Platelet function testing at low platelet counts: When can you trust your analysis?

Area of Science:

• Hematology
• Clinical Chemistry
• Biomedical Engineering

Background:

• Thrombocytopenia necessitates reliable platelet function testing (PFT).
• Flow cytometry-based PFT (FC-PFT) is often preferred over aggregometry in low platelet count settings.
• The impact of low platelet counts on FC-PFT versus light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) remains underreported.

Purpose of the Study:

• To compare the effects of varying sample platelet counts (10, 50, 100, 200 × 10^9 L⁻¹) on platelet activation.
• To evaluate FC-PFT, LTA, and MEA under standardized conditions using identical anticoagulant and agonist concentrations.
• To determine the influence of platelet count on PFT results using ADP and PAR1-AP agonists.

Main Methods:

• Platelet activation was assessed using FC-PFT, LTA, and MEA.
• Platelets were stimulated with adenosine diphosphate (ADP) and protease-activated receptor-1 activating peptide (PAR1-AP).
• Specific platelet counts were achieved by adjusting ratios of platelet-rich and platelet-poor plasma, with or without red blood cells.

Main Results:

• FC-PFT showed reduced P-selectin exposure and PAC-1 binding at 10 × 10^9 L⁻¹ with PAR1-AP, but not with ADP.
• Aggregometry-based PFTs (LTA and MEA) demonstrated significant reductions (>50% at 50 × 10^9 L⁻¹ and >80% at 10 × 10^9 L⁻¹), irrespective of the agonist.
• Platelet count-dependent effects in FC-PFT with PAR1-AP were mitigated by apyrase, suggesting purinergic signaling as a key factor.

Conclusions:

• FC-PFT is generally more robust than aggregometry at low platelet counts.
• Optimization of experimental parameters is crucial for FC-PFT to mitigate platelet count-related variability.
• Standardized methods are needed for accurate platelet function assessment in thrombocytopenic patients.