Evaluating Stored Platelet Shape Change Using Imaging Flow Cytometry

Abstract

Platelets are routinely stored at room temperature for 5-7 days before transfusion. Stored platelet quality is traditionally assessed by Kunickis morphology score. This method requires extensive training, experience, and is highly subjective. Moreover, the number of laboratories familiar with this technique is decreasing. Cold storage of platelets has recently regained interest because of potential advantages such as reduced bacterial growth and preserved function. However, platelets exposed to cold temperatures change uniformly from a discoid to a spherical shape, reducing the morphology score outcomes to a binary "spheres versus discs" during cooling. We developed a simpler, unbiased screening tool to measure temperature-induced platelet shape change using imaging flow cytometry. When reduced to two dimensions, spheres appear circular, while discs are detected on a spectrum from fusiform to circular. We defined circular events as having a transverse axis of > 0.8 of the longitudinal axis and fusiform events [≤] 0.8 of the longitudinal axis. We show that most EGTA-treated platelets and spherical beads are detected in the gate for circular events, while fresh platelets are found mainly in the gate for fusiform events. Using this assay, mouse and human platelets show a temperature and time-dependent, two-dimensional shape change from fusiform to circular, consistent with their three-dimensional change from discs to spheres. The method we describe here is a valuable tool for detecting shape change differences in response to agonists or temperature and will help screen for therapeutic measures to mitigate the cold-induced storage lesion. Plain language summaryO_ST_ABSWhat is the context?C_ST_ABSO_LIPlatelets for transfusion are currently stored for 5-7 days at room temperature, increasing the risk for bacterial growth C_LIO_LICold storage reduces the risk for bacterial growth but reduces circulation time C_LIO_LIStored platelet quality can be assessed by the light microscopy-based Morphology Score, first described in the 1970s C_LIO_LIDownsides of the Morphology Score include subjectivity, extensive training, and reduced availability in platelet laboratories. C_LI What is new?O_LIIn this study, we provide data showing that the Morphology score is reduced to a binary spheres versus discs response in cold-exposed platelets C_LIO_LIWe developed an imaging flow cytometry-based approach to quantify platelets response to cold based on the two-dimensional projection of the three-dimensional shapes, i.e., fusiform (discoid) versus circular (discoid and spherical) C_LIO_LIWe provide validation of this approach in mouse and human platelets C_LIO_LIWe provide evidence for increased temperature sensitivity in mouse platelets C_LI What is the impact?O_LIThis study provides an easy and unbiased tool for laboratories working on circumventing the cold-induced storage lesion or documenting spherical shape change in general C_LI