You have to add thrombin

You have to add either thrombin or an activator such as thromboplastin. Commercial thromboplastins e.g. Innovin are available.

Also you can add calcium alone or with tissue factor or kaolin. Besides thrombin you can use thrombin-like enzymes from snake venoms

10 mM CaCl2 (final concentration) is fine. It should clot in less than 1 h. Alternatively, you may add also some tissue factor (commercial thromboplastin, diluted 1/100 or 1/1000). If it does not clot, it means there is a problem somewhere (presence of heparin? serum instead of plasma?).

Impossible to do what you want, for the simple reason that your blood is collected on an anticoagulant, so there is no callot. But you can use tests as PT, PTTA or others

Hello,

How concentrated is your CaCl2 solution ? If I remember correctly, 10mM final concentration of CaCl2 is enough to produce a clot in PPP, so maybe your problem comes from the dilution of plasma. As others proposed, tissue factor should do the trick.

A contact activator such as kaolin or silica may be helpful too (if the material in which you try to make your clot aren't reactive enough), as are platelet phospholipids.

Hello Camille you are right .I agree with you, phospholipids problem

@Yiannis M Sarigiannis

Yes, that might be easier than adding kaolin or silica.

You can just add Ca++ 0,025M is usually used

Citrate acts as an anticoagulant by chelating the calcium. Restoring the calcium will activate the factors to propagate clot formation.

A plasma clot is formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma

I would try CaCl + procoagulant phospholipid (comm preparations are available from Stago etc); if this is not doing it I would add lipidated TF

Just add Ca2+ to a final concentration of ~ 10 mM

A too high Ca concentration will be preventing clotting here, so you need to titrate the dose (remember that the physiological Ca concentration is 1.2 mM, a bit higher will still do, but too high will give problems). However, it's not possible to do a direct calculation, but I send a paper where performed these types of experiments (however with 129 mM citrate) , and where I state that

Calcium chloride (0.5 mol/l) was added to a final

concentration of 10 mmol/l in blood and 18 mmol/l in

plasma, which gave a calcium ion activity of 1.2 mmol/l in

serum after clotting (determined potentiometrically with

an ICA 2 Ionised Calcium Analyser from Radiometer,

Copenhagen, Denmark).

so perhaps that could be a guide to where you should start looking at least. Regarding clotting initiators etc, it depends a bit on what you want to do with the product, so if you give some more information regarding this, it could be helpful  in order to be able to give further advice.

I have not much to add to what has been sais but it is rather simpler thant all has been said.

Citrate chelates the calcium and prevents the clotting.to get a clot may add cacl2 in order to get around 2.5 mM total calcium or if you have the possibility to check that 1.2 mM free ionized calcium. Not crucial however.

If you use plastic tubes go take a coffee, it would not clot because you need either an activator such as a glass tube or added thrombin. Physiologically platelets do they job but you are in PPP right ?

Question is it citrated plasma or ACD plasma

There is a very minor chance that you have collected blood from a donor that lacks activity of one of the upper factors of the intrinsic pathway (FXII, HK, PK or FXI) or has lupus anticoagulant activity. This can be without clinical symptoms but would help to explain the lack of spontaneous clotting of the plasma after recalcification. To rule out something like that, I would work with TF/phospholipids as was suggested by Dr. Bogdanov

Or your patients were on heparin, which prevents coagulation. Normally, high concentrations of Ca induce finally clotting. What is a purpose of clotting generation? To analyse clotting time ot to analyse a clot? You must be awae that every additional clotting activator may influence both.

If calcium is not working the plasma may contain heparin or another anticoagulant. Ususally 1 drop of a commercial fibrinogen reagent =thombin at ~50-100 NIH/mL) will cause a few mL of plasma to clot within seconds. If heparin is present it can be first neutralised with protamine sulphate solution.

Nornally the citrated platelet poor plasma is made by adding 9 parts of whole blood to 1 part of 0.105 M sodium citrate solution.  In order to neutralize this amount of citrate for every 200microliter of plasma you can add 100 microliter of 0.025M calcium chloride solution. 

You seem to have added high concentrations of citrate to make the platelet poor plasma and hence it may need high concentrations of calcium chloride solution.  Please appropriate volumes and concentrations of calcium chloride for appropriate volumes of citrated platelet poor plasma.

If there is no heparin in blood or polyanion (e.g. polypropylene) in the reaction tube or well then 10 parts of citrated plasma are recalcified with 1 part 250 mM CaCl2. The clot is generated within 30-60 min (37°C).

Thanks for all the answers. I wasn’t aware that too high Ca2+ prevents clotting. I’ve now tried titrating with CaCl2 and my PPP plasma is now clotting in concentrations from about 8 mM to 30 mM at 37C. Anything higher seems to prevent clotting.

A very small quantity of reptilase is sufficient, even with many of heparine, but the clot is little bit different (fibrinopeptide B not attacked)

Dear Thomas, in my experience a concentration of 23mM CaCl2 is the optimal concentration to clot PPP in order to counter-balance the anticoagulant activity of citrate; so this seems to match your observations. To accelerate the coagulation you may add a little glass beads (or metal beads) as is done in coagulation assays. The clot will form in about 10 min at RT. Higher CaCl2 concentrations will indeed progressively inhibit the coagulation. Alternatively you may add purified thrombin to have a final concentration close to 10 IU/ml, and the plasma will clot quickly. Best, Thierry

In my previous answer (adding Ca2+ to final conc = 10 mM) I forgot that you were using PFP. In this case, you also need phospholipids (PL) to a final conc = 4 uM (PS:PC:PE=1:3:1), since you don't have platelets anymore. Without PL, PFP may still clot, but very slowly.

You have have to add to 300µM of Cacl2 at 25 mM to  300 µM of platelet poor plasma (PPP) obtained after centrifugation of plama rich platelet during 30 minutes. The water may be at 37 °C during the incubation of PPP and calcium. After formation of clot you may incubate the PPP clot formed  in bath at 37 °C during 20 minutes until generation more thrombin and factor Xa associated to  PPP clot. Then you stop the enzymatic reaction  in bath glass at 0°C, you can separate the PPP clots from the serum. You congelate the serum for dosage the different factors . And you can mesure directly clots associated thrombin using by adding 200µl of CBS 3447 at 25 mM to 200 µl EDTA tris buffer containing clot incubated in water bath at 37 °C after 20 min incabation in water you stop the enzymatic rection by adding 50 µl of pur acetique acid pure and you transfere the tubes containing the clot and the serum in water bath and you remove the clots from the tubes. Like Ihave published. The clot  associated factor Xa  is read by using a CBS 3139 after activation of clot bound factor Xa , like I have published.

Dr S. Meddahi

Ca 10% for low clotting

Thrombin for quick clotting

1. Add 125 microliters of 0.9% saline to a well or tube.

2. Add 25 microliters of thawed plasma

3. Add 50 microliters of 30 mM CaCl2

Follow clotting spectrophotometrically at 405 nm. If no absorbance change by 30 min, something is wrong with your plasma. Freeze thaw, or excessive calcium will prevent clotting.

Hi Thomas,

This is what you have to do to make a clot using citrated blood as you have.  Take 200ul of citrated blood, incubate at 37 degree centigrade for 3 minutes and then  add 100ul of Thrombin 5 Units/ml.  You should get the clot in nearly 20 seconds.  You may also use Thrombin 10 Units/ml which should give you a clot in 10 seconds.  Using this proportion of blood sample tp Thrombin (2:1), you can generate clot using increased volumes of blood.  Good luck.

Omer

How do I clot EDTA blood. I need the serum for ferritin estimation

Hi Thomas and Vivek, your citrated or EDTA plasma can be clotted by addition of 1 part 250 mM CaCl2 to 10 parts of plasma. You need about 30 min at 37°C. Be sure that the tube you use is not too anticoagulant (polypropylene or irradiated polystyrene often inhibit thrombin generation).