I'm using dried blood spots (whole blood, not separated) to measure some markers of inflammation eg CRP. I want to compare the results from the dried blood spots to frozen plasma.
The problem is this: I add 10ul of blood to filter paper and let it dry etc. Then puch out a 3mm circle of the dried blood for analysis. This 3mm circle is extracted in 50ul of extraction buffer and then assayed. However, I don't know how much blood I have actually extracted from the dried blood spot and it has also been diluted in extraction buffer.
I want to compare the results to a known volume of frozen plasma. So how do I do this?
I was thinking of adding an internal standard to the sample before adding the blood to filter paper, that way I will know how much I have extracted as I can measure the amount of internal standard.
What could I use for the internal standard? It must be cheap, stable at room temperature and easy/cheap to measure. It must also not react with the blood.
I was thinking maybe a protein from another species e.g. horse myoblobin (could measure by ELISA although this is expensive) or a stable dye that can be measured using a spectrophotometer (we don't have access to mass spec).
any thoughts or suggestions would be greatly appreciated!