scholarly journals Ex Vivo Evaluation of Red Blood Cell Adhesion and Whole Blood Thrombosis in Pyruvate Kinase Deficiency

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 923-923
Author(s):  
Patrick C. Hines ◽  
Xiufeng Gao ◽  
Andrew Herppich ◽  
Wendy Hollon ◽  
Meera B. Chitlur ◽  
...  
Keyword(s):  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Research Funding ◽  
Thrombin Generation ◽  
Healthy Controls ◽  
Cell Disease ◽  
Adhesive Properties ◽  
Advisory Committees

Abstract Introduction Pyruvate Kinase Deficiency (PKD) is an inherited glycolytic enzymopathy that is characterized by a life-long chronic hemolytic anemia with severe comorbidities. Hypercoagulability due to increased platelet activity caused by nitric oxide sequestration by cell free hemoglobin has been well-described not just in PKD, but in other hemolytic anemias as well, such as e.g., sickle cell disease (SCD). Hypercoagulability is often accompanied by a cascade of pathophysiological events leading to cell oxidative damage, endothelial activation, and changes in both cell stability and adhesive properties. Increased red blood cell (RBC) adhesion and hypercoagulability may impair microvascular blood flow. Despite these well-recognized rheological changes that are similar to those that occur in other hemolytic anemias, the relationship between baseline erythrocyte adhesion and thrombosis potential have not been well-studied in PKD. Methods 10 PKD subjects and 5 healthy controls were recruited under the IRB-approved protocol from Wayne State University. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) was performed by flowing whole blood (1:1 dilution) through a microfluidic channel for 3 minutes (1 dyne/cm 2 shear stress, 1.67Hz pulse frequency). Flow adhesion avidity of the whole blood sample to VCAM-1 (FAAv-WB-VCAM), representing the strength of the RBC-VCAM-1 adhesive interactions, was assessed by quantifying adhesion following sequential increase in shear (5, 10, 20 dyne/cm 2). Thrombin generation assay was conducted using platelet poor plasma with and without thrombomodulin and microparticles (MP) as previously published [1]. Clotting time - reported as lag time (LT), time to peak (ttPeak) and peak height (velocity and amount of net thrombin production), and endogenous thrombin potential (ETP), representing number of substrates potentially convertible by thrombin, were measured. Significance was at p < 0.05. Results FA-WB-VCAM at baseline sample hematocrit was significantly elevated (Figure 1) in PKD subjects (808±377 cells/mm², n=10) compared to healthy controls (6±4 cells/mm², n=4) and even to our previously reported steady state levels in sickle cell samples (290±50 cells/mm² [2]. Thrombin generation profiles were similar between PKD subjects and healthy controls with the exception of the thrombin generation index (PPP+TP/PPP)*100ETP that was significantly (p<<0.01) elevated in citrated plasma of PKD subjects (92.9±6.8) as compared to healthy controls (68.6±11.9). For PKD subjects, FA-WB-VCAM correlated significantly with platelet counts (R²=0.81, p<0.05), and FAAv-WB-VCAM was negatively correlated with platelet (P=0.03, R 2=0.5), but not with erythrocyte-derived microparticles (MP). Platelet-derived MP strongly correlated with thrombin generation (ETP, p<0.01, R 2=0.76) but not with LT or ttPeak of thrombin generation. Red blood cell MP were significantly (p=0.02) decreased in splenectomized patients (200±170, n=7) vs. non-splenectomized subjects (2090±1860, n=3). LT and ttPeak were significantly longer in PKD subjects with thrombosis history than without. Conclusions PKD subjects in this study had elevated RBC adhesive properties similar to that observed in SCD, confirming that pathologic RBC membrane damage resulting in increased adhesion is a common feature of hemolytic anemias. The hemoglobin level of 7.8±1.1 g/dL (mean±SD) for PKD patients was within 6 to 11 g/dl range of hemoglobin levels typical for SCD. There was no significant difference in any other measured parameters (thrombin generation, adhesion avidity, microparticles data). Thrombin generation in PKD subjects was not consistent with hypercoagulability. Based on these observations, pathologic RBC adhesion may be both a novel a mechanism driving hypercoagulability in individuals with PKD. Further studies to determine whether RBC-modifying therapies may decrease thrombosis risk in PKD are warranted. 1. Zia A, Callaghan MU, Callaghan JH, et al. Hypercoagulability in adolescent girls on oral contraceptives - global coagulation profile and estrogen receptor polymorphisms. Am J Hematol, 2015;90:725-31 2. Pittman DD, Hines PC, Beidler D, et al. Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers. Blood. 2021;137(15):2010-20 Figure 1 Figure 1. Disclosures Hines: Functional Fluidics: Current holder of stock options in a privately-held company. Gao: Functional Fluidics: Current Employment. Herppich: Functional Fluidics: Ended employment in the past 24 months. Kwiatkowski: Imara: Consultancy, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Sangamo: Research Funding; Bioverativ: Research Funding; Vertex: Research Funding; Silence Therapeutics: Consultancy; bluebird bio: Consultancy, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Chiesi: Research Funding; CRISPR: Research Funding. Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company.

scholarly journals Whole Blood Adhesion to VCAM-1 and P-Selectin and RBC Mechanical Fragility Can be Compromised in Long COVID-19 Patients with Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 959-959
Author(s):  
Michael Tarasev ◽  
Marta Ferranti ◽  
Cidney Allen ◽  
Xiufeng Gao ◽  
Kayla Topping ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Membrane Stability ◽  
Cell Disease ◽  
Adhesive Properties ◽  
Rbc Membrane ◽  
Privately Held

Abstract Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe vascular complications associated with endothelial dysfunction and systemic inflammation. COVID19-specific IgG are detectable within a week of infection. Long COVID-19 has been described in patients continuing to exhibit symptoms after the virus is no longer detectable in the respiratory secretions, including fatigue, dyspnea, headache, and brain fog. The recent FAIR Health study reviewed a total of 1,959,982 COVID-19 patients for the prevalence of long COVID symptoms and reported that 23.2% had at least one post-COVID symptom [1]. The underlying biologic mechanisms of long COVID remain unclear, thus treatments are limited to symptomatic relief and supportive care. Many long COVID symptoms are consistent with systemic inflammation and impaired oxygen delivery observed in individuals with sickle cell disease (SCD), in turn associated with elevated blood cell adhesion and decreased red blood cell (RBC) stability. The aim of this study was to determine if deleterious changes in in blood cell properties related to adhesion and membrane stability under stress can be associated with the symptoms of long COVID-19. In this work we evaluated 7 SCD patients that were diagnosed with SARS-Cov-2 and tracked their recovery using semiquantitative IgG and blood cell function assays. Methods: Blood samples were collected by the Foundation for Sickle Cell Disease (SCD) Research from SCD (homozygous SS, n=6) patients coming for regular or urgent clinic visit with SARS-CoV-2 serological and blood cell functions tests performed per the standard of care. Semiquantitative IgG assay was performed using DXi-80 (Beckman Coulter). Flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM)and P-Selectin (FA-WB-Psel) substrates were determined by counting the cells that remain adherent in a microfluidics channel after perfusion with whole blood 1:1 diluted with HBSS buffer and washed by reversed flow at 1 dyne/cm 2. Red blood cell mechanical fragility (RBC MF) was measured as hemolysis induced by an oscillating cylindrical magnet with periodic non-invasive probing of cell-free hemoglobin fraction. Six individuals with SCD recovering from SARS-Cov-2 with biomarker data available both before and for more than 3 months after the infection (179±62 days) were included in the study. Results: IgG levels varied from less than 0.1 to 37, with positive values being defined as IgG > 1. The median estimated half-life of IgG decline was 53 days ranging from 25 to 90 days (the last, for the hospitalized patient). Averaged for IgG positive (IgG+) and IgG negative (IgG-) conditions, combining pre- and post-infection IgG- conditions, values of patient hemoglobin (Hb), FA-WB-VCAM, FA-WB-Psel, and RBC MF cell properties lacked statistical significance (under both a paired t-test and population statistics). Hb levels remained essentially unchanged regardless of the time from infection or IgG status. However, FA-WB-VCAM, FA-WB-Psel, and RBC MF were all significantly elevated after SARS-Cov-2 seroconversion and remained elevated despite declining IgG levels (e.g., Fig. 1). These increases in biomarker values were statistically significant for both FA-WB-VCAM and RBC MF, and were approaching significance for FA-WB-Psel (p<0065). These increases were highly patient-specific with potential return to pe-infection values observed in some cases at about 5-6 months after the infection. A qualitative review of the medical records indicated a new subjective report of fatigue in 5 of 6 patients. Longer observations are required to determine if abnormal blood cell adhesive properties and RBC membrane instability are mechanisms of long-COVID-19 pathophysiology. Conclusions: Whole blood adhesion to both p-selectin and VCAM-1 as well as RBC membrane stability can be significantly impaired in convalescent SARS-Cov-2 patients suggesting an association with long COVID-19. New and emerging treatments that modify whole blood adhesive properties and RBC membrane stability should be investigated for their potential to accelerated recovery from long COVID-19. Health F. A Detailed Study of Patients with Long-Haul COVID: An Analysis of Private Healthcare Claims; White Paper. June 15, 2021 Disclosures Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company. Ferranti: Functional Fluidics: Current holder of stock options in a privately-held company. Allen: Functional Fluidics: Current Employment. Gao: Functional Fluidics: Current Employment. Topping: Functional Fluidics: Current Employment. Ferranti: Functional Fluidics: Current Employment. Makinde-Odesola: Functional Fluidics: Other: conduct research for academic program. Hines: Functional Fluidics: Current holder of stock options in a privately-held company.

scholarly journals Whole blood viscosity and red blood cell adhesion: Potential biomarkers for targeted and curative therapies in sickle cell disease

2020 ◽  
Vol 95 (11) ◽  
pp. 1246-1256 ◽  
Author(s):  
Erdem Kucukal ◽  
Yuncheng Man ◽  
Ailis Hill ◽  
Shichen Liu ◽  
Allison Bode ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Blood Viscosity ◽  
Whole Blood ◽  
Cell Disease ◽  
Whole Blood Viscosity ◽  
Potential Biomarkers

Clinical Effects of Chronic Red Blood Cell Exchange and Different Types of Red Cell Concentrates in Patients with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4823-4823
Author(s):  
Sergio Cabibbo ◽  
Agostino Antolino ◽  
Giovanni Garozzo ◽  
Carmelo Fidone ◽  
Pietro Bonomo
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Iron Overload ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Clinical Effects ◽  
Red Cell ◽  
Blood Components ◽  
Cell Disease

Abstract For patients with severe SCD not eligible for hydroxyurea, two major therapeutic options are currently available: blood transfusion, and bone marrow transplantation. Either urgent or chronic red blood cell transfusion therapy, is widely used in the management of SCD but determines a progressive increase of ferritin level and is also limited by the development of antibodies to red cell antigens. The introduction of chronic red blood cell exchange and prestorage filtration to remove leucocytes and the use of techniques for multicomponent donation could be a good solutions. Thus, the aims of our studies were to evaluate the clinical effects of the different blood components in terms of annual transfusion needs and the intervals between transfusion, moreover we evaluated the efficacy of chronic red blood cell exchange (manual or automatic with cell separator) in preventing SCD complications and limiting iron overload. In our center we follow 78 patients affected by Sickle Cell Disease. We selected 36 patients occasionally treated with urgent red blood cell exchange because they had less than 2 complications/Year, and 42 patients regularly treated with chronic red blood cell exchange because they had more than 2 complications/Year with Hospital Admission. Moreover among these we selected 10 patients for fulfilling the criteria of continuous treatment at the Centre for at least 48 months with no interruptions, even sporadic and absolute transfusion dependency. All 10 patients were evaluated for a period of 4 years, during which two different systems of producing RCC were used. In the second two the patients were transfused with RCC obtained from filtering whole blood prestorage or with RCC from apheresis filtered prestorage. These products differed from those used in the preceding two years, during which the leucodepletion was obtained by bed-side filtration For all the patients we performed 782 automatic red blood cell exchanges and 4421 units of RCC were transfused. The exchange procedures were extremely well-tolerated by the patients and adverse effects were limited to symptoms of hypocalcaemia during automatic red blood cell exchange with cell separator. After every red blood cell exchange we obtained HbS level < 30%. The10 patients selected received respectively a mean of 6.9 and 6.1 units of RBCs exchanged per automatic procedure, in the first two years and in the second two years. Alloantibody developed in 14 patients but only 2 clinically significant and about the observed frequency of transfusion reactions it was very low. All patients treated with chronic red blood cell exchange had an improvement of the quality of life with a reduced number of complications/year (<2/year) and good compliance and moreover patients had limited iron overload making chelating therapy easier. In conclusion this study was focused on the most suitable characteristics of blood components for use in sickle cell disease patients and the choice of systematically adopting prestorage filtration of whole blood, enabled us to have RCC with a higher Hb concentration than standard. Moreover chronic manual or automatic red blood cell exchange as an alternative approach to simple long-term RBC transfusions give many advantages by being more rapid and tolerable as well as clinically safe and effective and minimize the development of iron overload especially when procedure was carried out with an automatic apparatus. To note that the clinical advantages for patients derived from good selection of the donor and good practices in the production of the blood components

scholarly journals Red Blood Cell Adhesion in Adult Patients with Sickle Cell Disease, at Baseline and with Pain, Measured on SCD Biochip Microfluidic Assay

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2386-2386
Author(s):  
Alexandra Boye-Doe ◽  
Jane Little
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Research Funding ◽  
Adult Patients ◽  
Cell Disease ◽  
University Hospitals ◽  
Western Reserve

Abstract Red Blood Cell Adhesion in Adult Patients with Sickle Cell Disease, at Baseline and with Pain, Measured on SCD Biochip Microfluidic Assay Alexandra Boye-Doe1, Erina Quinn1, Charlotte Yuan1, Umut A. Gurkan, PhD1, and Jane A. Little, MD2 1Case Western Reserve University, Cleveland, OH; 2Division of Hematology/Oncology, Case Western Reserve University/ University Hospitals Seidman Cancer Center, Cleveland, OH Background: Despite being monogenic, sickle cell disease (SCD) has a variable phenotype, in which clinical complications and manifestations evolve as patients age. In children, pain generally resolves between crises, whereas adults may experience acute, chronic, or acute-on-chronic pain. We have taken a multifaceted approach to characterize adhesion and inflammation during self-identified pain episodes in adults with SCD in order to better understand pain syndromes in adults and the potential for more specifically targeted therapies. In this pilot study, we assessed changes in red blood cell (RBC) adhesion to the subendothelial protein laminin (LN) during crisis in adults with SCD self-reporting for pain crisis on whom we had baseline adhesion data from a routine clinic visit. Methods: Surplus blood from patients' routine bloodwork was used. Crisis samples were collected from patients at the Acute Care Clinic or Emergency Department at University Hospitals Cleveland Medical Center (UHCMC) when patients presented for management of pain. Baseline samples were collected during routine visits to the Sickle Cell Clinic. This study was approved by the IRB at UHCMC. Within a 24-hour period, RBC adhesion to LN was quantitated by microscopy after passage of unprocessed whole blood through a LN-coated microfluidic adhesion assay, the SCD biochip [1]. Samples were analyzed for hemoglobin (Hb) phenotype by high-performance liquid chromatography (HPLC) in the clinical lab. Correlative clinical data, including, baseline lab values, and medical history, were obtained from the patients' medical records and used to characterize our results. Data from people with multiple samples were used as median values. Results: Blood samples from 19 unselected patients with sickle cell hemoglobin SS (HbSS) were obtained at crisis, and compared with baseline samples obtained from 2014 to 2018 (n = 67 samples). 2 groups were identified: Group 1 with increased adhesion (>25% rise from baseline, n= 10) during crisis, and Group 2 with decreased adhesion (>25% fall from baseline, n=8) or no change (<25% change) (n=1) during crisis (Fig. 1). Time between a patient's initial crisis event and when they presented for pain management varied, possibly affecting observed adhesion. Nonetheless, patients showing an increased adhesion in crisis also showed a decrease in Hb (p = 0.039) between their baseline analyses and the crisis visit. A decrease in Hb may associate with increased adhesion, if the latter contributes to crisis-related hemolysis. However, other markers of hemolysis did not change. By contrast, patients with decreasing RBC adhesion showed a decrease in absolute reticulocyte count (ARC, p = 0.019) at their crisis visit. Statistical analysis of HbS, HbA, and HbF levels n = 64 showed no significant change between baseline or crises. Discussion: A decrease in adhesion during crisis may reflect the presence of sickled RBCs that adhere preferentially to other basement membrane proteins such as fibronectin or thrombospondin. In addition, inflammatory white blood cell adhesion, possibly central to vaso-occlusion, is not evaluated in this study. We are currently examining cytokine and monocyte profiles from people with SCD presenting for management of pain, so that we may better understand inflammatory mediators of pain. Our data are the first to try to understand RBC adhesion in people with SCD who present for management of pain, whether this as an isolated or common event for that person. We found that unselected adults with SCD who presented for management of pain had heterogeneous changes in RBC adhesion, which may reflect differences in underlying mechanism. The pathophysiologic heterogeneity of pain in adults with SCD will be important to understand as anti-adhesion therapies are being developed and adopted clinically. Disclosures Little: Doris Duke Charitable Foundations: Research Funding; NHLBI: Research Funding; Hemex: Patents & Royalties: Patent, no honoraria; PCORI: Research Funding.

scholarly journals Rate of Sickle Hemoglobin Recovery in Sickle Cell Disease Patients Undergoing Red Blood Cell (RBC) Exchange Transfusion Is Associated with Age of Patients and Number of RBC Units Transfused

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2170-2170
Author(s):  
John Chinawaeze Aneke ◽  
Aliraza Rajabali ◽  
Nafanta Fadiga ◽  
Stéphanie Forté ◽  
George A. Tomlinson ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Research Funding ◽  
Exchange Transfusion ◽  
Rate Of Change ◽  
Cell Disease ◽  
Sickle Hemoglobin ◽  
Indirect Bilirubin

Rate of Sickle Hemoglobin Recovery in Sickle Cell Disease Patients Undergoing Red Blood Cell (RBC) Exchange Transfusion is Associated with Age of Patients and Number of RBC Units Transfused Introduction: Automated and manual red blood cell exchange (RBCX) transfusions are useful in the primary and secondary prevention of sickle cell disease (SCD) complications (Ware et al., 2012). The ability to consistently maintain sickle hemoglobin (HbS) below target (30% or 50% depending on indication) is quite variable (Kuo et al., 2012). With emerging indications such as silent cerebral infarction, it is imperative that effective means of chronic transfusion to maintain appropriate hematological and clinical targets be identified. We hypothesize the rate of HbS recovery is dependent on the individual's hemolytic and erythropoietic rate. The purpose of this study is to evaluate the effect of the rate of erythropoiesis and hemolysis on HbS recovery in SCD patients undergoing RBCX. Methods: Fifteen (15) patients were prospectively recruited from the adult SCD transfusion program (9 automated, 6 partial manual), from December 2018 to July 2019, and followed through one exchange cycle (4 weeks). Automated and partial manual exchange transfusion protocols have been previously described elsewhere (Canadian Haemoglobinopathy Association Consensus Statement on the Care of Patients with Sickle Cell Disease in Canada, Version 2.0, Ottawa; 2015). Exclusion criteria included active hydroxyurea or erythropoietic stimulating agents use, reported ill health in the preceding 4 weeks, co-morbid hemolytic condition or non-HbSS genotype. Hemoglobin, hematocrit, HbS, lactate dehydrogenase (LDH), reticulocyte count, indirect bilirubin, and serum erythropoietin level were determined for each patient: pre- and post- first exchange, weekly for 3 weeks and pre- second exchange (the 4th week). Descriptive variables were either expressed as means ± SD or median (IQR), based on normality, while linear regression was performed for continuous variables. Co-variates were included in multivariable analysis if P < 0.10. Multivariable linear regression was conducted to examine the potential association between the change in HbS over one RBCX cycle and age of patients, pre-RBCX hematocrit, LDH, and number of RBC units transfused. Results: We identified 36 eligible patients from the Program database, after which 15 consented to participate in the study. Mean age was 32.9 ± 12.3 years, consisting of 7 males and 8 females. There was an association between the rate of change in HbS and age of patients (p=0.035), pre-RBCX hematocrit (p=0.030) and number of transfused RBC units (p=0.030). LDH showed a trend towards reduced rate of change in HbS (p=0.069). Rate of change in HbS was not associated with automated vs. partial RBCX (Figure), female vs. male patients, pre-RBCX HbS, erythropoietin, indirect bilirubin, reticulocyte and age of transfused RBCs. Age of patients (p<0.001) and number of units transfused (p=0.010) were independently associated with the rate of change of HbS, after adjusting for hematocrit and LDH. For every decade increase in age, the rate of HbS recovery was 3% lower in one RBCX cycle. For each additional unit of RBC exchanged, the rate of HbS recovery was 0.78% higher in one RBCX cycle. Conclusion: This is the first study to evaluate the determinants of variability in the rate of HbS recovery in SCD patients on RBCX. Age of patients and number of RBC units transfused may underlie the significant variability in achieving HbS targets in SCD patients on RBCX. We failed to show a significant difference in the rate of change in HbS between automated and partial manual exchange transfusion. Reduction in HbS recovery with advancing age may be related to erythropoietic reserve in the patients' marrow. However, the influence of units of RBC recovery appeared paradoxical, and the finding may be spurious due to small sample size. It may therefore be possible to appropriately titrate the number of RBC units with advancing age of SCD patients with a view to achieving desired HbS targets. Disclosures Patriquin: Ra Pharma: Consultancy, Research Funding; Apellis: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding. Kuo:Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy.

scholarly journals Hypoxia Impact on Red Blood Cell-Mediated Microvascular Occlusion and Adhesion in Sickle Cell Disease and Sickle Cell Trait

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 966-966
Author(s):  
Yuncheng Man ◽  
Zoe Sekyonda ◽  
Karamoja Monchamp ◽  
Ran An ◽  
Erdem Kucukal ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Sickle Cell Trait ◽  
Single Gene ◽  
Cell Disease ◽  
The Impact ◽  
Rbc Deformability

Abstract Introduction: Sickle cell disease (SCD) is a recessively inherited anemia caused by a single gene mutation leading to sickle hemoglobin production. Sickle cell trait (SCT) is the carrier state. Abnormal hemoglobin polymerization and resultant red blood cell (RBC) sickling, decreased deformability and increased adhesion, are well-known features of homozygous SCD. However, the overall pathophysiological impact of SCT on the RBC remains incompletely characterized. Here we use microfluidic techniques designed by us, the OcclusionChip and SCD Biochip (previously published), and commercially available ektacytometry to investigate hypoxia impact on RBC biophysical properties in SCT. Methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS, SCT (HbAS), and non-anemic controls (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using standard soft lithography protocols [1]. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the OcclusionChip device with a constant inlet pressure. Following a wash step, the OcclusionChip microchannel was imaged, and Occlusion Index (OI), a standardized generalizable parameter we developed, representing the overall microcapillary network occlusion, was quantified. SCD Biochip microchannels were fabricated by lamination and were functionalized with human laminin (LN-511) [2]. Undiluted whole blood was injected into the microchannel at 1 dyne/cm 2, a shear stress value typically observed in the post-capillary venules. Following a wash step, the SCD Biochip microchannel was imaged, and the number of adherent RBCs in a 32-mm 2 window was quantified. For hypoxia experiments, a hypoxic setup was fabricated for blood deoxygenation (pO 2 ~45 mmHg) [3, 4]. Ektacytometry measurements were performed according to the manufacturers' specifications (Lorrca Maxsis). Data are reported as mean ± standard deviation (SD). Results: We initially analyzed RBC-mediated microvascular occlusion under normoxia or hypoxia using the OcclusionChip (Figure 1A). Under normoxia, HbSS-containing RBCs had relatively greater OI values compared to HbAA- and HbAS-containing RBCs (Figure 1B, P = 0.057 for HbSS vs HbAA and P = 0.060 for HbSS vs HbAS). However, exposure to hypoxia led to significantly elevated OI values in the HbAS- and HbSS-containing RBCs (Figure 1B, 0.05 ± 0.02% vs 33.62 ± 18.31%, P = 0.015 for HbAS, and 0.27 ± 0.24% vs 49.37 ± 24.47%, P = 0.001 for HbSS, normoxia vs hypoxia). Negligible occlusion was observed in HbAA-containing RBCs (Figure 1B). We then analyzed RBC adhesion to LN under normoxia or hypoxia using the SCD Biochip (Figure 1C). Hypoxia led to greater number of adherent RBCs on LN in the HbSS-containing RBCs (Figure 1D, 141 ± 91 vs 497 ± 392, P = 0.089, normoxia vs hypoxia), but this effect was not present in HbAA- or HbAS-containing RBCs (Figure 1B, 2 ± 1 vs 3 ± 1, P &gt; 0.05 for HbAA, and 10 ± 7 vs 12 ± 3, P &gt; 0.05 for HbAS, normoxia vs hypoxia). Further, under normoxia, we found that the HbAS-containing RBCs had slightly greater number of adherent RBCs on LN compared to the HbAA-containing RBCs (Figure 1D, P = 0.057 for HbAA vs HbAS). As previously reported, HbSS-containing RBCs showed greatest adhesion to LN under normoxia compared to the HbAA- and HbAS-containing RBCs (Figure 1D, P = 0.027 for HbSS vs HbAA and P = 0.033 for HbSS vs HbAS)., Finally, we preformed Lorrca oxyscan and found that ektacytometry is less sensitive to RBC deformability change under hypoxia in SCT (Figure 1E). Conclusions: Findings in this study suggest that although RBCs from subjects with SCT are deformable under normoxia and are able to clear narrow capillaries similar to normal RBCs, hypoxia changes deformability, presumably due to hypoxic polymer formation, and could contribute to microvascular occlusion in SCT. The OcclusionChip is a single cell-based technology, and may be more sensitive to single RBC deformability. Future studies will prospectively focus on analyzing RBC adhesion on activated microvascular endothelial cells in physiologic flow to further interrogate the impact of hypoxia on pathophysiology in SCT. References: [1] Man et al., LabChip, 2020, 20, 2086-2099. [2] Kim et al., Microcirculation, 2017, 24, e12374. Figure 1 Figure 1. Disclosures An: Hemex Health, Inc.: Patents & Royalties. Kucukal: BioChip Labs: Current Employment, Patents & Royalties. Nayak: BioChip Labs: Patents & Royalties. Little: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Patents & Royalties. Gurkan: Dx Now Inc.: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Biochip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties.

scholarly journals Five-Year Outcomes of Chronic Red Blood Cell Exchange Transfusion Program for Patients with Sickle Cell Disease, the Experience of University of Nebraska Medical Center

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4988-4988
Author(s):  
Omar Abughanimeh ◽  
Steven Ebers ◽  
Mahammed Khan Suheb ◽  
Julie Eclov ◽  
Robin High ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Emergency Room ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Research Funding ◽  
Medical Center ◽  
Emergency Room Visits ◽  
Cell Disease ◽  
The Mean

Abstract Background: Red blood cell exchange (RBCX) is an effective therapy in treatment of acute and chronic complications of sickle cell disease (SCD). It involves exchanging patient's red blood cells (RBCs) with donor RBCs to significantly lower hemoglobin S concentration without subjecting the patient to the risk of iron overload. The University of Nebraska Medical Center (UNMC) established a chronic RBCX program in November 2015, which cared for patients with multiple hemoglobinopathies. In this study, we aim to evaluate some of the outcomes of patients with SCD who joined the program. Methods: This is a retrospective study based on review of medical records of patients with sickle cell disease. We reviewed the health records of patients with SCD who were enrolled in the chronic RBCX program between 11/2015-8/2020 at UNMC. We included patients with SCD, regardless of age, who underwent RBCX in the outpatient setting during the study period. Data were collected to assess if RBCX influenced the frequency of SCD crisis, emergency room visits, hospitalizations, and other sickle cell-related complications. Results: A total of 404 sessions of exchange transfusions were performed between November 2015 and August 2020 for 21 patients with SCD. The study included 9 adults (age ≥ 18 years) and 12 children with a median age of 12 years (2-31 years). During the study period, 3 adults left the program due to relocation out of state, patient's preference, or physician's decision. Table 1 summarizes the population demographic. The most common indication for enrollment in the RBCX program was recurrent sickle cell crisis (Figure 1). The mean number of emergency room visits before enrollment in the RBCX program was 22.5 visits (2-62 visits), which reduced after enrollment to 10.4 visits (0-65 visits), with a difference in mean of 12.1 visits (P=0.0021). The mean number of hospital admissions before enrollment in the RBCX program was 13.2 admissions(0-54 admissions), which also reduced to 6.7 admissions (0-50 admissions), with a significant difference in the means equal to 6. 6 admissions (P=0.0013) (Figure 2). Thirteen patients had a baseline ferritin &gt; 500 ng/ml at enrollment; all of them had a decrease in their baseline ferritin during the study, with 4 of them achieving a new baseline &lt; 500 ng/ml. Six patients had pre-existing antibodies at enrollment due to prior alloimmunization; however, no new alloantibodies were noticed after enrollment. The patients without preexisting antibodies were transfused with Rh and Kell matched blood. The patients with pre-existing antibodies were transfused with phenotypically matched blood. Three patients became pregnant during the study period, and their pregnancies were uncomplicated except for one patient with preeclampsia resulting in early delivery. There was no reportable death, acute chest syndrome, or stroke among the patients during the study period. Conclusion Outpatient chronic RBCX demonstrated safety and feasibility in both adults and children. It also showed promising outcomes in terms of reduction of sickle cell complications, number of emergency room visits and hospitalizations. These results can provide the basis for evaluating RBCX in a prospective study to better understand changes in quality of life and clinical outcomes of patients with SCD and limited therapeutic options. Figure 1 Figure 1. Disclosures Gundabolu: Pfizer: Research Funding; Samus Therapeutics: Research Funding; BioMarin Pharmaceuticals: Consultancy; Bristol-Myers Squibb Company: Consultancy; Blueprint Medicines: Consultancy.

scholarly journals Effects of Genotypes and Treatment on Oxygenscan Parameters in Sickle Cell Disease

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 811
Author(s):  
Camille Boisson ◽  
Minke A. E. Rab ◽  
Elie Nader ◽  
Céline Renoux ◽  
Celeste Kanne ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Cell Disease ◽  
Oxygen Gradient ◽  
Acute Complication ◽  
Adults And Children ◽  
The Difference

(1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/β+) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.

Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Yuncheng Man ◽  
Debnath Maji ◽  
Ran An ◽  
Sanjay Ahuja ◽  
Jane A Little ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Electrical Impedance ◽  
Cell Disease ◽  
Blood Disorders

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contributes to vaso-occlusion and disease pathophysiology. However, there are few...

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