4:139.
Background and Objectives: Previous studies have shown that storage causes RBC membrane damage
and subsequent potassium leakage to extracellular environment, with the effects exacerbated by RBC irradiation.
While damage to RBC appears to worsen with storage time (ST), ST alone has not been shown to fully account
for this phenomenon. It is therefore important to study the extent to which other time-independent factors can
affect RBC membrane integrity. RBC mechanical fragility (MF) is evaluated as a surrogate measure of RBC
membrane integrity due to its potential to reflect aggregate biochemical and biomechanical changes associated
Materials and methods: Samples from 45 units non-irradiated and 58 units of irradiated leuko reduced RBC
units were subjected to shear stress using a bead mill at different durations at a fixed intensity (50 Hz); induced
hemolysis was ascertained via spectral analysis. Profile curves characterized the relationship between stress
duration and induced hemolysis, from which specific parameter values were interpolated.
Results: There was high variability among RBC MF parameters. MF profiles were significantly variable
among both irradiated and non-irradiated stored RBC units, and in some, within the same units which resulted in
distinguishable subpopulations. RBC base-line hemolysis (hemolysis before stress application) MF variation was
largely independent of ST. Donor blood type appeared to influence MF parameters and base-line levels.
Conclusion: RBC membrane properties, as defined by MF, vary markedly across RBC units. This variability
is largely independent of ST. MF could potentially be used clinically to assess RBC membrane
Investigating Direct Non-Age Metrics of Stored Blood Quality Loss
Alfano K, Tarasev M. Investigating Direct Non-Age Metrics of Stored Blood Quality Loss. The Internet Journal of Medical
Technology. 2011 Volume 5 Number 1.
Long storage times for blood products are often unavoidable. Product age is essentially the only indicator used today for Red
Blood Cell (RBC) quality loss during storage. Much controversy persists over the impact of RBC age on transfusion outcomes,
as studies on this remain inconclusive. Such inconsistency may arise from unit-to-unit variability, which likely introduces some
age-independence to RBC state. Thus, quality metrics other than storage time could aid with inventory management and/or
treatment decisions. RBC membrane mechanical fragility is proposed here as one such candidate in vitro metric: it aggregately
reflects a range of biochemical and biomechanical changes associated with storage lesion, and can provide a more
comprehensive characterization of particular units than other properties. Preliminary data suggest this property can vary
substantially among units of equal age, and further work now in progress is investigating its correlation to post-transfusion red
cell survival in vivo.
Central Nervous System Events in Children with Sickle Cell Disease Presenting Acutely with Headache.
Hines PC, McKnight TP, Seto W, Kwiatkowski JL. Central nervous system events in children with sickle cell disease presenting acutely with headache. J Pediatr. 2011 Sep;159(3):472-8. doi: 10.1016/j.jpeds.2011.02.009. Epub 2011 Mar 25. PMID: 21439575.
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.
Novel Epinephrine and Cyclic AMP-Mediated Activation of B-CAM/Lu-Dependent Sickle (SS) RBC Adhesion.
Hines PC, Zen Q, Burney SN, Shea DA, Ataga KI, Orringer EP, Telen MJ, Parise LV. Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion. Blood. 2003 Apr 15;101(8):3281-7. doi: 10.1182/blood-2001-12-0289. Epub 2002 Dec 27. PMID: 12506027.
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.
Sickle Cell Adhesion to Laminin: Potential Role for the α5 Chain
Lee SP, Cunningham ML, Hines PC, Joneckis CC, Orringer EP, Parise LV. Sickle cell adhesion to laminin: potential role for the alpha5 chain. Blood. 1998 Oct 15;92(8):2951-8. PMID: 9763582.
Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.