A Flow cytometer-based platelet aggregation assay permits platelet function testing in small blood volumes
S. Liedtke, J. Wesche, K. Althaus, T. Thiele, A. Greinacher (Greifswald, Germany)
Time: 17:15 - 18:15
Objective: Platelet aggregation assays require high volumes of platelet rich plasma or whole blood. Especially for pediatric patients, platelet function testing requiring small sample volumes is desirable. A low volume flow cytometer-based aggregation assay was introduced by De Cuyper et al. (2013). We compared this flow cytometer-based aggregation assay with light transmission aggregometry (LTA).
Methods: 200µL citrated whole blood was aliquoted into two 100µL tubes. Platelets were stained with FITC- or PE- conjugated CD31, respectively. Samples were washed twice, resuspended in 100µL hirudinized buffer (PBS with Ca+Mg, containing 20% normal plasma) and mixed. Anti P-selectin antibodies (CD62P) were added and 20 µL of each mix were incubated with increasing concentrations of ADP (0.078-40 µM), TRAP (1-40 µM), arachidonic acid (0.375-5 mM), epinephrine (0.04-20 µM), or collagen (1-10 µg/mL). Platelet activation was stopped using 0.5% paraformaldehyde after 2, 4, 6, 8, 10, 12 and 20 min. FITC- and PE- double positive events, platelet-counts and CD62P expression were analyzed by flow cytometry. LTA was performed with platelet rich plasma.
Results: Maximal aggregation and activation signals were observed after 2 min. Optimal agonist concentrations were for ADP: 20 µM; arachidonic acid: 0.75 mM; epinephrine: 2.5 µM; collagen: 5 µg/ml; TRAP: 20 µM. Platelets counts were decreased from 1500±300 (PBS) to 230±33 (ADP), to 440±250 (arachidonic acid), to 450±150 (epinephrine), to 173±42 (collagen), to 170±33 (TRAP). Aggregates (double stained events) increased from 5±2 (PBS) to 20.4±6.5 (ADP), to 20.3±4.6 (arachidonic acid), to 18.5±2.7 (epinephrine), to 19.2±6 (collagen), or to 16.25±7.2 (TRAP). CD62P mean fluorescence intensity increased from 5±2.8 (PBS) to 16.9±10.2 (ADP), to 19.8±12.4 (arachidonic acid), to 11.9±8.2 (epinephrine), to 32±21,5 (collagen), to 29±20 (TRAP). Patients with impaired platelet function in LTA were assessed by flow cytometry. Both tests correlated in 0/10 (TRAP), 2/7 (ADP), 1/9 (arachidonic acid), 4/11 (collagen) and 5/12 (epinephrine) cases so far.
Conclusion: The most sensitive agonists were ADP, epinephrine and collagen, but the flow cytometer-based assay is not directly comparable with the LTA. However, it allows small volume analysis of functional aggregation and could be used to interpret the potential of different agonists or stimuli within this assay.