scholarly journals A Method to Measure Voxelotor Exposure in People with Sickle Cell Disease Using Capillary Zone Electrophoresis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2025-2025
Author(s):  
Susanna A Curtis ◽  
Elana M Friedman ◽  
Caterina Minniti ◽  
Annie Ngyuen Dang ◽  
Mira Pochron ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Capillary Zone Electrophoresis ◽  
Sickle Cell ◽  
Whole Blood ◽  
Blood Concentration ◽  
Research Funding ◽  
Cell Disease ◽  
Calculated Concentration ◽  
Zone Electrophoresis ◽  
Capillary Zone

Abstract Background: Voxelotor (Oxbryta®) is a small molecule that binds to the alpha chain of hemoglobin (Hb) and increases the affinity of Hb for oxygen which reduces sickle Hb polymerization. It was approved by the FDA in 2019 for the treatment of sickle cell disease (SCD). Currently, the only method available to estimate the concentration of voxelotor in the blood is to obtain exposure measurements which are only available in select research laboratories. A method to measure voxelotor at a standard laboratory would allow clinicians to assess compliance and may be useful in determining optimal dosing. Case studies have reported that voxelotor binding to Hb interferes with capillary zone electrophoresis (CZE). As previously reported, in CZE the characteristic peaks of hemoglobin A2 (HbA2) and hemoglobin F (HbF) split in the presence of voxelotor. Interestingly, it does not cause the hemoglobin S peak to split. We posited that we could use this split to estimate the presence of voxelotor and whole blood concentration. Biophysical measurements of voxelotor binding to Hb were also measured in these samples. Methods: Patients were enrolled prospectively in an IRB approved protocol. Voxelotor was initiated at 1500 mg daily on day 0 and samples were taken at day 0 (pre-dose), 14, 30, and 60. Samples had Hb variants quantified by CZE using the Capillarys 2 FlexPiercing Instrument (Sebia, Georgia). Hematology parameters were measured with the Sysmex XN-1000 automated analyzer (Sysmex, Illinois). To determine whole blood concentration of voxelotor, samples were sent to Worldwide Clinical Trials where a validated liquid chromatography-tandem mass spectrometry method was used. Voxelotor's interference with HbA2 on CZE is dependent on the HbF percentage, therefore samples from patients with SCD were combined into three pooled samples (5-10 samples per pool) of HbF percentages spanning 5-30%. Three hundred µL aliquots of each pool were spiked with voxelotor in DMSO in triplicate to different concentrations between 0 and 600 µMol/L and were incubated at room temperature for 1 hour before being tested with CZE. Samples were then analyzed for voxelotor interference resulting in split peaks of HbF and HbA2. HbA2 interference percent (%VarA2) was calculated as the reported value of the HbA2 split peak over the total HbA2 value (both split and parent peak) times 100. F% was used as directly reported by the instrument without consideration of voxelotor interference. Results were then analyzed in Excel (Data Analytics package) using a multiparameter regression to generate a line of best fit. To allow for logarithmic fit when examining the correlation of calculated concentration with increase in Hb due to voxelotor, samples with negative Hb rises were excluded and concentrations which resulted as negative values were changed to 0.01 µM. Results: Of 20 patients which have been enrolled to date, 9 patients have completed the study and their data was used for these analyses. Using the CZE method described above the concentration of voxelotor was quantifiable using the following equation. Equation 1: uM voxelotor = -99.13 + 7.10*%HbF +12.52*%VarA2 The calculated concentrations of voxelotor based on CZE results had a strong correlation with whole blood concentration (R 2 = 0.85, p <0.001). (Figure 1) When calculated concentration was compared to change in Hb at days 14, 30, and 60 there was a significant positive logarithmic correlation between concentration and change in Hb (R 2=.56, p<0.01). (Figure 2) Conclusions: Using equation 1, CZE can be used to detect the presence of voxelotor and estimate its whole blood concentration. This will allow clinicians to have a better understanding of how their patients are using voxelotor. Additionally, higher calculated whole blood concentrations correlated with higher increases in Hb. It was previously shown that patients who receive higher doses of voxelotor have on average larger increases in Hb. If it could be shown that increasing concentration in an individual on voxelotor is associated with an increased Hb for that individual, then our method could also be used to help clinicians select and adjust doses of voxelotor in a similar manner to how HbF is used in hydroxyurea dosing. Figure 1 Figure 1. Disclosures Curtis: GBT: Consultancy. Minniti: CSL Behring: Other: Endpoint adjudicator; Bluebird Bio: Other: Endpoint adjudicator; F. Hoffmann-La Roche: Consultancy; Chiesi: Consultancy; Novo Nordisk: Consultancy; Forma: Consultancy; Novartis: Consultancy; GBT: Consultancy. Ngyuen Dang: GBT: Current Employment. Pochron: GBT: Current Employment. Campbell: GBT: Research Funding; Sebia: Research Funding.

Variable Expression of Fetal Hemoglobin In Sickle Cell Disease Patients From Oman

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4808-4808
Author(s):  
Salam Alkindi ◽  
Muna Almaskari ◽  
Shoaib Alzadjali ◽  
Rhea Misquith ◽  
Rajagopal Krishnamoorthy ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Research Funding ◽  
Fetal Hemoglobin ◽  
Case Control ◽  
Chromosome 2 ◽  
Cell Disease ◽  
Sultan Qaboos University ◽  
Variable Expression

Abstract Abstract 4808 Although sickle cell disease (SCD) is a recessively inherited monogenic disorder, its clinical expression is variable from patient to patient, even within the same family; and, both heritable and environmental factors significantly contribute to this inter-patient difference in disease severity. The most prominent genetic modifiers include co-inherited alpha thalassaemia, over expression of fetal hemoglobin (HbF) in adulthood, and the excessive production of insoluble bilirubin. The purpose of this study was to ascertain the emerging role of HbF by studying several known SNP's in Chromosome 2, 6 and 11 that are involved in modulating the fetal hemoglobin levels in patients from Oman. This case-control study was conducted at Sultan Qaboos University Hospital from February to May 2010. Eighty eight SCD patients were consecutively studied along with forty four Omani blood donors who were selected randomly to complete a 2:1 case-control enrollment after an informed consent. Whole blood samples were collected in EDTA tubes from SCD patients and donors. DNA was extracted from the whole blood after Complete Blood Count and High Performance Liquid Chromatography were performed on the patient and donor samples. The molecular analysis included genotyping of BCL11A, HBSIL-MYB, HBG2 SNP's by direct DNA sequencing with 3100 genetic analyzer (ABI systems, Illinois, USA) using Tag SNP's in each category namely rs11886868, rs4671393 (BCL11A-Ch.2), rs7776054, rs9399137, rs4895441 (HBSIL-MYB-Ch.6), & rs7482144 (HBG2-Ch.11) to correlate these SNP's with the HbF expression in the SCD patients. A stepwise regression analysis result of the 6 SNP's genotyped at the BCL11A, HBSIL-MYB, and HBG2 gene locus were correlated to the HbF levels in our SCD study cohort and is shown below. At each step, the least significant SNP was removed, until all SNP's left in the multivariate model were significant. The model at step 4 has the best goodness of fit value, as assessed by the likelihood ratio test, than models 1, 2 & 3. [Table] Step 1: HbF levels~rs7482144 + rs7776054 + rs9399137 + rs4895441 + rs11886868 + rs4671393 Step 2: HbF levels~rs7482144 + rs7776054 + rs9399137 + rs11886868 + rs4671393 Step 3: HbF levels~rs7482144 + rs9399137 + rs11886868+ rs4671393 Step 4: HbF levels~rs7482144 + rs9399137 + rs4671393Table:Fetal hemoglobin association results for SNPs at the BCL11A, HBS1L-MYB, and HBG2 gene loci in the sickle cell cohort from Oman.SNP'sChromosomeMAF# (allele)Effect sizeStandard Errorp-valuers7482144110.262 (A)4.1610.6890.000*rs939913760.0875 (C)5.5631.2660.000*rs467139320.59 (A)-1.6610.8360.05*#MAF, minor allele frequency. Minor alleles (positive strand) are given in parenthesis.*A p value of <0.05 was considered as highly significant. Our study has demonstrated a significant association between the BCL11A, HBSIL-MYB, HBG2 SNP's and HbF levels. Together these 6 SNP's accounted for ~46% variation in the HbF levels in our SCD study subjects and in part explains the clinical heterogeneity seen in these patients. Three SNP's one each in Chromosome 2, 6 and 11 namely rs4671393, rs9399137 and rs7482144 respectively demonstrated the strongest effect on HbF levels. Disclosures: Alkindi: Sultan Qaboos University: Employment, Research Funding. Krishnamoorthy: INSERM U763: Employment, Research Funding. Pathare: Sultan Qaboos University: Employment, Research Funding.

scholarly journals MetAP2 Inhibition Modifies Hemoglobin S (HbS) to Delay Polymerization and Improve Blood Flow in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2260-2260
Author(s):  
Melanie Demers ◽  
Sarah Sturtevant ◽  
Kevin Guertin ◽  
Dipti Gupta ◽  
Kunal Desai ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Research Funding ◽  
Oxygen Affinity ◽  
Enzyme Kinetic ◽  
Erythroid Cells ◽  
Cell Disease

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet). MetAP2 is the methionine aminopeptidase able to cleave iMet from Val1 on α-globin and βS-globin as the unfolded N-terminal peptides emerge from the ribosome. Enzyme kinetic studies with pure MetAP2 and N-terminal octapeptides showed that βS-globin peptide is a 5-fold better substrate than α-globin peptide. Lentiviral shRNA knock-down of MetAP2 in differentiating erythroid HUDEP cells in vitro confirmed that α-globin is more extensively modified than βS-globin, consistent with the enzyme kinetic data. Selective MetAP2 inhibitors used to treat cultured human erythroid cells (HUDEP and PBMC derived CD34+) and Townes SCD mice in vivo confirmed that both α-globin and βS-globin domains of HbS are extensively modified by N-terminal iMet and acetyl-iMet. N-terminal retention of iMet and subsequent acetylation creates a mixture of modified HbS tetramers with combined modifications on both globins. Cation exchange chromatography separated nine different modified HbS variants from unmodified HbS as identified by LCMS. Purified samples of HbS modified by N-terminal iMet and acetyl-iMet had increased oxygen affinity as measured by decreased P50. Modified HbS containing the acetyl-iMet-βS-globin were found to have delayed polymerization under complete hypoxia (sodium metabisulfite triggered hypoxia in 1.8 M phosphate). Two modified HbS variants were further purified for X-ray crystallography studies (βS-globin / iMet-α-globin and acetyl-iMet-βS-globin / iMet-α-globin). Oxyhemoglobin structures of both modified HbS variants were in the R2-state previously described in structures of aldehyde modified HbS. This R2-state stabilizes the oxygenated R-state of HbS from conversion to the deoxygenated T-state that initiates HbS polymerization in sickle RBC. Treatment by selective irreversible covalent or reversible MetAP2 inhibitors resulted in high levels of HbS modification (>75%) in cultured erythroid cells (HUDEP and CD34+ cells). Dose dependent modification of HbS was observed in Townes sickle cell mouse blood RBC in vivo with total modification of HbS approaching 50%. In whole blood ex vivo studies, modification of HbS decreased RBC sickling under hypoxia (4% O2) and significantly increased the affinity of RBC for oxygen (decreased P50). Blood samples from MetAP2 inhibitor treated mice were analyzed for single-cell O2 saturation of the RBC and for the fractional flow velocity drop in whole blood rheology under decreasing partial oxygen pressures. In blood from vehicle treated sickle mice, a low-saturation peak of deoxy-HbS was observed in 7.8% O2, in contrast to blood from MetAP2 inhibitor-treated mice where the low-saturation peak was only observed in 6.4% O2. Similarly, in an assay of O2 dependent blood flow rheology, the half-maximum fractional velocity drop occurred at 5% O2 in control blood decreasing to 2% O2 in MetAP2 inhibitor treated blood. Our studies show that MetAP2 inhibition results in retention of iMet on βS-globin and α-globin and allows further acetylation of the retained iMet to create a mixture of N-terminal modified HbS tetramers. These modified HbS variants resist polymerization and RBC sickling under conditions of low O2 by delaying HbS polymerization and increasing O2 affinity. Our data suggests that MetAP2 may warrant further study as a potential therapeutic target for sickle cell disease. Disclosures Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.

scholarly journals Mechanism Underlying a Role for Factor XIII (FXIII) Polymorphism in Sickle Cell Disease-Associated Priapism

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2361-2361
Author(s):  
Marilyn J. Telen ◽  
Milena Batchvarova ◽  
Joan D Beckman ◽  
Martha Delahunty ◽  
Karen L Soldano ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Research Funding ◽  
Factor Xiii ◽  
Cell Disease ◽  
Risk Genotype ◽  
Transglutaminase Activity ◽  
Clot Contraction ◽  
Fxiii Activity

Abstract Background: The pathophysiology of priapism in sickle cell disease (SCD) is poorly understood. While blood stasis is essential to tumescence, most research in SCD-associated priapism to date has focused on the potential role of abnormal signaling pathways. We have previously observed that a coding sequence single nucleotide polymorphism (rs5988) of the transglutaminase factor XIII gene (FXIII) is strongly associated with SCD priapism, with an odds ratio of 2.52 [C.I. 1.27 -5.03] for the risk genotype (G/G, expressing only FXIII E652) vs the most common non-risk genotype (G/C, expressing both FXIII E652 and FXIII Q652). We therefore explored the effect of the rs5988 polymorphism on various aspects of FXIII function. Methods: Recombinant FXIII (rFXIII) E652 and rFXIII Q652 were expressed by 293 kidney cells and isolated from serum-free tissue culture supernatant. Before use in assays, activation of rFXIII by thrombin was confirmed by generating activated rFXIII (rFXIIIa) with thrombin (10 U/ml), followed SDS-PAGE, western blotting for FXIII, and densitometry for quantitation. Whole blood samples and plasmas were obtained from previously genotyped subjects with SCD under an IRB-approved protocol. Plasma FXIII and rFXIII transglutaminase activity was measured by the ability to catalyze 5-(biotinamido)pentylamine incorporation into a suitable substrate. Plasma FXIII antigen was assayed by ELISA. Clot contraction was measured after tissue factor-initiated clotting of recalcified whole blood. Clot resistance to lysis was measured after exposure to tissue plasminogen activator (tPA). Results: Transglutaminase activity of each rFXIII was measured using fibrinogen and fibronectin as substrates. At 20 minutes, rFXIIIa E652 showed 1.44-fold more transglutaminase (crosslinking) activity toward fibrin(ogen) than rFXIIIa Q652 (p=0.027), and a nonsignificant trend toward more activity (1.32-fold, p=0.079) toward fibronectin. Kinetic assays also showed that rFXIII E652 had significantly greater activity toward both matrices (p=0.006 and 0.012, respectively), suggesting the risk genotype (homozygosity for the G allele) enhances fibrin(ogen) and/or fibronectin crosslinking. FXIII activity in the plasma of 18 genotyped SCD patients (3 CC, 7 GC, 8 GG) demonstrated a consistent, but not significant, trend toward increased FXIII activity with increasing presence of G alleles (80.01% CC, 98.90% CG, and 107.20% GG). Although results were not adjusted for genotype at other loci reported to affect FXIII expression, there was no significant difference in FXIII antigen among genotypes. Compared to contracted whole blood clots from patients with only one or no risk alleles, clots from SCD patients with two copies of the risk allele (GG, expressing only FXIII E652) did not differ in either RBC retention within clots or clot mass (weight). Moreover, inhibition of transglutaminase activity with T101 significantly increased RBC release and decreased clot weight to a similar degree in contracted clots from patients with either the GC or GG genotype. In clots formed from FXIII-deficient plasma supplemented with rFXIIIa E652 or rFXIIIa Q652 and ABO-compatible donor RBCs, clots containing rFXIIIa E652 were 14% more resistant to lysis than clots containing rFXIIIa Q652 (p=0.016). Parallel studies with SCD patient plasma samples also showed that clots containing only FXIII E652 were more resistant to lysis than clots containing both FXIII E652 and FXIII Q652(p=0.0001). Conclusions: These data suggest the FXIII rs5988 polymorphism does not alter protein expression or clot contraction but may regulate clot stability via slightly increased transglutaminase activity and enhanced resistance to lysis. These effects may predispose patients to formation of microclots during tumescence, thus impairing blood egress and increasing risk of priapism. Further studies should be conducted to determine if anticoagulation or fibrinolytic treatments are viable preventative or treatment strategies for SCD patients with the risk FXIIIGG genotype and recurrent priapism. Disclosures Telen: Pfizer, Inc.: Consultancy, Research Funding. Wolberg:GlaxoSmithKline: Employment; Novo Nordisk: Research Funding.

scholarly journals COVID Symptoms and COVID Anxiety in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Wally R Smith ◽  
Benjamin Jaworowski ◽  
Shirley Johnson ◽  
Thokozeni Lipato ◽  
Daniel M Sop
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Telephone Survey ◽  
Cell Disease ◽  
Weighted Mean ◽  
The Us ◽  
Associated Symptoms ◽  
At Home

Background Even before the US upswing of the current COVID pandemic, the number of sickle cell disease (SCD) patients coming to hospitals and EDs appeared to fall drastically. This happened despite SCD patients having often been heavy utilizers of the ED and hospital for their iconic vaso-occlusive crises (VOC). Though ambulatory SCD clinics quick converted largely to telehealth in order to comply with stay-at-home orders designed to suppress person-to-person transmission, some SCD patients appeared to avoid care, delay care, or refuse doctors' invitations for care. Presumably patients did so out of COVID fears, but this has not been confirmed in the literature. Further, whether these patients had COVID symptoms but stayed at home has not been studied. As part of quality improvement (QI) to conduct COVID surveillance in an adult sickle cell program, we sought to explain and predict SCD health care utilization patterns we were observing, as well as to determine urgent physical and mental health needs of patients who appeared to be avoiding care. Methods Fifteen staff in the Adult Sickle Cell Medical Home at Virginia Commonwealth University, a large urban academic medical center, conducted a telephone survey ("wellness check"was used when we talked to patients) of all known adults with SCD over 19 days in 2020. A staff member confirmed the patient had SCD, asked permission to proceed, then asked about symptoms consistent with COVID-19. At the end of the telephone survey, respondents wer invited to complete an email survey of sickle cell and COVID-19 utilization attitudes (19-33 items, depending on the response pattern, either drawn from the National Health Interview Survey, from the Adult Sickle Cell Quality of Life Measurement quality of care survey, or drafted by the authors), the Sickle Cell Stress Survey-Adult (SCSS-A, a 10-item previously validated survey), and anxiety and depression (PHQ9 of the PRIME-MD). Results Of 622 adults approached by phone call, 353 responded to the following yes/no screening questions regarding the prior 14 days: fever over 100 F 0/353 (0.00%); cough 3/353(0.01%); difficulty breathing 0/353(0.00%); unexplained shortness of breath 2/353(0.01%); sore throat 2/353 (0.01%); unexplained muscle soreness 2/353(0.01%);contact with anyone who tested positive for COVID-19 2/353(0.01%); testing for COVID 19 6/353(0.02%). For QI purposes, we set a threshold of three or more COVID-associated symptoms or the presence of fever as criteria requiring intense telephone or in-person staff monitoring for the following week. Only three patients met criteria. A total of 219/353 had email surveys sent. Of 63 patients (28.8%) who returned email surveys by June 10, 2020, 35.9% had already managed a "pain attack" at home 4 or more times in the prior 12 months, and 45.5% of these said their bad ER experiences were very or somewhat important in that decision. In the prior 14 days, although 30/64 reported a crisis for at least one day, only 4/64 had visited the Emergency Department for pain. On a 0-10 scale, 21/61 patients endorsed "0" for worry that they would be COVID-infected by going for medical care (weighted mean 3.9), but 18/59 endorsed "10" for worry they were more at risk of COVID because of SCD (weighted mean 6.31), and 22/60 endorsed "10" for worry they would fare worse than others if COVID infected (weighted mean 6.97). Many patients forwent "needed" care (16/62) or delayed "needed" care by at least a day (36/61). Eleven patients met criteria for moderately severe to severe depression on the PHQ-9, and 28/63 somewhat or strongly agreed with the statement "death is always on the back of my mind" on the SCSS-A. Conclusions In adolescents and adults with SCD, many were already reticent to come to the ED for pain, but a significant portion reported delays or avoidance of needed care during the early stages of the US COVID pandemic, and few reported using the ED despite over half reporting at least one crisis day in 14. Patients nonetheless reported very few COVID-associated symptoms. Fears of COVID infection/susceptibility may limit visits for needed sickle cell care among adults. Acknowledgements: Mica Ferlis RN, FNP, Caitlin McManus, RN, FNP, Emily Sushko, RN, FNP, Justin West, RN, Kate Osborne, RN, Stefani Vaughan-Sams, Marla Brannon, BS, Nakeiya Williams, BS Disclosures Smith: GlycoMimetics, Inc.: Consultancy; Emmaeus Pharmaceuticals, Inc.: Consultancy; Novartis, Inc.: Consultancy, Other: Investigator, Research Funding; Global Blood Therapeutics, Inc.: Consultancy, Research Funding; Shire, Inc.: Other: Investigator, Research Funding; NHLBI: Research Funding; Patient-Centered Outcomes Research Institute: Other: Investigator, Research Funding; Health Resources and Services Administration: Other: Investigator, Research Funding; Incyte: Other: Investigator; Pfizer: Consultancy; Ironwood: Consultancy; Novo Nordisk: Consultancy; Imara: Research Funding; Shire: Research Funding.

scholarly journals Clinical and Biomarker Predictors for Avascular Necrosis in Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3091-3091
Author(s):  
Michael Rabaza ◽  
Maria Armila Ruiz ◽  
Liana Posch ◽  
Faiz Ahmed Hussain ◽  
Franklin Njoku ◽  
...  
Keyword(s):  
Risk Factors ◽  
Sickle Cell Disease ◽  
Board Of Directors ◽  
Avascular Necrosis ◽  
Sickle Cell ◽  
Research Funding ◽  
Medical Attention ◽  
Cell Disease ◽  
Advisory Committees ◽  
Early Management

Abstract Introduction Sickle cell disease (SCD) affects 1 in 365 African Americans and approximately 25 million people world-wide. A common skeletal system complication is avascular necrosis (AVN), which can cause substantial pain and a reduced quality of life. While early management of AVN is focused on increasing range of motion with physical therapy and pain relief, there are no clear predictors for who is more likely to develop AVN and earlier institution of these preventive measure could help decrease disease progression. Vascular endothelial growth factor (VEGF) is a biomarker of endothelial injury and may indicate reduced vascular supply to the femoral or humeral head. Here we describe potential risk factors and biologic pathways for AVN in SCD, as understanding these may lead to improvements in future monitoring, early detection, and early intervention practices. Methods We investigated clinical and laboratory risk factors associated with AVN in a cohort of 435 SCD patients from our center. Blood samples, clinical, and laboratory data were collected at the time of enrollment during a clinic visit. Genotyping for alpha thalassemia was performed by PCR and the serum concentration of VEGF was measured by ELISA. AVN status was confirmed by review of the medical record and available imaging. We conducted a cross-sectional analysis comparing categorical and linear variables by AVN status using the chi-square and Kruskal-Wallis test, respectively. The independent association of the clinical and laboratory variables with AVN status was determined by logistic regression analysis. The initial model included variables with a P-value &lt; 0.1 on univariate analysis and the final model was ascertained by stepwise forward and backward selection. Median values and interquartile range (IQR) are provided. Results The median age of the cohort was 32 (IQR, 24 - 43) years, 57% (250/435) were female, and 46% (198/435) were on hydroxyurea. AVN was observed in 34% (149/435) of SCD patients. SCD patients with AVN were older, had more frequent vaso-occlusive crises requiring medical attention, and had a higher body mass index (Table I) (P ≤ 0.002). We measured VEGF in 241 of the SCD patients with serum samples available at the time of enrolment. Serum VEGF concentrations trended higher in SCD patients with versus without AVN (420 vs. 359 pg/mL, respectively; P = 0.078). In the multivariate analysis model, AVN was independently associated with increased number of vaso-occlusive crises (OR 1.1, 95% CI: 1.0 - 1.14; P = 0.02), AST concentration (natural log OR 0.5, 95% CI: 0.2 - 0.9; P = 0.03), VEGF concentration (natural log OR 1.4, 95% CI: 1.0 - 1.9; P = 0.047), and tobacco use (OR 1.9, 95% CI: 0.9 - 3.7; P = 0.078). Discussion In conclusion, we demonstrate a high prevalence of AVN in an adult cohort of SCD patients. The presence of AVN was independently associated with a greater frequency of vaso-occlusive pain episodes, which may demonstrate a shared pathophysiology between AVN and vaso-occlusion that merits further investigation. We demonstrate that serum VEGF concentrations are higher in SCD patients with AVN and may be a clinical tool to identify those at high-risk and for earlier intervention for this complication. Figure 1 Figure 1. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Alkaline Phosphatase Is Associated with Red Cell Alloimmunization in the Pulmonary Hypertension and Hypoxic Response (PUSH) Sickle Cell Disease Cohort

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Victoria Brooks ◽  
Oluwalonimi Adebowale ◽  
Victor R. Gordeuk ◽  
Sergei Nekhai ◽  
James G. Taylor
Keyword(s):  
Risk Factors ◽  
Alkaline Phosphatase ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Severe Disease ◽  
Case Control ◽  
Red Cell ◽  
Cell Disease ◽  
History Of

Background: Blood transfusion is a common therapy for sickle cell disease (SCD). Although, highly effective, a major limitation is development of alloantibodies to minor blood group antigens on donor red cells. Alloimmunization has a prevalence of 2-5% for transfusions in the general population, but it is significantly higher in SCD. Risk factors for alloimmunization have been poorly characterized, although number of lifetime transfusions is an important risk factor. Alloimmunization has been clinically observed in children with a prevalence of about 7%. With development of each antibody, blood donor matching becomes increasingly difficult and expensive with an increased risk for transfusion reactions and diminished availability of compatible red cell units for treatment of SCD. The ability to identify risk factors for developing alloantibodies would be beneficial for clinicians. To identify markers for alloimmunization in SCD, we have analyzed children and adults who developed this complication. Methods: We analyzed The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study, which enrolled n=468 pediatric and n=59 adult SCD subjects. In both children and adults, alloimmunization cases were defined as a history of at least 1 alloantibody. Controls in both cohorts were defined as subjects with no history of alloantibodies and receipt of more than 10 lifetime red cell transfusions. All others within the study who did not meet these criteria were assigned to a third comparison group. To identify differences between cases, controls and all others, we performed univariate analyses (using ANOVA or Kruskal Wallace where appropriate) for clinical parameters and laboratories. Case control comparisons were also performed for selected variables and plasma levels for 11 cytokines. Results were further analyzed using regression modeling. Results: The overall prevalence of alloimmunization was 7.3% among children (34/468 subjects; median age 12, range 3-20 years) compared to 28.8% in adults (17/59 subjects; median age 37, range 18-73 years). When only considering those with &gt;10 lifetime transfusions, the prevalence was considerably higher at 29.3% and 54.8% in children and adults, respectively. At the same time, 8 pediatric (23.5%) and 5 adult (29.4%) alloimmunization cases had received fewer than 10 transfusions. In a 3-way pediatric cohort comparison (cases, controls and all others), risk factors associated with alloimmunization included SS genotype, older age and markers of more severe disease (higher ferritin, WBCs, platelets and total bilirubin). Comparison of cases to controls showed alkaline phosphatase (P=0.05) was significantly lower in cases, whereas AST (P=0.02) was significantly higher even with adjustment for age. Levels of plasma cytokines MCP-1 (P=0.01) and IFNgamma (P=0.08) were lower in cases from a subset of the pediatric cohort. In adults, only 4/59 (6.8%) subjects had never received a lifetime transfusion (all non-SS). In the adult 3-way comparisons, only SS genotype and higher ferritin were associated with alloimmunization. The adult case control analysis showed higher absolute monocyte count (P=0.02), absolute eosinophil count (P=0.04) and absolute basophil count (P=0.008) in association with alloimmunization cases. In addition, alkaline phosphatase was again significantly lower among cases (P=0.02) as seen in the pediatric cohort. There were no significant differences in cytokine levels among adults. Conclusions: When considering only transfused SCD patients, the prevalence of alloimmunization is higher than 30%. As seen in prior studies, higher lifetime red cell transfusions are an important risk factor especially among adults where most patients have received transfusions. Children who develop alloantibodies appear to have laboratory markers of more severe disease, but this is not observed in adults. A novel association observed across both pediatric and adult subjects is a significantly lower serum alkaline phosphatase in those with alloantibodies. The results of this study suggest a need for improved tracking of red cell transfusion therapy in the US for SCD patients due to a high prevalence of alloimmunization. Further study is also needed to elucidate the significance of the alkaline phosphatase association. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Ironwood: Research Funding; Imara: Research Funding.

scholarly journals Cardiac Injury and Microvascular Ischemia in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2286-2286
Author(s):  
Kiranveer Kaur ◽  
Ying Huang ◽  
Subha Raman ◽  
Eric H. Kraut ◽  
Payal Desai
Keyword(s):  
Sickle Cell Disease ◽  
Myocardial Perfusion ◽  
Sickle Cell ◽  
Cardiac Mri ◽  
Research Funding ◽  
Cardiac Injury ◽  
Microvascular Disease ◽  
Elevated Troponin ◽  
Cell Disease ◽  
Troponin Elevation

Introduction: Myocardial ischemic injury remains an under recognized problem in patients with sickle cell disease (SCD), for which the exact prevalence remains undefined. SCD patients are known to have microvascular disease, impaired myocardial perfusion reserve and lack of typical epicardial vessel involvement based on prior data. Previous study at our institution has demonstrated that 3/22(13%) patients with clinically stable sickle cell disease had impaired myocardial perfusion reserve but no epicardial coronary artery disease. In this study, we will aim to learn prevalence of cardiac injury and microvascular ischemic disease. We will also evaluate for impact of these findings on overall survival (OS) of SCD patients. Methods: We conducted a retrospective chart review of patients with SCD seen at OSU Wexner Medical Center from July 2005 to July 2015 to identify patients who had elevated troponin-I level or cardiac MRI performed for chest pain. Clinical and laboratory data around the time of cardiac MRI and troponin elevation was collected. Abnormal MRI was defined in three ways: 1) Microvascular disease was defined by presence of subendocardial or myocardial perfusion defects and myocardial scarring. 2) Myocardial disease otherwise includes other findings suggestive but not specific for myocardial ischemia including left ventricular dysfunction, midmyocardial fibrosis, inflammation and regional wall motion abnormalities. 3) Abnormal MRI includes patients described in either 1) or 2). Kaplan-Meier (KM) method was used to evaluate the impact of microvascular disease defined in all 3 ways on OS. Proportional hazards model was fit to estimate the association between troponin elevation and OS, where troponin elevation was treated as a time-dependent variable and OS was measured from time of birth. Results: Sixty-nine (51% male; genotype Hb SS 75%, SC 16%, and Sβ-thal 9%) of 373 SCD patients had either abnormal troponin and/or had cardiac MRI done. Median age was 34 years (range 19-67 years). Of 238 patients who had troponin-I measured over this period, 18 % (n=42) had elevated troponin. 24 of 47 patients with cardiac MRI showed abnormalities described above specific for microvascular disease (n=14, 30%) and myocardial disease otherwise (n=10, 21%). We identified 22 patients with troponin measurement within 30 days before cardiac MRI. Elevated troponin levels predicted MRI abnormalities with sensitivity of 71% (95% confidence interval (CI) 42-92%) and specificity of 63% (95% CI 24-91%). The degree of troponin elevation did not correlate with the MRI abnormality. Hazard ratio of death in patients with elevated troponin was 5.1 (95% CI 2.7-9.6; p<0.0001). While the KM survival curves show lower OS in patients in abnormal MRI (p=0.74) and microvascular disease (p=0.42; Figure 1) group compared with normal MRI, the comparisons were not statistically significant. There was no difference in OS for patients with nonspecific myocardial disease findings (p=0.59). Conclusion: Over a 10-year period, the prevalence of cardiac injury as measured by elevated troponin was 18% (42/238) in patients with atypical chest pain. Among 47 patients who had cardiac MRI performed, 51% were abnormal with 30% having findings specific for microvascular cardiac disease. Troponin elevation appears to significantly increase the risk of all-cause mortality. Patient with microvascular and myocardial ischemic disease tend to have lower OS, but it did not reach statistical significance. This could be one of the potential contributing factors to high early mortality and sudden deaths in SCD patients. Further studies will be needed to elaborate on disease modifying interventions that impact survival in these patients. Disclosures Desai: Novartis: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Potomac: Speakers Bureau; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Board.

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 &gt; 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&lt;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.

Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis

2003 ◽  
Vol 1014 (1-2) ◽  
pp. 93-101 ◽  
Author(s):  
Chien-Yuan Kuo ◽  
Hsin-Lung Wu ◽  
Hwang-Shang Kou ◽  
Shyh-Shin Chiou ◽  
Deng-Chyang Wu ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
Capillary Zone Electrophoresis ◽  
Whole Blood ◽  
Human Whole Blood ◽  
Zone Electrophoresis ◽  
Capillary Zone

scholarly journals Featured Article: Depletion of HDL3 high density lipoprotein and altered functionality of HDL2 in blood from sickle cell patients

2017 ◽  
Vol 242 (12) ◽  
pp. 1244-1253 ◽  
Author(s):  
Eric Soupene ◽  
Sandra K Larkin ◽  
Frans A Kuypers
Keyword(s):  
Sickle Cell Disease ◽  
High Density Lipoprotein ◽  
Sickle Cell ◽  
Acute Phase ◽  
Whole Blood ◽  
Density Lipoprotein ◽  
High Density ◽  
High Density Lipoproteins ◽  
Mrna Levels ◽  
Cell Disease

In sickle cell disease (SCD), alterations of cholesterol metabolism is in part related to abnormal levels and activity of plasma proteins such as lecithin cholesterol acyltransferase (LCAT), and apolipoprotein A-I (ApoA-I). In addition, the size distribution of ApoA-I high density lipoproteins (HDL) differs from normal blood. The ratio of the amount of HDL2 particle relative to the smaller higher density pre-β HDL (HDL3) particle was shifted toward HDL2. This lipoprotein imbalance is exacerbated during acute vaso-occlusive episodes (VOE) as the relative levels of HDL3 decrease. HDL3 deficiency in SCD plasma was found to relate to a slower ApoA-I exchange rate, which suggests an impaired ABCA1-mediated cholesterol efflux in SCD. HDL2 isolated from SCD plasma displayed an antioxidant capacity normally associated with HDL3, providing evidence for a change in function of HDL2 in SCD as compared to HDL2 in normal plasma. Although SCD plasma is depleted in HDL3, this altered capacity of HDL2 could account for the lack of difference in pro-inflammatory HDL levels in SCD as compared to normal. Exposure of human umbilical vein endothelial cells to HDL2 isolated from SCD plasma resulted in higher mRNA levels of the acute phase protein long pentraxin 3 (PTX3) as compared to incubation with HDL2 from control plasma. Addition of the heme-scavenger hemopexin protein prevented increased expression of PTX3 in sickle HDL2-treated cells. These findings suggest that ApoA-I lipoprotein composition and functions are altered in SCD plasma, and that whole blood transfusion may be considered as a blood replacement therapy in SCD. Impact statement Our study adds to the growing evidence that the dysfunctional red blood cell (RBC) in sickle cell disease (SCD) affects the plasma environment, which contributes significantly in the vasculopathy that defines the disease. Remodeling of anti-inflammatory high density lipoprotein (HDL) to pro-inflammatory entities can occur during the acute phase response. SCD plasma is depleted of the pre-β particle (HDL3), which is essential for stimulation of reverse cholesterol from macrophages, and the function of the larger HDL2 particle is altered. These dysfunctions are exacerbated during vaso-occlusive episodes. Interaction of lipoproteins with endothelium increases formation of inflammatory mediators, a process counteracted by the heme-scavenger hemopexin. This links hemolysis to lipoprotein-mediated inflammation in SCD, and hemopexin treatment could be considered. The use of RBC concentrates in transfusion therapy of SCD patients underestimates the importance of the dysfunctional plasma compartment, and transfusion of whole blood or plasma may be warranted.

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