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Published on: May 16, 2013
Platelet Depletion Alters MCP-1 Measurements in Plasma From Children With Dengue
Héctor Felipe Ocampo-Amariles1, Sebastián Castro-Trujillo1, Carlos F Narváez1,2
1Medical Program, Faculty of Health Sciences, Universidad Surcolombiana, Neiva, Huila, Colombia, usco.edu.co.
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.
Insights
Residual platelets in plasma can skew dengue cytokine measurements. Effective platelet depletion significantly increases MCP-1 levels, highlighting the need for standardized methods in dengue research.
Area of Science:
- Immunology
- Hematology
- Virology
Background:
- Platelets are recognized contributors to the cytokine storm in dengue.
- Residual platelets in plasma can lead to biased cytokine measurements.
- Current plasma isolation methods in pediatric dengue studies lack standardization.
Purpose of the Study:
- To compare Monocyte Chemoattractant Protein-1 (MCP-1) levels in regular plasma (RP) and platelet-poor plasma (PPP) in children with dengue.
- To evaluate the influence of platelet depletion on cytokine quantification in dengue.
- To assess the variability in plasma isolation and platelet depletion methods in pediatric dengue research.
Main Methods:
- Systematic review of plasma isolation methods in pediatric dengue studies.
- Collection of paired RP and PPP from 16 hospitalized children with dengue.
- Platelet quantification using flow cytometry and cytokine measurement via cytometric bead array.
Main Results:
- Only 40% of reviewed articles reported plasma isolation methods, and 5% performed platelet depletion.
- Effective platelet depletion (verified by flow cytometry) was achieved in 90% of PPP samples.
- MCP-1 levels were nearly 5-fold higher in PPP compared to RP (p=0.008), while IFN-γ showed minimal change.
Conclusions:
- Standard plasma isolation methods do not consistently remove platelets.
- Platelet depletion protocols can artifactually elevate platelet-associated cytokines like MCP-1.
- Standardized reporting of platelet depletion methods is crucial for dengue immunology research.
