scholarly journals Clinical Testing of Hemechip in Nigeria for Point-of-Care Screening of Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1095-1095 ◽  
Author(s):  
Muhammad Noman Hasan ◽  
Arwa Fraiwan ◽  
Priyaleela Thota ◽  
Tolulope Oginni ◽  
Grace Mfon Olanipekun ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Research Funding ◽  
Point Of Care ◽  
Cost Effective ◽  
Test Results ◽  
Clinical Testing ◽  
Cell Disease ◽  
Blood Samples ◽  
The World ◽  
Hemoglobin Variants

Abstract In sub-Saharan Africa, nearly a quarter of a million babies are born with sickle cell disease (SCD) each year. An estimated 50-90% of these babies die before age 5 due to lack of early diagnosis and timely treatment. The World Health Organization estimates that more than 70% of SCD related deaths are preventable with simple, cost-effective interventions, such as early screening followed by affordable and widely available treatment regimens. Here, we present the early clinical testing results of HemeChip, which is the first single-use cartridge-based microchip electrophoresis hemoglobin screening platform. HemeChip was developed by Hemex Health, Inc., based on technology licensed from Case Western Reserve University. HemeChip allows affordable, objective, quantitative screening of hemoglobin variants at the point-of-care. HemeChip works with a drop of finger or heel-prick blood and separates hemoglobin variants on a piece of cellulose acetate paper that is housed in an injection molded plastic cartridge with a precisely controlled electric field. HemeChip works with a portable reader to produce easily understandable, objective, and quantitative descriptions of the hemoglobin types and percentages present in a blood sample. The HemeChip reader guides the user step-by-step through the test procedure with animated on-screen instructions to minimize user errors. Hemoglobin identification and quantification is automatically done with a custom software on the reader. HemeChip reader records and analyzes the hemoglobin electrophoresis real-time, and it can wirelessly transmit the test results to a central electronic database, if needed. HemeChip prototype units have been clinically tested and benchmarked against the clinical standard technique in Kano, Nigeria, where the SCD prevalence is the highest in the world. We tested a total of 248 subjects (228 children aged 6 weeks to 5 years in Kano, Nigeria; and 20 adults in Cleveland, Ohio, United States) under institutional review board approval, using both HemeChip and the clinical standard laboratory method, High Performance Liquid Chromatography (HPLC, VARIANT™ II, Bio-Rad Laboratories, Inc., Hercules, California). HemeChip tests were done on eHealth Africa campus in Kano, Nigeria, by trained local healthcare workers using blood samples collected at the nearby Aminu Kano Teaching Hospital. Clinical standard (HPLC) testing was done independently by the International Foundation Against Infectious Disease in Nigeria (IFAIN, Abuja, Nigeria) for the blood samples obtained in Kano or by the University Hospitals Cleveland Medical Center Clinical Laboratories (Cleveland, Ohio) for the blood samples obtained in Cleveland. Test results included the following: homozygous SCD (HbSS), heterozygous sickle hemoglobin C disease (HbSC), heterozygous sickle trait (HbAS), and normal (HbAA). HemeChip identified the subjects with HbSS with 100% accuracy, HbSC with 100% accuracy, HbAS with 98.2% accuracy, and HbAA with 96.4% accuracy in comparison to HPLC (Table 1). Overall accuracy of HemeChip was 97.2% in comparison to HPLC for the subjects tested. HemeChip sensitivity was 100% for all hemoglobin variants tested (Table 2), and specificity was 96.4% for HbSS vs. HbAA, 98.2% for HbSS vs. HbAS, 100% for HbSC vs. HbAS, and 100% for HbAS vs. HbAA. Bland-Altman analysis indicated strong agreement between the quantitative HPLC and HemeChip results for hemoglobin percentages, with a mean bias of -3.2%. HemeChip enables, for the first time, accurate, cost-effective identification and quantification of hemoglobin variants at the point-of-need. HemeChip has been developed based on a versatile, mass-producible microchip electrophoresis platform technology that may address other unmet needs in biology and medicine that require rapid, decentralized hemoglobin or protein analysis, identification, and/or quantification. Disclosures Thota: Hemex Health Inc: Employment. Little:PCORI: Research Funding; Hemex: Patents & Royalties: Patent, no honoraria; NHLBI: Research Funding; Doris Duke Charitable Foundations: Research Funding.

scholarly journals Advancing Healthcare Outcomes for Sickle Cell Disease in Nigeria Using Mobile Health Tools

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2173-2173
Author(s):  
Arwa Fraiwan ◽  
Muhammad Noman Hasan ◽  
Ran An ◽  
Amy J. Rezac ◽  
Nicholas J. Kocmich ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mobile Health ◽  
Sub Saharan Africa ◽  
Diagnosis And Treatment ◽  
Test Results ◽  
Cell Disease ◽  
The World ◽  
Equity Ownership ◽  
Sub Saharan

Nigeria leads the world in the number of cases of sickle cell disease (SCD). An estimated 150,000 babies are born annually in Nigeria with SCD, a heredity disorder, and 70-90% die before age 5. Only a small portion of affected infants and children in sub Saharan Africa (SSA) reach adolescence. Over 650 children die per day in sub-Saharan Africa from SCD. These dismal statistics are in sharp contrast to outcomes in high-income countries (HICs) where more than 90% of SCD patients reach adulthood. The World Health Organization (WHO) estimates that 70% of deaths could be prevented with a low cost diagnostic and treatment plan. Meaningful preventive care and treatment cannot be implemented without a structured plan for early diagnosis and patient tracking.Early diagnosis requires improved access to parents and guardians of children with SCD, and gaining this access remains a challenge in most of SSA. In 2015, Nigeria's Kano state government, with support from foreign partners, established a community-based program for newborn registration. This platform provides unique access to newborn babies in one of Nigeria's most populous cities, but still lacks a functioning patient testing, tracking, and monitoring system, which we plan to address in our ongoing study. This study will introduce mobile health in a low-income country with low literacy rate and hopefully accustom that segment of the population to more varied mobile health applications that will ultimately improve their health in the long run. Our current operational platform in Kano, Nigeria provides access to a large population with a high prevalence of SCD. We have previously completed pilot testing of 315 subjects for SCD using our microchip electrophoresis test. We are planning to test up to 4,500 additional subjects less than 5 years of age at Murtala Muhammed Specialist Hospital. The hospital staff includes 97 physicians and 415 nurses and outpatient clinics serve about 30,000 patients monthly. The maternity department has a 200-bed capacity and the antenatal clinic performs about 1,000 deliveries and serves an average of 3,000 mothers monthly. Enrollment is planned to start on September 15, 2019 and medical staff are currently being trained to run the tests. Our study is registered in the United States National Library of Medicine's ClinicalTrials.gov (Identifier: NCT03948516). Our technology is uniquely paired with an automatic reader and an Electronic Medical Record (EMR) and patient management solution to record POC test results, register new cases, and track patients for follow-up (Fig. 1). The reader enables automated interpretation of test results, local and remote test data storage, and includes geolocation (Global Positioning System) (Fig. 2). The system will generate reports for all cases of SCD, track hospital visits, appointments, lab tests, and will have mobile and dashboard applications for tracking patients and samples. The application will be installed on mobile devices provided to users. The proposed system will be compliant with the existing privacy standards to handle medical data (e.g., HIPAA in the US and GDPR in the EU). All communications between the parties will be secured via end-to-end encryption as a safeguard. We anticipate that our project will increase the rates of screening, diagnosis and timely treatment of SCD in Kano State of Nigeria. The project's broader impact will likely be the ability to track and monitor screening, disease detection, diagnosis and treatment, which can be scaled up to the whole nation of Nigeria, then to sub-Saharan Africa. The data obtained and analyzed will be the first of their kind and will be used to inform the design of programs to improve access to, and availability of, effective care for this underserved populations. The importance of increased access to diagnosis and treatment should not be underestimated - it is crucial for realizing effective management of people with SCD. The impact can be enhanced by complementing diagnosis and patient tracking with education for the families so they can provide or seek the necessary preventative treatment. Identification of the location of the patients in need would help identify the areas where family, parent, caregiver education should be provided. Disclosures Fraiwan: Hemex Health, Inc.: Equity Ownership, Patents & Royalties. Hasan:Hemex Health, Inc.: Equity Ownership, Patents & Royalties. An:Hemex Health, Inc.: Patents & Royalties. Thota:Hemex Health, Inc.: Employment. Gurkan:Hemex Health, Inc.: Consultancy, Employment, Equity Ownership, Patents & Royalties, Research Funding.

International Multi-Site Clinical Validation of Point-of-Care Microchip Electrophoresis Test for Hemoglobin Variant Identification

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3373-3373
Author(s):  
Arwa Fraiwan ◽  
Muhammad Noman Hasan ◽  
Ran An ◽  
Julia Z. Xu ◽  
Amy J. Rezac ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Low Income ◽  
Research Funding ◽  
Point Of Care ◽  
Low Income Countries ◽  
Clinical Validation ◽  
Test Results ◽  
Blood Samples ◽  
Hb E ◽  
Equity Ownership

Introduction: Nearly 24% of the world's population carry hemoglobin (Hb) gene variants, with the large majority of affected births occurring in low-income countries. The most prevalent structural Hb variants are the recessive β-globin gene mutations, βS or S, βC or C, and βE or E1. Hb S mutation is prevalent in sub-Saharan Africa and in Central India. Hb C is common in West Africa, and Hb E is common in Southeast Asia and in India. Homozygotes or compound heterozygotes with βS (e.g., Hb SS or SC) have sickle cell disease (SCD), a chronic sickling disorder associated with pain, chronic multi-organ damage, and high mortality. While Hb EE causes only a mild microcytic anemia, Hb E in combination with β-thalassemia can lead to transfusion dependent thalassemia. Though carriers are typically asymptomatic, they may pass the mutations to their offspring. Screening is needed so that these disorders can be diagnosed early and managed in a timely manner2. For example, in low-income countries, due to lack of nationwide screening and comprehensive care programs, up to 80% of babies born with SCD are undiagnosed and less than half of them survive beyond 5 years of age2. The unmet need for affordable, portable, accurate point-of-care tests to facilitate decentralized hemoglobin testing in resource-constrained countries is well-recognized 2,3. Here, we present international multi-site clinical validation results and high diagnostic accuracy of the 'HemeChip' (Fig. 1), an affordable, 10-minute point-of-care microchip electrophoresis test for identifying common Hb variants S, C, and E. Methods: Institutional Review Board approvals were obtained at each study site, and blood samples were collected as part of the standard clinical care. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. 315 children (6 weeks to 5 years of age) were tested in Kano, Nigeria. Study participants were enrolled at three hospitals, Amino Kano Teaching Hospital, Murtala Mohammed Specialist Hospital, and Hasiya Bayero Pediatric Hospital. 124 subjects (7 weeks to 63 years old) were included in the study at Siriraj Thalassemia Center in Bangkok, Thailand. 298 subjects (8 months to 65 years old) were tested at a referral testing facility of ICMR-National Institute of Research in Tribal Health, located at Late Baliram Kashayap Memorial Medical College, Jagdalpur, Chhattisgarh, India. Blood samples were tested with both HemeChip and the standard reference methods, high performance liquid chromatography or cellulose acetate electrophoresis. Reference test results were not available to the HemeChip users. Similarly, HemeChip test results were not available to the users of the standard reference tests. Clinical validation studies presented here were performed with a fully functional, portable HemeChip prototype developed at Case Western Reserve University (Fig. 1A). A commercial product has been developed based on this technology by Hemex Health Inc. under the product name, GazelleTM(Fig. 1B). Results and Discussion: Among the total 768 tests performed with HemeChip in all test sites, 732 were valid tests, as defined by the Standards for Reporting Diagnostic Accuracy (STARD)4. HemeChip correctly identified all subjects with Hb SS, Hb SC, Hb AS, Hb AE, and Hb EE with 100% accuracy (Table 1). Nine subjects with normal Hb (Hb AA) were identified as HbSS in Nigeria. No subjects with disease were identified as normal or trait by HemeChip. Three subjects with compound heterozygous Hb Sβ-thalassemia (2 subjects with Hb Sβ+-thalassemia, 1 subject with Hb Sβ0-thalassemia) were identified as Hb SS. Sensitivity was 100% for all Hb types tested. Specificity was 98.7% for Hb SS versus other Hb types, and 100% for all other Hb types tested. HemeChip displayed an overall diagnostic accuracy of 98.4% in comparison to standard reference methods for the Hb variants tested in all clinical testing sites (Table 1). HemeChip is a versatile point-of-care system that enables affordable, accurate, decentralized hemoglobin testing in resource-limited settings. References: 1. Weatherall DJ, Clegg JB. Bull World Health Organ. 2001;79(8):704-712. 2. Mburu J, Odame I. International Journal of Laboratory Hematology. 2019;41(S1):82-88. 3. Alapan Y, Fraiwan A, Kucukal E, et al. Expert Review of Medical Devices. 2016;13(12):1073-1093. 4. Bossuyt PM, Reitsma JB, Bruns DE, et al. BMJ : British Medical Journal. 2015;351:h5527. Disclosures Fraiwan: Hemex Health, Inc.: Equity Ownership, Patents & Royalties. Hasan:Hemex Health, Inc.: Equity Ownership, Patents & Royalties. An:Hemex Health, Inc.: Patents & Royalties. Thota:Hemex Health, Inc.: Employment. Piccone:Hemex Health, Inc.: Patents & Royalties. Little:Hemex Health, Inc.: Patents & Royalties; GBT: Research Funding. Gurkan:Hemex Health, Inc.: Consultancy, Employment, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Accuracy of a Rapid and Simple Point-of-Care Test for Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2182-2182 ◽  
Author(s):  
Patrick T. McGann ◽  
Beverly A. Schaefer ◽  
Mary C. Paniagua ◽  
Thad A. Howard ◽  
Russell E. Ware
Keyword(s):  
Whole Blood ◽  
Point Of Care ◽  
Limited Resource ◽  
Dried Blood Spots ◽  
Cell Disease ◽  
Blood Samples ◽  
Low Concentrations ◽  
Hemoglobin Variants ◽  
Whole Blood Samples ◽  
Blood Spot

Abstract Sickle cell disease (SCD) is a common and life-threatening inherited disorder of hemoglobin, affecting over 400,000 newborns annually. The majority of these births occur in low-resource countries, particularly in sub-Saharan Africa, where limited access to accurate diagnostics results in early mortality. Accurate diagnosis of SCD currently relies upon analytical techniques that are relatively expensive and tedious, requiring equipment, electricity, and laboratory expertise. In Africa, outside of referral and regional hospitals, the availability of accurate SCD diagnostics is extremely scarce. A simple, rapid, accurate, and inexpensive point-of-care (POC) method for diagnosing SCD in limited resource settings would represent a tremendous advance for the management of SCD worldwide. We evaluated a novel point-of care SCD immunoassay (Sickle SCANTM, Biomedomics, Inc., Research Triangle Park, NC) that utilizes lateral flow technology and antibody-mediated detection of hemoglobin variants, with the goal of determining the accuracy, sensitivity, specificity, and ease of identifying the presence of hemoglobin A (HbA), hemoglobin S (HbS), and hemoglobin C (HbC) using blood samples from patients with a variety of hemoglobin patterns. Blood samples collected in EDTA were first tested by HPLC to determine the percentages of normal and abnormal hemoglobins and were then tested using Sickle SCAN. Mixtures of specific hemoglobin combinations were also created to determine the sensitivity of the POC assay for detecting low concentrations of HbA, HbS, and HbC. The Sickle SCAN kit includes tubes prefilled with 1.0mL of buffer, which lyses erythrocytes and releases hemoglobin. Whole blood samples were tested by adding 5µL of whole blood from the EDTA tube to the prefilled buffer container, mixing by inverting the tube three times, discarding 3 drops of the mixed solution and then applying 5 drops to the testingcartridge. Dried blood spot samples were also tested by dropping a 3mm punch into the prefilled buffer container, mixing, discarding 3 drops, and applying 5 drops to the testing cartridge. Five minutes after sample application, two independent and masked clinicians visually scored each sample for the presence/absence of each potential band (HbA, HbS, HbC, and Control). Figure 1 illustrates the visual results of common hemoglobin patterns. A total of 50 samples were evaluated using the POC device, including 32 whole blood samples, 7 dried blood spots, and 11 samples artificially created to contain known low concentrations of HbA, HbS, and HbC. Temperature and stability were evaluated using 10 additional samples that were stored at 37C for up to 30 days. In order to identify potential interference by hemoglobin variants, samples included many different types of hemoglobin (HbA2, Hb Bart's, HbD, HbE, HbF, Hb Lepore, Hb Hope, Hb I-Texas, and Hb G-Philadelphia). From whole blood samples, HbA, HbS, and HbC were easily detected in both heterozygous and homozygous samples, but the intensity of individual bands did not correlate with actual percentages. Newborn samples with high fetal hemoglobin (HbF) were also easily and accurately analyzed for the presence of HbA, HbS, and HbC, with no obvious interference from HbF. The presence of common variant hemoglobins also did not cross-react, but both observers noted a faint HbA band for a newborn sample with a HbFE pattern. For samples artificially created to contain low concentrations of HbA, HbS, or HbC, these hemoglobin were detected at concentrations of <5%. Dried blood spot samples also yielded clear positive bands, without loss of sensitivity or specificity. Devices stored at 37C and blood samples stored at 4C for up to one month gave identical results to those stored at room temperature. These analyses indicate that the Sickle SCAN POC device was simple, robust, and highly sensitive and specific for detecting HbA, HbS, and HbC, even in very low percentages. The device easily and rapidly detected common hemoglobins, but was not quantitative. Specificity was excellent even in the presence of HbF and common variants, with the possible exception of HbE. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high-HbF phenotypes, suggests that this device is suitable for large-scale screening of SCD in limited resource settings. Figure 1. Figure 1. Disclosures Ware: Eli Lilly: Other: DSMB membership; Biomedomics: Research Funding; Bayer Pharmaceuticals: Consultancy; Bristol Myers Squibb: Research Funding.

scholarly journals Heterogeneous Hypoxia-Mediated Neutrophil and Red Blood Cell Adhesion to E-Selectin in Microscale Flow

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3671-3671 ◽  
Author(s):  
Yuncheng Man ◽  
Erdem Kucukal ◽  
Shichen Liu ◽  
Deepa Manwani ◽  
Jane Little ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Research Funding ◽  
Neutrophil Adhesion ◽  
Cell Disease ◽  
Blood Samples ◽  
Group 2 ◽  
Group 1

Abstract Hypoxia promotes red blood cell (RBC) sickling, oxidative stress, systemic endothelium activation, vascular inflammation, and activation of coagulation in sickle cell disease (SCD), which results in a malicious cycle contributing to the progression of vaso-occlusion and other consequent clinical manifestations, such as acute chest syndrome and ischemic injuries. In the multistep and multicellular paradigm of vaso-occlusion, recruitment of neutrophils and adhesion of sickle RBCs to activated endothelial cells are critical in initiating this cascade of events. To better understand the role of hypoxia in this pathophysiological process, we assessed the adhesion profiles of RBCs and neutrophils to immobilized e-selectin utilizing blood samples from a clinically diverse patient population with SCD. Blood samples were collected from 11 subjects with homozygous SCD (HbSS) and 5 normal subjects (HbAA). Prior to the experiments, whole blood samples were mixed with Hank's balanced salt buffer solution modified with calcium and magnesium (1:1 v/v). A total volume of 25 µl blood sample was perfused through each e-selectin immobilized microchannel under both normoxic and hypoxic (7.5% oxygen level) conditions using SCD Biochip microfluidic adhesion assay [1, 2]. Blood perfusion was followed by a rinse with Hank's buffer solution at 1 dyne/cm2 corresponding to the typical shear stress levels observed in post-capillary venules. Thereafter, neutrophil adhesion, neutrophil rolling, neutrophil-platelet aggregation, and RBC adhesion data were obtained and analyzed. E-selectin functionalized microchannels supported neutrophil adhesion as well as neutrophil rolling when flowing normal blood samples, where we observed higher adhesion and rolling rates in hypoxia (Fig. 1A). SCD subjects were categorized into two distinct groups based on their adhesion profiles: Group 1: hypoxia-enhanced neutrophil adhesion without significant RBC adhesion (N=7), and Group 2: hypoxia-reduced RBC adhesion with marginal neutrophil adhesion (N=4) (Fig. 1B). We find that both normal and SCD neutrophil adhesion to e-selectin is significantly enhanced under hypoxic conditions (Fig. 1C, p<0.05, paired t-test). Moreover, we observed significantly increased neutrophil-platelet aggregates and an increase in the percentage of adhered neutrophils involved in neutrophil-platelet aggregation induced by hypoxia (Fig. 1D&E), suggesting that hypoxia is strongly associated with neutrophil-platelet aggregation driven vaso-occlusive events in Group 1. Furthermore, rolling velocity of 20 neutrophils from each SCD subjects under shear stress was measured, and hypoxia-mediated neutrophil rolling behavior was determined (Fig. 1F). A unique adhesion profile was observed in Group 2, in which the number of adhered RBCs was significantly reduced in response to hypoxia (Fig. 1G, p<0.05, Mann-Whitney non-parametric analysis). Here, we report two different adhesion profiles among SCD sub-populations using an e-selectin functionalized microfluidic model. We observed elevated numbers of adherent neutrophils, decreased neutrophil rolling velocities, and enhanced neutrophil-platelet aggregation induced by hypoxia in one group, while lowered number of adhered RBCs mediated by hypoxia in the other group. We speculate that there may be two distinct mechanisms that initiate vaso-occlusive events in SCD: in Group 1, neutrophils are responsive to endothelial activation. In this group, neutrophil recruitment and the resulting neutrophil-platelet aggregates and other complexes may precipitate vaso-ooclusion, which is strongly susceptible to hypoxia. In Group 2, in whom neutrophil recruitment is less effective, vaso-occlusion may be induced by vascular RBC adhesion, in which hypoxia may be a less proximate trigger. References: Alapan, Y., C. Kim, A. Adhikari, K.E. Gray, E. Gurkan-Cavusoglu, J.A. Little, and U.A. Gurkan, Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease. Transl Res, 2016. 173: p. 74-91.e8. Kim, M., Y. Alapan, A. Adhikari, J.A. Little, and U.A. Gurkan, Hypoxia-enhanced adhesion of red blood cells in microscale flow. Microcirculation, 2017. 24(5). Disclosures Little: NHLBI: Research Funding; Hemex: Patents & Royalties: Patent, no honoraria; PCORI: Research Funding; Doris Duke Charitable Foundations: Research Funding.

Development of a Novel, Portable Cost-Effective Sickle Cell Disease Diagnostic Test Suitable for Neonatal Screening in Low Resource Countries

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4844-4844
Author(s):  
Michael Ryan Cleary ◽  
Tej Vaddi
Keyword(s):  
Developing Countries ◽  
Sickle Cell Disease ◽  
Diagnostic Test ◽  
Peripheral Blood ◽  
Neonatal Screening ◽  
Sandwich Elisa ◽  
Cost Effective ◽  
Cell Disease ◽  
Blood Samples ◽  
Positive Line

Abstract Background: Sickle cell disease (SCD) is common throughout sub-Saharan Africa, where 75% of the 300,000 global births of affected children live. A significant proportion of these children die before adulthood, with preventable conditions such as pneumococcal infection. Introduction of neonatal screening in the developed world has cut down the mortality rate for SCD from 16% to >1% by allowing targeted interventions. A low-cost, rapid and point-of-care diagnostic test is needed to improve the feasibility of neonatal SCD screening in developing countries due to limited economic constraints. Lateral flow-immunoassay (LFIA) devices offer an attractive approach to develop such a test. LFIA tests are widely used for the diagnosis of infectious and non-infectious diseases in developing countries and offer advantages of stability, precise performance, interpretation by minimally trained users without requiring refrigeration during shipping and storage. Methods: The conventional antibody (ab) based diagnostic approach to SCD is to utilize antibodies specific to the mutated hemoglobin (Hb), but such a strategy requires multiple antibodies, each specific to a given mutated form. To design a simple, cost-effective diagnostic, an alternative strategy of utilizing a single anti-human HBB ab that does not recognize any mutated form was adopted. In this device, a positive line would denote normal adult Hb and an absence of a positive line would denote a diagnosis of SCD. A number of monoclonal anti-human HBB abs were initially screened in direct enzyme-linked immunoassays (ELISA) using different purified hemoglobins (HbA0, HbS and HbA2) as antigens to identify candidates to develop the LFIA device. Based on the results from direct ELISAs, appropriate capture and detection ab pairs were generated to develop sandwich ELISA, which can be translated to a sandwich LFIA format. Results of ELISAs using pure Hbs were confirmed by using diluted peripheral blood samples from healthy and SCD patients. Strip-based LFIA was designed with 4 antibodies A) a device control using anti-mouse ab that confirms appropriate lateral flow B) a Hb control ab that recognizes all forms of HBB to confirm the presence of enough adult Hb in the sample, C) an HBB ab that only recognizes HbA0 but not HbS and HbA2 and D) a gold conjugated alpha chain specific ab as a labelled recognition element. The incorporation of gold nanoparticles as labels allows visual examination of colors at the test and control lines, resulting in qualitative or semi-quantitative analysis. Results: The results from the direct ELISA screening identified two potential antibodies that can be used in the LFIA, SCAD001 and SCAD003. SCAD001 was the most differentiating with a 10-fold difference between HbA0 vs. HbA2 or HbS, which allows its use as a test line. The optical density (OD) reading for SCAD001 with HbA0 HbA2 and HbS were 0.867, 0.07 and 0.08, respectively (Figure 1a). SCAD003 was able to bind all three variants of Hb with high OD readings of 3.7, 3.6, and 3.7, respectively, suggesting its potential to be used as a gold conjugation ab. In the sandwich ELISA format, SCAD001 demonstrated excellent selectivity for HBA0 over other variants of Hb (1.4 vs .04 for HbA0 and HbS, respectively). A pilot LFIA test device was then designed using SCAD001. Representative results as shown in Figure 2 from an LFIA test using SCAD001 demonstrate that the positive line is evident with HbA0 but not with HbA2 and HbS. These devices are currently being scaled up to be evaluated in different test conditions. Results from the current experiments using peripheral blood samples from patients with different forms of SCD and a comparison of current standard of care diagnostic vs LFIA device will be included in the presentation. Conclusion: These results demonstrate the feasibility of a novel approach of designing an LFIA device based on anti-HBB ab that selectively identifies normal Hb but not other variants. With its simple design and a single differentiating ab this device has the potential to be further developed as a neonatal screening test in low resource countries. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Correction of Point-of-Care International Normalized Ratio (INR) Values in Patients with Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Syeda Rahman ◽  
Andrew Srisuwananukorn ◽  
Robert E. Molokie ◽  
Michel Gowhari ◽  
Franklin Njoku ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Correction Factor ◽  
Research Funding ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
International Normalized Ratio ◽  
Cell Disease ◽  
Discovery Cohort ◽  
Advisory Committees

Background: With a narrow therapeutic range, warfarin use needs to be closely monitored to minimize the risk of bleeding or thrombosis. Point-of-care (POC) International Normalized Ratio (INR) tests using finger stick blood samples provide instantaneous results and improve testing efficiency; however, the test accuracy of POC-INR may be affected by various factors such as anemia (1, 2). Thrombosis and warfarin use are prevalent in patients with sickle cell disease (SCD) (3), but evidence on the accuracy or correction of POC-INR in this patient population is lacking. This study evaluates correlation of POC-INR to clinical laboratory INR (CL-INR) in patients with SCD. Methods: POC-INR and CL-INR values measured within 12 hours of each other in patients with SCD treated at the UI-Health from 2015-2017 were collected. A total of 28 INR pairs were used to form a discovery cohort. A cohort of non-SCD African American (AA) patients with POC-INR and CL-INR values measured within 12 hours who were treated at UIC-Health during the same period were utilized as a control group. Patient demographic and clinical characteristics were recorded to calculate a correction factor. Additional 28 POC- and CL-INR pairs in patients with SCD were used to form a validation cohort. Descriptive statistics, Kruskal-Wallis test, Chi-square test, and linear regression were used for data analysis. The study was approved by the UIC Institutional Review Board (protocol #2020-0191). Results: In the discovery cohort with 100% AA patients, POC-INR in an acceptable range of the CL-INR, defined as an INR difference within ±0.5 INR of CL-INR when POC-INR &lt; 2 or ±30% of CL-INR when POC-INR ≥ 2 (4), was present in 6 of 7 (86%) of patients when INR &lt; 4 compared to 5 of 21 (24%) when INR ≥ 4. For POC-INR ≥ 4, a correction factor of 0.7 was derived using a linear regression model (95%CI: 0.685-0.752, P&lt;0.001, R square: 0.991), which significantly improved the in-range POC-INR from 24% to 100% in the discovery cohort (P&lt;0.001). In the validation cohort, the in-range POC-INR increased from 19% to 95% (P&lt;0.001) after applying the correction factor of 0.7 (Figure 1). When compared to the non-SCD AA cohort, the patients with SCD were younger (38 vs 59 years, P&lt;0.001) and more anemic (HCT 27% vs 37%, P&lt;0.001). However, there was no significant difference in the in-range POC-INR percentage (37% vs 42%, P = 0.547) (Table 1). The correction factor for POC-INR ≥ 4 in the non-SCD AA cohort was the same as the SCD cohort as 0.7. Summary: POC-INR over-estimates a simultaneous CL-INR when POC-INR ≥ 4 in patients with SCD, and a correction factor significantly improved the agreement with CL-INR. Despite the difference in anemia, the accuracy of POC-INR and correction factor were similar between SCD patients and non-SCD AA patients. An institution-specific correction factor for POC-INR ≥ 4 should be applied similarly to both SCD and non-SCD patients. Reference: 1. R. W. Hoel et al., Correlation of point-of-care International Normalized Ratio to laboratory International Normalized Ratio in hemodialysis patients taking warfarin. Clin J Am Soc Nephrol4, 99-104 (2009). 2. C. E. DeRemer, B. McMichael, H. N. Young, Warfarin Patients With Anemia Show Trend of Out-of-Range International Normalized Ratio Frequency With Point-of-Care Testing in an Anticoagulation Clinic. J Pharm Pract32, 499-502 (2019) 3. A. Srisuwananukorn et al., Clinical, laboratory, and genetic risk factors for thrombosis in sickle cell disease. Blood Adv4, 1978-1986 (2020). 4. W. Plesch et al., Results of the performance verification of the CoaguChek XS system. Thromb Res123, 381-389 (2008). Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding; Ironwood: Research Funding; Novartis: Consultancy. Saraf:Novartis, Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Boards, Speakers Bureau; Pfizer, Global Blood Therapeutics, Novartis: Research Funding.

scholarly journals Geospatial Mapping of Sickle Cell Disease in Northwest Tanzania: The Tanzania Sickle Surveillance Study (TS3)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3662-3662 ◽  
Author(s):  
Emmanuela E. Ambrose ◽  
Luke R. Smart ◽  
Mwesige Charles ◽  
Arielle G. Hernandez ◽  
Adolfine Hokororo ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Sickle Cell Trait ◽  
Surveillance Study ◽  
Cell Disease ◽  
Public Health Infrastructure ◽  
Lake Zone ◽  
Hemoglobin Variants ◽  
Geospatial Mapping

Abstract Tanzania ranks third in Africa for the estimated number of annual births with sickle cell disease, but these estimates are based on sparse data from small studies reported over the past 50 years. A recently completed surveillance study from Uganda documented substantial variation in the prevalence of sickle cell trait and disease across the country. Tanzania lacks a national newborn screening program, and no contemporary multi-regional screening of infants has been undertaken. We designed and conducted a prospective study to determine the prevalence of sickle cell trait and disease by region and district in northwest Tanzania, where the prevalence of sickle cell is thought to be highest. The study used existing public health infrastructure while building local capacity for accurate diagnosis of sickle cell disease. Secondary objectives included characterization of hemoglobin variants and exploration of associations between sickle cell trait, sickle cell disease, malaria, and HIV. The Tanzania Sickle Surveillance Study (TS3) is a prospective cross-sectional study of HIV-exposed infants born in 9 regions across the Lake Zone of northwest Tanzania. In Tanzania, the HIV early infant diagnosis (EID) program collects dried blood spots (DBS) from all children born to HIV-infected mothers. DBS are transported to a central laboratory for prompt detection of HIV vertical transmission. In northwest Tanzania, the DBS are transported to Bugando Medical Centre, a teaching and consultancy hospital in Mwanza, where they are tested for HIV and then stored on-site, and thus available for further testing. Isoelectric focusing (IEF) equipment was donated to Bugando Medical Centre along with reagents and supplies. Two laboratory staff were trained by a board certified hematologist, and then attended a two day seminar by the IEF manufacturer. One pediatrician completed a 2-month observership at Cincinnati Children's Hospital. All DBS samples were tested by IEF using appropriate controls. Completed gels were scored independently by two Tanzanian staff members as normal, disease, trait, variant, or uninterpretable. DBS samples scored as disease or variant were repeated for confirmation and preserved for later genotyping. Regular Skype calls were convened with US-based collaborators to improve quality and interpretation. HIV test results were obtained from the local EID program. Between February 2017 and May 2018, 232 IEF gels were completed by the local staff. After children >24 months of age were excluded to obtain a more accurate newborn prevalence, the median age of children tested was 52 days (IQR 41-93 days), and a total of 17,278 unique DBS samples were scored. The quality of laboratory testing was extremely high with only 20 samples scored as uninterpretable and 54 with missing results, and the primary analysis was performed on the 17,204 remaining samples. The overall prevalence of sickle cell trait and disease in the entire cohort was 20.3% and 1.2%, respectively, with a 0.1% prevalence of hemoglobin variants. This corresponds to an allelic frequency of 0.114 for the sickle gene mutation and demonstrates perfect Hardy-Weinberg equilibrium. No HbC or other common beta-globin variants were identified. Geospatial mapping revealed some variation across regions, with sickle trait ranging from 16.6% to 22.5% and disease ranging from 0.5% to 1.5%. Analysis of individual districts with >100 samples revealed wider geographic variability, with sickle trait ranging from 15.2% to 27.8% and disease ranging from 0.0% to 4.3%. Co-morbidity between HIV and sickle cell disease was analyzed to compare it with the effect on mortality previously observed in Uganda. The prevalence of sickle cell disease was the same among HIV-infected and HIV-negative children (1.2%), suggesting no difference in mortality. The prevalence of sickle cell trait and disease among infants born in northwest Tanzania is very high, exceeding 20% trait and 1.2% disease. All regions in the Lake Zone are affected possibly due to lack of immigration to the area and similar environmental exposures. Targeted newborn screening can be started in high prevalence districts, using existing public health infrastructure with minimal start-up cost and training. Future work will evaluate the correlation between historical malaria prevalence and sickle cell prevalence, and identify hemoglobin variants. Disclosures Ware: Addmedica: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Agios: Other: advisory board; Global Blood Therapeutics: Other: advisory board; Biomedomics: Research Funding; Nova Laboratories: Consultancy.

scholarly journals Sickle Cell Anemia Presenting with Vaso-Occlusive Pain: Should We Screen for COVID-19?

2021 ◽  
pp. 1-4
Author(s):  
Mohammad Ali ◽  
Lina Okar ◽  
Nabil E. Omar ◽  
Jabeed Parengal ◽  
Ashraf Soliman ◽  
...  
Keyword(s):  
High Risk ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Signs And Symptoms ◽  
Risk Groups ◽  
Position Statement ◽  
Interesting Case ◽  
International Federation ◽  
Cell Disease ◽  
The World

Despite the widespread of coronavirus disease-19 (CO­VID-19) infection around the world, there are very scarce reported literature about the care of patients with a known diagnosis of hemoglobin disorders such as sickle cell disease (SCD) or thalassemia and confirmed COVID-19 infection. Thalassemia International Federation issued a position statement to include patients with thalassemia and SCD among the high-risk groups of patients. Here, we present an interesting case of a 42-year-old patient know to have SCD presenting with Vaso-occlusive (VOC) pain episode in the absence of COVID-19 signs and symptoms, who tested positive for COVID-19 infection and had a smooth recovery. This case highlights the importance of screening SCD patients presenting with VOC-related events even in the absence of COVID-19 signs and symptoms.

scholarly journals Sickle cell disease pain management and the medical home

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 433-438 ◽  
Author(s):  
Jean L. Raphael ◽  
Suzette O. Oyeku
Keyword(s):  
Pain Management ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Medical Home ◽  
Cost Effective ◽  
Cell Disease ◽  
Patient Centered ◽  
Centered Care ◽  
The Individual ◽  
Effective Models

Pain is the most common cause for hospitalization and acute morbidity in sickle cell disease (SCD). The consequences of SCD-related pain are substantial, affecting both the individual and the health care system. The emergence of the patient-centered medical home (PCMH) provides new opportunities to align efforts to improve SCD management with innovative and potentially cost-effective models of patient-centered care. The Department of Health and Human Services has designated SCD as a priority area with emphasis on creating PCMHs for affected patients. The question for patients, clinicians, scientists, and policy-makers is how the PCMH can be designed to address pain, the hallmark feature of SCD. This article provides a framework of pain management within the PCMH model. We present an overview of pain and pain management in SCD, gaps in pain management, and current care models used by patients and discuss core PCMH concepts and multidisciplinary team–based PCMH care strategies for SCD pain management.

scholarly journals Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 208-208
Author(s):  
Ping Zhang ◽  
John D Belcher ◽  
Julia Nguyen ◽  
Fuad Abdulla ◽  
Gregory M Vercellotti
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Western Blot ◽  
Human Plasma ◽  
Research Funding ◽  
Heme Binding ◽  
Cell Disease ◽  
Inflammatory Signaling ◽  
Tlr4 Signaling ◽  
Potential Source

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p&lt;0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p&lt;0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p&lt;0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p&lt;0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p&lt;0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p&lt;0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p&lt;0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p&lt;0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p&lt;0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document