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

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.