The World Health Organization has recently recognized sickle cell disease as a major global health concern. The highest rates of sickle cell disease and sickle cell trait are seen in malaria-endemic developing countries, which also have the most severe resource constraints, especially on clinical laboratory infrastructure. Daktari Diagnostics, a 50-person, venture-backed start-up company focused on the pressing needs for innovative global health technologies is partnering with the Whitesides laboratory at Harvard University and the University Teaching Hospital in Lusaka, Zambia, to develop a novel polymer system into a low-cost, rapid, and simple-to-use assay for the screening and diagnosis of sickle cell disease in resource-limited settings. The sickle cell assay-named Mpana, an acronym for MultiPhase ANAlyzer, and also a Swahili word meaning "a broad, wide, open channel"-is based on self-assembling polymer technology developed by the Whitesides group for industrial and medical applications, and recently shown in preliminary studies in Zambia to be useful for the identification of sickle cells in blood with high sensitivity and high specificity. Moreover, by design these polymer systems are robust and have strong material properties that immediately suggest a path to a commercial product that can meet the difficult constraints necessary for success in resource-limited settings- long shelf-life in environmental extremes, minimal power requirements enabling battery operation, simplicity of use, and, importantly, low cost. The Mpana project described here will transition the promising prototype from the academic setting into a commercial manufacturing environment. For the operator, the final Mpana assay will involve loading capillary blood from a heel stick or finger prick into a small microhematocrit tube pre-filled with multiphase polymers that form step-gradients based on density-and inserting the loaded tube into a small battery-operated centrifugation instrument with a simple user interface. Inside the tube, under centrifugation, dense sickle cells settle to a specific location in the densty gradient, forming a band that can be easily read visually or optically. After approximately 12 minutes, the Mpana system will report out the presence of sickle cell disease, sickle cell trait, o a normal red blood cell profile, and will differentiate HbSS from HbSC disease. Results will be reported either automatically, or by a visual readout, and will be quality controlled through QC reagents included within the microhematocrit tube. To develop the final Mpana assay, we will optimize the current multi-phase polymers developed by the Whiteside group for stability and manufacturability. We will also develop a robust anti-coagulation tube coating and final tube material that will enable easy blood collection for the operator, as well as improve the sensitivit and specificity of the system through the elimination of RBC and platelet aggregation, which can confound the density-based assay performance. In addition, we will develop control microbeads of a fixed, narrow density as on-board QC material, used to confirm the stability of the density gradient. On the instrument side, we will convert what has been an expensive, table-top centrifuge used in the preliminary Harvard studies in Zambia, into a low-cost, portable, battery-operated centrifuge system, a core Daktari expertise for developing simple instrumentation that has been applied previously by the Daktari engineering team to the development of a handheld, battery-operated CD4 system. As a final set of preliminary studies, we will evaluate the performance of the commercial prototype in the context of difficult sample specimens, including specimens containing interfering substances, severe anemia, and a variety of hemoglobinopathies, first in de-identified samples obtained from the Medical University of South Carolina, and finally in an initial pilot study at the University Teaching Hospital in Zambia. We expect the final deliverable from this two-year Phase I SBIR program will be a simple, low-cost instrument and assay tube capable of being used at the primary care level to rapidly and inexpensively identify children with sickle cell disease and sickle trait, at a level of accuracy demonstrably matching or exceeding the expectations of the RFA.
Sickle cell disease has been transformed in the United States and developed countries, due to neonatal screening and effective interventions. However, more than 200,000 children born with sickle cell disease in Africa each year will die in infancy or earl childhood, due to the painful and debilitating consequences of undiagnosed sickle cell disease-infections, respiratory distress, and sickle cell crisis. Most sickle cell- endemic developing countries also have the most severe resource constraints. Current methods to diagnose sickle cell disease, which rely on complex laboratory methods, cannot be used in clinics in rural Africa;as a result, hundreds of thousands of children are born each year with sickle cell disease, go undiagnosed, and do not receive effective interventions-like penicillin-until it is too late. In ths proposal, we will develop a simple, rapid, accurate, inexpensive test for sickle cell disease, the Daktari Mpana assay, which can enable millions of children worldwide to be screened for sickle cell disease, and to be then reached with simple and effective interventions to help them survive this difficult condition. (End of Abstract)