Hi Deblina, are you using blood collected  in EDTA or heparin/EDTA tubes?

If there is heparin in the blood collection tubes then added Ca++ will have little effect.

If there is no heparin, transfer the blood with the Ca++ to a non-treated glass tube and let it sit for ~30min at RT. If there is heparin, add 0.1% protamine (final conc.)

Hi Steingrimur , Thank you so much for your reply. I am getting the blood in EDTA tubes. I am not sure about heparin. I will figure it  out. I do not have much knowledge in this field. So could you please tell me the name of the chemical(e.g. CaCl2 or CaCO3 etc) with the quantity (e.g. x mol/l or y gm/l)? As I have mentioned that the quantity of blood I want to clot is 1 ml to 1.5 ml. 

Thanks so much for your help......

Hi Deblina, was the blood originally collected into EDTA vacutainer tubes? When filled with blood, these tubes should have ~ 5mM EDTA.

1 EDTA molecule binds 2 Ca++.

The Ca++ conc. in blood is ~2 mM.  So if you add CaCl2 to blood make it 50mM, then that should swamp out the EDTA and activate the intrinsic coagulation pathway.

That being said, are you working with 1-1.5 ml aliquots of blood from a filled EDTA vacutainer? Or did you collect 1-1.5 ml of blood into a vacutainer that was designed to hold more volume of blood?

If you are collecting ~1 ml of blood into EDTA vacutainers designed to hold greater volumes, then that will increase the conc. of EDTA in your sample correspondingly.

Hi Steingrimur,

Thanks for your reply. The sample do not contain heparin. 3 ml blood is collected in EDTA tube which contains 5.4 mg of K2 EDTA. I am using 1 to 1.5 ml of that EDTA blood sample. What is the amount of CaCl2 I should use?

Thanks for your help.....

Deblina

Hi Deblina,

Thanks for your question, I wonder did you get the clot from that blood? I have the same question with you.

Same question here. I did some study and it is ok to create blood clot using 25mM CaCl2 (final concentration) in case of Citrate treated vacucontainer tube. Good to know how to prepare in EDTA tube.

I agree with Steingrimur Stefansson , the blood clots by the method Steingrimur has mentioned. I recently tried the same and got positive results thanks for sharing.