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Paraformaldehyde fixation induces a systematic activation of platelets
Volker Schmidt1, Thomas Hilberg, Gabriele Franke
1Department of Sports Medicine, Friedrich-Schiller-Universität Jena, Germany.
Platelets
|August 29, 2003
Summary
Paraformaldehyde (PFA) fixation in platelet assays can artificially stimulate platelets, altering activation marker detection. For accurate results, label platelets immediately after blood collection and analyze unfixed samples within 10 minutes.
Area of Science:
- Hematology
- Immunology
- Biochemistry
Background:
- Platelet activation assays are crucial for diagnosing and monitoring various hemostatic and thrombotic disorders.
- Paraformaldehyde (PFA) fixation is commonly used to stabilize platelets and prevent spontaneous activation during whole blood flow cytometric analysis.
- The precise impact of PFA fixation on platelet surface marker expression and activation state remains incompletely understood.
Purpose of the Study:
- To investigate the effects of paraformaldehyde (PFA) fixation on platelet surface marker expression in whole blood flow cytometry.
- To evaluate how PFA fixation influences the detection of different platelet activation epitopes, including CD62P, CD41, PAC-1, and CD42b.
- To determine optimal conditions for minimizing artifactual platelet activation during sample processing.
Main Methods:
- Direct immunofluorescence labeling and whole blood flow cytometry were employed.
- The study compared PFA fixation performed before and after antibody labeling.
- Mean fluorescence intensity (MFI) of four distinct platelet surface epitopes was measured in fixed versus unfixed samples.
Main Results:
- PFA fixation prior to antibody labeling significantly increased MFI for CD62P, CD41, and PAC-1, indicating artificial platelet stimulation.
- Conversely, PFA fixation decreased MFI for CD42b.
- Fixation after antibody labeling resulted in less pronounced changes, with significant increases in CD41 and PAC-1, and a moderate rise in CD42b.
Conclusions:
- Paraformaldehyde (PFA) induces systematic platelet stimulation, leading to altered detection of activation markers.
- The findings suggest that PFA fixation is not a neutral process and can introduce artifacts in platelet activation assays.
- Minimal in vitro platelet activation is achieved by immediate antibody labeling of unfixed samples and analysis within 10 minutes at 4°C in the dark.