Purpose: Severe thrombocytopenia frequently occurs after xenotransplantation of porcine cells or organs, and when pig organs are perfused with human blood. We developed a flow cytometry-based technique to accurately quantify platelets and to distinguish them from similarly-sized particles found in blood during cross-species organ perfusion studies.

Methods: Three pairs (n=6) of wild type pig lungs were perfused with heparinized human whole blood. Blood perfusate was left untreated (n=3) or treated with a thromboxane synthase inhibitor and histamine receptor blocker (n=3). Platelet counts measured by an Heska (Hematru) traditional hemocytometer (HC), and a refined flow cytometric method (FC) using calibrated beads. FC results analyzed by FlowJo were compared with HC results. Imaging flow cytometry using Image StreamX Mark II imaging flow cytometry (Amnis Corporation) was employed to visualize and identify blood elements.

Results:  Platelet counts measured by FC showed a decreasing trend over time while platelet counts by HC showed an increasing trend and large variations within 4hrs hour of lung perfusion (Figure 1). FC analysis revealed an increasing population of “platelet-sized events” which were negative for human platelet markers (CD41, CD61). Using human leukocyte (CD45), erythrocyte (CD235a), and pig-specific (CD41, CD45, CD31) antibodies a majority of platelet-sized CD41-negative events express the CD235a red blood cell marker (Figure 2A and 2B). Platelets, intact RBC and platelet-sized RBC fragments were distinguishable by AMNIS (Figure 2C).

Conclusions:  Flow cytometric counting improves the accuracy of platelet enumeration. AMNIS image analysis reveals that red blood cell fragments are falsely detected as platelets by hemocytometers, causing overestimation of platelet counts by that method.