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.
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.
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.
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
Background: 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.
Objective: This work studies effects of different beads in creating qualitatively as well as quantitatively different shear stresses when oscillated in a sample containing RBC.
Methods: Identical, diluted RBC samples were stressed via bead milling using different beads, with hemolysis profiles developed in each case.
Results: 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.
Conclusion: 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.
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.
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.
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.
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.
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.
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.
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 with storage.
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
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.
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.