BIOLOGICAL ASSAY OF HUMAN ANTIHAEMOPHILIC FRACTlON The potency of human antihaemophilic fraction 15 determined by comparing the amount necessary

assay of human antihaemophilic fraction described below the estimated potency is not less than 80% and not more than 125% of the stated potency. The fiducial limits of error are not less than 60% and not more than 156% of me stated potency BIOLOGICAL ASSAY OF HUMAN ANTIHAEMOPHILIC FRACTlON The potency of human antihaemophilic fraction 15 determined by comparing the amount necessary to reduce We clonting time of a test mixture containing substances that cause clotting of blood with Use amount of the Standard Preparation necessary to produce the same effect under the conditions of the following method of assay standard PreparationThe Standard Preparation is the 4th Intermational Standard for Blood coagulation factor VIIC: concentrate, human, established in 1989, consisting of an intermediate purity concentrate of human blood clotting factor. VIII (supplied in ampoules containing 6.3 Units of clotting factor Vlll). or another suitable preparation the potency of which has been determined in relation to the International Standard. The Unit is the specific anohaemophific factor contained in such an amount of the Standard Preparation as the Ministry of Health & Family Welfare, Govt. of India may from time to time indicate as the quantity exactly equivalent to the Unit accepted for international use. Special ReagentsNormal serum reagent Collect normal human blood in a dry, sterile, glass bottle, shake continuously until coagulation is complete, incubate at 37" for 3 hours, maintain at 4" overnight, remove the serum, store at-20*, dry from the frozen state ana keep in a vacuum desiccator. over pnosphorus pentoxide Dissolve a quantity of the dried serum calculated to have been obtained from 1 ml. of the serum in sufficient imidazone buffer pH 7.4 to produce 10 ml and allow to stand at A* fbr 16 to 24 hours Phospholipid: Wash a quantity of normal human or bovine brain freed from meninges and blood vessels and macerate in a suitable blender. Weigh 1000 to 1300 gm of the macerate and measure its volume (V). Extract with three quantities, each of 4V ml, of acetone, filter by suction and dry the precipitate at 37* for 18 hours Extract the dried precipitate with two quantities, each of 2V ml, of a mixture of two volumes of light petroleum (boiling range 30' to 40") and 3 volumes of light petroleum (boiling range 40* to 60"), filtering each extract through a filter paper previously washed with the light petroleum mixture Combine the extracts and evaporate to dryness at 45* at a pressure not exceeding 0.7 kPa. Dissolve the residue in 0.2V ml of ether and allow to stand at 4* until a deposit forms Centrifuge and evaporate the clear supernatant liquid under reduced pressure until the volume is about 100 ml per kg of the original macerate allow to stand at 4* until a precipitate forms (12 to 24 hours) and centrifuge. To the clear supernatant liquid add 5 times its volume of acetone, centrifuge, discard the supernatant liquid, dry the precipitate and store protected from light in a vacuum desiccator Phospholipid reagent Suspend 0.125 g of pnospholipid in 5 ml of water, shake and stir until a uniform suspension is obtained Prepare a dilution with saline solution that will give minimum cloting times consistent with the largest clotting time differences between consecutive dilutions of the Standard Preparation and the preparation being examined The concentration usually lies between 50 and 250 ug per ml The diluted suspension may be kept at -20* for 6 weeks Clotting factor V solution: Prepare from fresh oxalated bovine plasma by fractionation at 4* with a saturated solution of ammonium sulphate prepared at A" Use the fraction precipitating between 38% and 50% saturation (which contains clotting factor V not signpficantly contaminated with clotting factor VIII). analysed to remove ammonium sulphate and diluted with saline solution to grve a solution containing between 10% and 20% of the amount of clotting factor V present in fresh normal human plasma Determine the clotting factor V content of the solution as fallows Prepare two dilutions in imidazole buffer pH 74 to contain. 1 volume of the solution' being examined in 10 volumes and 20 volumes respectively Test each dilution as follows. mix 0.1 ml each of substrate plasma deficient in clotting factor V, the dilution being tested, thrombokinase extract and 0.025M calcium chloride Record as the cloning time the interval between the addition of the calcium chloride solution and the first indication of hbnn formation, which may be observed visually or by -mechanical means Similarly determine the clonting times, in duplicate, for four dilutions of pooled normal human plasma in imidazole buffer pH 7 A -containing 1 volume in 10 volumes (equivalent to 100% of clotting factor V). in 50 volumes 120%), in 100 volumes (10%)L and in 1000 volumes (1%), respectively.To calculate the result, plot the mean of the ctoltting times for each driution of human plasma on