Platelet Depletion Alters MCP-1 Measurements in Plasma From Children With Dengue.

Abstract

Background: Platelets are increasingly recognized as contributors to the cytokine storm in dengue, yet their residual presence in plasma may bias cytokine measurements. We compared MCP-1 levels between paired regular plasma (RP) and platelet-poor plasma (PPP) in children with dengue and evaluated how depletion influences cytokine quantification.

Methods: We first conducted a systematic review of plasma isolation methods in pediatric dengue studies. We then collected paired RP and PPP from 16 hospitalized children with dengue (11 with warning signs and 5 with severe dengue). The PPP was obtained by double centrifugation and verified to contain < 10,000 platelets/μL. Absolute platelet counts were determined by flow cytometry using an antihuman CD41 antibody and bead-based quantification. After one freeze-thaw cycle, MCP-1 and IFN-γ (negative control for platelet secretion) were measured by cytometric bead array.

Results: Among 167 reviewed articles, only 40% reported plasma isolation methods and 5% performed platelet depletion, with wide variability in centrifugation protocols. In our cohort, 63% of RP samples exceeded the platelet-poor threshold compared with only 10% of PPP after double-spin depletion. After effective platelet removal, MCP-1 levels increased nearly 5-fold in PPP versus RP (p = 0.008), while IFN-γ showed only a very modest rise, indicating that platelets are highly susceptible to physical and mechanical factors during the depletion process.

Conclusion: Standard plasma isolation fails to consistently remove platelets, and subsequent depletion protocols can artifactually elevate the platelet-associated cytokines MCP-1. These findings underscore the need for standardized reporting of platelet depletion methods in dengue immunology research.