I am researching the optimum centrifuge speed/time when preparing PRP for immediate autologous PRP reinjection, and have found an unexpected increase in the supernatant plasma platelet concentration beyond that which is theoretically possible.
Using citrate as the anticoagulant and a spin of 150g for 10 mins, the RBC layer will have a proportion of the plasma mixed with it because the RBC packing is not complete. The residual plasma volume reduces as the centrifuge speed/time increases. I have measured that there is no significant concentration gradient of platelets within the supernatant plasma at lower speeds below 200g, so on first principles I would expect the platelet concentration to be the same in both the supernatant plasma and the plasma within the RBC fraction. However I have consistently noticed that the platelet concentration in the supernatant plasma at 150g for 10 mins gave a significantly higher concentration than was theoretically possible.
I then measured the platelet concentration within the RBC layer and from this calculated the concentration within the plasma fraction within the RBC layer and found that this was consistently depleted to 15-30% of the supernatant platelet concentration. This suggests that the platelets within the RBC layer are selectively being transferred to the supernatant plasma. One possible explanation for this might be the RBCs forming rouleaux as they are centrifuged down, thus displacing the platelets upwards.
I cannot find any reference to this effect in the literature.
Has anybody noticed this phenomenon before and does anybody have any theories to account for it?
Please read extra notes added below on 16/5/15
I have observed this phenomenon with murine prp and you are right. The normal protocol mentions using a centrifugation of 100g, but I was never satisfied with these results. At that time, I was working on murine PRP and considering the low quantity of Whole Blood derived from a single mouse, it was getting increasingly difficult to perform my studies, until I decided to experiment using different centrifugal accelerations and noticed that a centrifugal acceleration of 150g for 10 minutes yielded the highest concentration of PRP. I would assume it has probably some role to play with the physics and concentration gradients, however, I am not too sure about it.
I think the problem may be with the method used to count platelets. If you are using an automated hematology analyzer to cunt platelets in PRP, that could be the culprit. We use a particle counter to avoid the issues with the cell counters.
I agree with Trolio. Perhaps the analyzer counts fragments as platelets.
Thank you for the comments which are very valid. Indeed I had assumed initially that the results were an artefact. However I don’t think that platelet fragments are the explanation. My initial introduction was of necessity brief, so here are some more details of our results and experimental technique
Blood from 4 donors into 10ml tubes has been spun multiple times at 110g , 141g and 174g for 10 mins. Heights of the plasma and the RBC layer were recorded for later calculation of volumes. After centrifugation serial 1ml samples of the supernatant plasma were taken down to the buffy layer. The buffy layer was then taken along with the remainder of the supernatant plasma and the topmost element of the RBC layer to a volume of approx 1 ml so that the buffy layer did not contaminate the RBC layer measurements. All samples (up to 7 in number) were then passed through a Coulter Counter
The results show that at all speeds no effective concentration gradient of platelets in the supernatant layer was created. They also show that there is no significant change in the Platelet Distribution Width or the Mean Platelet Volume between the different samples and these are in line with an uncentrifuged citrated sample.
On a theoretical level I would not expect there to be high levels of platelet activation within the supernatant layer and indeed the measurements of the platelet parameters in the Buffy Layer and the RBC layer also suggest that there has not been any significant activation, where the shear forces during centrifugation will have the most effect.
Calculating the total number of platelets in the supernatant RBC layers was simple but could only be estimated in the Buffy Layer because the volume measurement was only approximate. However on averaging all the results it does seem to indicate that the total platelets in all layers of a centrifuged sample is in line with the total platelet numbers in an uncentrifuged citrated sample. I accept that experimental error makes this difficult to interpret and will be repeating the experiment to measure accurately the volume of the sample containing the buffy layer
Further comments please!!
In our experience with murine platelets 10 min centrifugation is too long, we got good PRP with 150g for 3-5 min. But, it could be because of low volume of (~1-1.5ml) of blood.
Preeti: could you send me your complete protocol for mice? we have a number of labs working with murine models, and your suggestion may help them. TNX. Bill
Working on human platelet aggregation I have observed this phenomenoni very frequently. My personal idea is that plasma viscosity and protein play a role. This idea become from the fact that to obtain a good murine PRP for my poupose, I simple dilute whole blood 1:1 with PBS before centrifugationand at 150g for 10 min in 1.5 ml tube.
dear chris,
questions: is your anticoagulation correct?
i work with 10% ACD-A determined to the total Bloodvolume/sample
a rest period of 2 hours before zentrifugation is cecommendable
the platelets need time fore desaggregation after collection
after collection the platelet are stressed and slope to clottadiucation
then you can try a centrifugation run by 380g, 15 min with a very slow acceleration and a mild deceleration
that brought that RBC sediments first complete before the platelets starts to the bottom
i have to admit though that my experiences come from work with 150ml-plastic bags
sorry fore my bad english
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue