While both anticoagulants work by chelating calcium, EDTA holds on to the calcium in a much stronger fashion.  Out of curiosity was your citrate buffer prepared at 10x?  If you are unable to use the standard blue top tubes for blood collection then you should ensure your citrate buffer be prepared fresh at 10x and filtered to ensure there are no particulate matter that could possibly activate platelets in the blood sample.  If you satisfy all those requirements and your sample is still clotting it may be spending to much time between the blood draw and addition of the citrate.  This is often the case for small samples obtained from mice/rats/rabbits.  If function testing is not required in these samples you could also use heparin.

Hi Jason Newton, Hopefully no error was made during blood collection (human blood), blood collected by a professional in a local clinic, no transportation issues too. I used 1X buffer, and it was freshly prepared. No, I didn't filtered the buffer, since no precipitate was formed during the buffer preparation. Does it needed to be filtered even when no precipitates were formed?! Is it because I used 1X? (My reference material has quoted for 1X buffer only!) 

Hi Niranjan,

if you are unable to get the blood drawn straight into vacutainers (optimum) then from my understanding you should generally use the same ratio.  Blue top tubes have 1mL 3.2% (some labs in the US mostly stick to the older concentration of 3.8%)citrate in them in the form of ACD or Na-Citrate with around 2.3% of that being in the form of free citrate ion.  That is 10x as it sits in the tube. Your ratio is right (1:10), and if your using a 3.8 or 3.2 % (10x) solution  then particulate matter would be my guess.  This puts the 1x working solution at .38 or .32 % respectively.  One last troubleshooting question- the blood was drawn by the clinic and added immediately to the tubes you prepared?  Even at times less than 20 seconds clotting can begin, and the calcium regulated enzymes you are trying to prevent from getting activated will already have enough free Ca to get kicked off.  Is budgeting an issue?  If you have the funds I suggest using commercially available collection tubes if possible.

https://www.bd.com/vacutainer/products/venous/ordering_info_tubes.asp#coag_citrate_glass

Jason, that explains so well. thank you. ya, this is kinda mini project. Immediate blood transfer was also ensured. we finished the experiment with EDTA. Initially We started with both EDTA and citrate buffer, with EDTA no clotting was seen, but with citrate there was thread like clotting. So as you said I think filtration might solve the problem. I'll try and get back. thank you for the info. 

EDTA chelates also several bivalen metal ions that are involved in blood coagulation, for example as part of the FV molecule (Mn) oder in the contact phase (Zn).

Hello Hans,

While EDTA does chelate magnesium (Mg) the effects Mg have on coagulation are relative to it's action as a cofactor to Vitamin K, as the proteases vitamin K controls would already be generated when the blood is drawn into the tube this most likely would only be an issue with in vivo administration and ex vivo it will be less of a problem.  And there is mounting evidence that Zn++ secreted by platelets during activation can act as a cofactor instead of Ca++ both EDTA as well as citrate (--) can handle it.  The issue with EDTA is that most of your classical and functional coagulation tests (PT, aPTT, TEG, ROTEM, ROTEG, Hemodyne, CAT, and platelet aggregometry) cannot be performed in samples collected in EDTA- the chelation effect is too strong to overcome with simple recalcification.  

As Hans and Jason mention the problem is that EDTA has a higher affinity for calcium than citrate. Thus EDTA can bind calcium-ions in the coagulation factors like FV and FVIII leaving them non-functional (which is not restored by recalcification). Citrate has a lower affinity for calcium and therefore only chelates the calcium in solution, which can be restored by recalcification. So for coagulation test you should trust the results taken in citrate 3.2 or 3.8%. 

Ethylenediaminetetraacetic disodium, dipotassium salt. It acts by a chelating effect on calcium (Ca ++) preventing blood clotting. It is used for: cell counts (red cells, white cells and platelets), hematocrit, hemoglobin concentration ([Hb]), blood smear, etc.Secuestra calcium and separates the coagulation cascade, preventing blood from clotting. Given the small amount of solution, drying it is not necessary, since virtually no thins the blood to be analyzed; excess adversely affects both erythrocytes and leukocytes, causing it to shrink and

causing changes in the way; therefore care should be taken to add the

right amount of blood to anticoagulant. salt is preferred

disodium dipotassium to since it is more soluble (10 times) and this

makes effective mixing of anticoagulant with blood.,

Tripotassium EDTA is dispensed as a liquid and THUS causes a slight dilution of the specimen. This salt has-been shown to Present Affect the red blood cell size and more at Increased Concentrations on storage than the dipotassium salt. THEREFORE, dipo-tassium EDTA is recommended as the anticoagulant of choice in specimen collection for blood cell counting and sizing. The amount of dipotassium EDTA used is 1.5-2.2 mg (3.7-5.4 pmol) per milliliter of bood, Avoids the deleterious effects of ethylenediaminotetraacetic acid (EDTA) on mean platelet volume and Present Prevents EDTA-induced platelet clumping. It is suitable for routine cell counting. The values of the common hematologic parameters agree Well with EDTA-treated Those from samples and are stable for at Least Six Hours after sampling.

Citrate, in the form of sodium citrate or acid citrate dextrose, is used to disrupt the coagulation cascade and prevent clotting. These citrate compounds bind to calcium in the blood. By reducing the amount of calcium, there will be no regulation of the link and the cascade can not begin.

Choice between citrate and EDTA depends on the parameters you want to study, if you dont want EDTA which is a stronger anticoagulant but a membrane lysing agent too, then try 3.8% trisodium citrate (1:8 v/v to blood) with gentle mixing every 2-3 minutes, or keeping at a slow shaker.

For blood coagulation assay its best to prepare a citrate buffer 0.109 M (3.2%) at pH 7.0 as indicated below. In 1 litre volumetric flask, put 400 ml of dH2O and

dissolve 31.57 g of trisodium citrate and 11.92 g N-2-hydroxyethylpiperazine-N.sub.1 -2-ethanesulfonic acid (HEPES) hemisodium salt and fill up to 1 litre with dH2O. Mix well (ideally using magnetic stirrer). Adjust pH to 7.0 (with 0.5 M HCl or 0.05 M NaOH as appropriate based on the initial pH).

For assay the proper ratio is 1 part citrate:9 parts whole blood when hematocrit

is within 25–55%.