Hello,
I am currently working on a project of comparing blood samples of children with ADHD and ASD. We plan to do the following:
1. receive blood from the hospital of control blood, ADHD blood, and ASD blood
2. receive another batch of control blood and ASD blood from another lab co-working with us
3. centrifuge to separate RBC and serum
4. deplete using 14 protein depletion spin column
5. digest
6. mass spec analysis
Here's the problem, usually the blood we receive from the university hospital is in the blood collection tubes (IDK what they're called) and we centrifuge on the same day and aliquot the serum in eppendorf tubes and freeze them (-20C), but the lab we got them from, they gave us blood in eppendorf tubes in the frozen state, without separating the serum, which I heard is bad for a number of reasons but I heard RBCs can break and contaminate into the serum which can interfere with the identification of low-abundance proteins (Where we can likely find our markers)
Two questions:
1. is there a precedent where the researchers used frozen whole blood for serum biomarker discovery proteomics? How can this situation be fixed (besides getting new samples)?
2. (For my personal curiosity) what other detriments are there when performing serum proteomics analysis with frozen whole blood?
I appreciate it guys!
Andrew
Hello Andrew Kim
It would be better to discard the frozen blood sample.
Freezing blood is usually not recommended. Blood contains on an average approximately 50% water. On freezing, the water will cause ice crystals to form, which will subsequently damage the blood cells. Red blood cell lysis will cause spilling of the cells’ contents contributing extraneous protein to plasma and serum samples thereby interfering with biomarker detection and validation. RBC is mainly comprised of hemoglobin, catalase, peroxiredoxin and carbonic anhydrase-1.
Proteins in serum is less stable when contaminated with contents of lysed RBCs suggesting that the proteases present in contaminated serum may induce protein degradation. Thus, contamination of serum could potentially impact the observed proteome.
Best.
Hello! Od course the best option is to work with not frozen blood sample, but in our lab we used ID-CellStab (from Bio-Rad) for blood frozen in -80 degree C. It is not ideal (partial lysis of the blood cells), but good option when the sample must be storage.
Krzysztof Mikolajczyk I will definitely keep that in mind for future studies I do with serum! Thanks!
question: do you treat this buffer then to the blood before they freeze? or are you talking about blood that is already frozen (without the cellstab buffer)? If it is the latter case, do you treat the buffer on frozen blood and let it thaw?
Thanks again!
You should do it according to manufacturer instruction. You should wash the blood sample with this buffer and then frozen it at proper concentration.
Good luck!
Krzysztof Mikolajczyk oooh so this buffer is used to "wash off" RBCs or any RBC lysates... Gotcha! thanks!
Malcolm Nobre thank you always for the thorough explanation! Always helpful!
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