Methods : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [3]
Imaging by DHM (HoloMonitor M4; Phase Holographic Imaging AB, Lund, Sweden) was performed as described elsewhere [12]. The data were analyzed using specialized software, HoloStudio M4 (Phase Holographic Imaging AB). For surface roughness and area analysis, after a series of images were captured, the grayscale images were converted to the black-and-white format by the Otsu method, and the cell identification and segmentation in the images were adjusted either automatically or manually.
Based on the accumulated data on the cell refractive index, the average refractive index for cultured monolayer cells was fixed at 1.38 (Phase Holographic Imaging AB, personal communication). This value was applied to platelets. The refractive index of the surrounding medium is 1.34 and should not excessively deviate (± 0.08) from the cell refractive index.
On the basis of our preliminary data, we focused on the parameters related to the cell area and roughness and examined at least 4000–8000 platelets in each platelet population.
This parameter was determined by means of a Coaguchek XS system (Roche Diagnostics International Ltd., Basel, Switzerland). For citrated samples, 500 μL of whole-blood samples was pre-warmed at 37 °C for 5 min, mixed well with 10 μL of 10% CaCl2 by gentle inverting, and incubated for 5 min prior to the analysis.
Next, PRP fractions were centrifuged at 1060×g for 5 min to fractionate platelet-poor plasma (PPP). PRP or PPP (1.5 mL) was mixed with 10% CaCl2 in the ratio mentioned for whole-blood samples on a watch glass or in a plastic dish. To activate platelets, small cut pieces of a collagen sponge composed of collagen microfibers (Integran®; Koken, Tokyo, Japan) were added to PRP in the plastic dish. Time for complete clot formation was determined.
The data were expressed as mean ± standard deviation (SD). For multigroup comparisons, statistical analyses were conducted to compare the mean values by one-way analysis of variance (ANOVA), followed by Dunn’s multiple-comparison test (SigmaPlot 12.5; Systat Software, Inc., San Jose, CA, USA). Differences with P values of < 0.05 were considered statistically significant.
Serial posts:
- Abstract : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- Background : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- Background : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- Methods : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- Methods : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- Methods : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [3]
- Results : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- Results : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- Discussion : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- Discussion : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- Discussion : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [3]
- Conclusions : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- Abbreviations : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- References : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- References : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- References : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [3]
- Acknowledgements : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- Author information : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [1]
- Author information : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma [2]
- Ethics declarations : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- Rights and permissions : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma
- About this article : Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma