Platelet Detection in Unaltered Whole Blood

Abstract

Abstract Platelets are non-nucleated cellular fragments circulating in the peripheral blood, derived from megakaryocytes. They are critical in the maintenance of hemostasis. Alterations in their function are associated with a range of clinical conditions. Using flow cytometry, light scatter signals from platelets typically overlap signals from red blood cells (RBC). Since RBC outnumber platelets by more than an order of magnitude in whole blood, platelets are typically studied by depleting samples of RBCs by centrifugation methods or selective RBC cell lysis. These sample preparation procedures require manipulations that may affect platelet health and function, and result in loss of cells. Additionally, lysed samples require labeling of the platelets with a fluorescent marker to adequately resolve the platelets from debris/noise. We present a novel technique for resolving leukocytes and platelets from RBC in whole blood using orthogonal light scatter from 2 lasers. This method exploits the differences in light-scattering properties between RBCs and platelets and white blood cells (WBC). Hemoglobin readily absorbs 405 nm laser light, reducing the scatter signal from RBC relative to WBCs and platelets, resulting in a reproducible scatter pattern when analyzing human whole blood with both blue (488 nm) and violet (405 nm) side scatter. Using this approach, a distinct platelet population can be discerned in unaltered whole blood, requiring only dilution of whole blood in buffer. Combining this approach with acoustic cytometry allows great flexibility in determining optimal dilution and sample collection rates for minimal coincidence and rapid analysis. For research use only. Not for use in diagnostic procedures.