163 - Developmental differences in platelet phenotypes defined using spectral flow cytometry

Publication Number: 2157.163

Andrew L.. Frelinger III, Boston Children's Hospital, Boston, MA, United States; Benjamin Spurgeon, Versiti, Milwaukee, WI, United States; Erin Soule-Albridge, Boston Children's Hospital, Boston, MA, United States; Jacqueline Perry, Boston Children's Hospital, Westbrook, ME, United States; Martha Sola-Visner, Harvard Medical School, Boston, MA, United States; Patricia Davenport, Boston Children's Hospital, Boston, MA, United States

Background: Thrombocytopenic neonates often receive platelet (PLT) transfusions to prevent bleeding. However, a randomized trial found higher morbidity/mortality in neonates transfused at a higher vs. lower threshold. The mechanisms underlying this harm may be due in part to functional differences between the transfused (adult) and the recipient's (neonatal) PLTs. Recently, PLT subpopulations with different functional specifications (proinflammatory, proaggregatory and procoagulant) have been identified in adults.

Objective: To compare the functional profile of PLTs derived from cord blood (CB) vs adult blood (AB).

Design/Methods: CB and AB were collected from full-term cesarean sections and adult volunteers, respectively. CB and AB PLTs, resting or stimulated with various agonists (ADP, TRAP, CRP, and rhodocytin), were stained for 15 surface markers (PAC-1, CD61, CD42a, CD29, CD31, CD32, CD62P, CD63, CD107a, CD154, annexin V, TLR9, TLT-1, CD36 and GPVI) and analyzed by spectral flow cytometry. The machine-learning algorithm FAUST was used for unbiased identification of functionally distinct PLT subpopulations.

Results: CB (n=13, 7 male) and AB (n=13, 4 male) did not differ with respect to PLT count, immature PLT fraction, or MPV. At baseline, only CD42a and CD32 expression differed between CB and AB PLTs (1.7-fold higher on CB platelets). In all PLTs, increasing agonist concentrations led to dose-dependent increases in PAC-1, CD62P, CD63, CD107a, CD154, TLT-1 and TLR9, but the EC50s for TRAP- and CRP-induced expression were 3- and 9.2-fold higher for CB vs AB PLTs, while that for ADP was similar - suggesting that the developmental differences in activation are agonist, concentration, and pathway dependent. With maximal activation, CB PLTs had equal CD63, CD107a, CD154, TLT-1 and TLR9 expression levels, but persistently lower PAC-1 and CD62P compared to AB PLTs. FAUST analysis identified 12 PLT subpopulations based on the expression patterns of annexin V, PAC-1, CD62P, CD107a, CD63 and TLT-1. Developmental differences in subpopulations were observed, with CB PLTs having a greater abundance of a proinflammatory population (FAUST07) while AB PLTs had a greater abundance of proaggregatory/proinflammatory platelets (FAUST10) (Fig.1).

Conclusion(s): Spectral flow cytometry and FAUST analysis provided new insights into neonatal PLT function and identified developmental differences in PLT subpopulations. How these differences impact the neonatal responses to PLT transfusions is under investigation.

Fig. 1. Platelet subpopulations
FAUST analysis of PLT populations from washed PLTs isolated from fresh cord blood (CB) or adult blood (AB) (n=13 per group).