Is it possible to seperate serum from plasma? I read you can add excess calcium ions and protamine suphate to do so but how much of these do you need to add? Does anyone have a protocol?
Serum and plasma are individually derived from whole blood. Serum is obtained after you clot the whole blood and spin it down. Plasma is obtained from anti-coagulated whole blood after removing the blood cells. I guess you could clot the plasma (your question above) and call it serum when you centrifuged out the clotted material. From a operational (hematological) definition, this would really not be 'serum'. Unfortunately, this still does not answer your question.
It is a bit difficult to separate out serum from plasma because calcium ions or prolamine sulfate can not separate serum from plasma. Serum does not contain prothrombin and fibrinogen. Therefore, they have to be separated out first. If it is not absolutely mandatory, then it is much better option to separate serum from the whole blood.
(Blood plasma) - (fibrinogen & clotting factors) = Blood serum. Protamine sulfate alone might work to clot the blood, and that has been extensively studied in heparinzed patients. You might need more, depending on your goal. See this patent:Inventor: Frank M. La Duca
Original Assignee: International Technidyne Corp.
Primary Examiner: S. Saucier
Current U.S. Classification: 435/269; 210/632; 435/2
International Classification: G01N 3386
From another viewpoint - Can I assume that you have collected plasma but require serum? If you are interested in antibodies present in the blood then my work has suggested that serum and plasma are both comparable with regard to studying antibody content. Hope this helps.
@Frank Church: For our analysis we maybe able to work with theserum derived from plasma. Do you know of any method or protocol than can be utilized to do this?
@Subhabrata Moitra: Do you mean protoamine? Do you have a protocol stating quantities or calcium and protoamine to add?
As Frank Church says serum is derived from plasma after the blood has clotted. Therefore if blood is drawn and left to stand in a vial without anticoagulant then it will activate the coagulation pathway and serum is created. The fibrinogen converts to fibrin. All that needs to be done then is remove the fibrin usually by centrifugation and the serum is left standing on top. If calcium is to be used then it really depends on the anticoagulant used - usually excess calcium chloride will cause citrates plasma (or whole blood) to clot and centrifugation will again result in being able to remove the serum. If heparinised blood is used then adding excess thrombin or protamine sulphate will cause the blood to clot and again centrifugation will allow the serum to be removed.
Yes Ca will work if the anticoagulant was EDTA and you could add thrombin - all details will be in a standard text such as 'Pracrical Haematology' by Dacie & Lewis
If the anticoagulant is EDTA or sodium citrate, add Calcium to clot the plasma and then centrifuge. The dosage of calcium is to aim the clotting of plasma only, it can be calculated as the same concentration as the physiological concentration in our plasma, or it can be excess. If the anticoagulant is heparin, just add protamine sulphate as the same concentration of heparin to neutralize the heparin effect, and after clotting, centrifuge to get serum.
Best regards,
I would like recommended to you preparation of serum from plasma by defibrination of sample. You must add minimally 2 IU of thrombin to 1 ml of sample and after 2 minutes perform separation of clot by centrifugation at 4500 rpm minimally 5 min.
LS
As others suggest, use thrombin followed by calcium to convert plasma to serum (calcium alone - as calcium chloride, if I remember correctly - may do in some cases, depending on what was used as anticoagulant). I used to have a protocol but it is many years since I did this and I can't find it. Once the clot has formed (leave it to shrink) you will need to centrifuge the serum + clot to get the serum layer, free of fibrin - or you could carry out the reaction by tumbling in a vessel with some glass beads which will attract the fibrin as they fall from one end of the vessel to the other when tumbled (we used to use a wheel rotator). This "tumbling on glass beads" can also be used for whole blood instead of anticoagulants - defibrinated blood is quite good if you want to isolate mononuclear leukocytes which are relatively free of platelets (which stick to the fibrin clot on the glass beads) - and you also get serum from the sample.
Add 20% w/v CaCl2 into plasma to a ratio of 1:100 (v:v of CaCl2:plasma). Mix and lt clot at 4C overnight. Centrifuge to pellet down the clotting protein then you will get serum.
If you allow the blood to clot you will get serum, not plasma. If you use an anticoagulant, you will get plasma, not serum. The reaction is not reversible since clotting removes all fibrinogen by converting it to fibrin. However, if you use heparin as an anticoagulant, it inhibits this conversion. If you remove heparin by using protamine sulphate, then coagulation will occur and is enhanced by the presence of calcium ions.
Plasma is the liquid part of the blood.It is a solution in water of many compounds. It includes soluble proteins. also. Now blood serum is blood plasma from which the fibrinogen has been removed.
The main solids of plasma are the proteins. It is possible to separate out the individual proteins from mixtures by different methods. 'Salting out' methods use different concentrations of salt solutions(e.g.; ammonium sulfate,sodium sulfate and sodium sulfite) at which different proteins are precipitated and can be removed.
Methods like electrophoresis and ultracentrifugation and ethanol fractionation ( Cohn) have been utilized.
Add 20% w/v CaCl2 into plasma to a ratio of 1:100 (v:v of CaCl2:plasma). Mix and lt clot at 4C overnight. Centrifuge to pellet down the clotting protein then you will get serum