Plasma Substrate R1

Plasma Substrate R1 Substrate plasma R1

Use water-repellent equipment (made from materials such as suitable plastics or suitably siliconised glass) for taking and handling the blood. Collect a suitable volume of blood from each of an appropriate number of sheep. (A volume of 285  ml of blood added to 15  ml of anticoagulant solution is considered suitable; smaller volumes may be collected. Whatever volume is collected it is considered advisable to use at least 5 sheep.) Take the blood, either from a live animal or immediately after slaughter, using a needle attached to a suitable cannula which is long enough to reach the bottom of the collecting vessel. Discarding the first few millilitres and collecting only free-flowing blood, collect the blood into sufficient of an anticoagulant solution containing 8.7  g of sodium citrate and 4  mg of aprotinin in 100  ml of water to give a final ratio of blood to anticoagulant solution of 19:1. During and immediately after collection swirl the flask gently to ensure mixing, but do not allow frothing to occur. When collection is complete, close the flask, cool to 10° to 15° and then pool the contents of all the flasks with the exception of any that show obvious haemolysis or clots and keep the pooled blood at 10° to 15°.

As soon as possible and, in any case, within 4 hours of collection, centrifuge the pooled blood at 1000 to 2000  g at 10° to 15° for 30 minutes. Separate the resulting supernatant liquid and centrifuge it at 5000  g for 30 minutes. (More powerful centrifugation, for example at 20,000  g for 30 minutes, may be used if necessary to clarify the plasma at this stage but filtration procedures should not be used.) Separate the resulting supernatant liquid and, without delay, mix thoroughly and distribute the resulting substrate plasma into small stoppered containers in portions sufficient for a complete heparin assay (for example, 10 to 30  ml). Without delay, freeze rapidly at a temperature below –70° (for example, by plunging the containers into liquid nitrogen) and store at a temperature below –30°.

The prepared plasma is considered suitable for use as substrate plasma in the assay for heparin if, under the conditions of the assay, it gives a clotting time appropriate to the method of detection used and if it provides reproducible, steep log dose-response curves. When required, thaw a portion of substrate plasma in a water bath at 37°, gently swirling until thawing is complete; once thawed it should be kept at 10° to 20° and used without delay. The thawed substrate plasma may be lightly centrifuged if necessary; filtration procedures should not be used.