A No-Lysis Flow Cytometry Protocol to Characterize Heterotypic Aggregates with Red Blood Cells in Sickle Cell Disease
Authors: Butler Megan , Huq Muhammad , Schilke Robert , Woolard Matthew , Stokes Karen 
Published: May 2023

Sickle Cell Disease (SCD) is an inherited hemoglobinopathy characterized by a pro-inflammatory, pro-thrombotic phenotype. Platelet-leukocyte aggregates (PLAs), a hallmark of thrombo-inflammatory diseases, are elevated in SCD. While neutrophils play a role in aggregate formation, little is known about other leukocyte subpopulations. Additionally, SCD red blood cells (RBCs) can become adherent but evidence showing how they participate in aggregate formation is limited. The first aim of this study was to develop a flow cytometry protocol to test our hypothesis that red blood cell aggregate formation with different leukocyte subpopulations and/or platelets is elevated in SCD. The second aim of this study was to compare the PLA findings from our new no-lysis protocol with those from a standard lysis protocol.Systemic blood was collected from the tail vein of C57Bl/6J (WT) and Townes (SCD) mice (n=6-10/grp). Flow cytometry analysis was performed with lysed and non-lysed blood. PLAs were identified by double-positive staining for CD45+ (leukocytes) and CD41+ (platelets). Leukocyte subpopulations were identified as Ly6G+ (neutrophils-PNAs), CD115+ (monocytes-PMAs), or Ly6G-/CD115- (primarily lymphocytes-PLyAs). RBC aggregates were further identified as Ter119+. Student’s t-test was used for statistical analysis with significance set at p<0.05.Total platelet and leukocyte counts (including individual neutrophil, monocyte, and lymphocyte subpopulations) were significantly increased in SCD mice. Total PLAs were significantly higher in SCD versus WT mice with the increase being driven by the significant increases in PNAs and PMAs in the lysis protocol whereas in the no-lysis protocol it was driven by significant increases in PNAs and PLyAs. Overall numbers of circulating PLAs were about 5-fold higher for both WT and SCD mice in the lysis protocol compared to the no-lysis protocol. RBC-leukocyte-aggregates (RLAs), RBC-platelet-aggregates (RPAs), and RBC-platelet-leukocyte-aggregates (RPLAs) were all significantly increased more than two-fold in SCD mice compared to WT mice.Our findings of increased circulating PLAs and PNAs in SCD mice reflect what is seen in human SCD. The elevated numbers of PLAs in both WT and SCD mice from the lysis protocol compared with the no-lysis protocol suggests that the additional lysis and processing steps introduces an artifact of excessive aggregation, with the no-lysis protocol potentially being more representative. Furthermore, using our new no-lysis protocol we were able to detect increased numbers of all aggregates involving RBCs in SCD mice. We have characterized a model to further investigate the mechanisms underlying the systemic thrombo-inflammatory phenotype of SCD, and better understand RBC interactions with leukocyte subpopulations, platelets, and PLAs. Funded by T32HL155022 from the NIH NHLBI, awarded to A. Wayne Orr and Karen Y. Stokes, Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center Shreveport, and by 5R01HL134959 from the NIH NHLBI awarded to Karen Y. Stokes. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.