withdrawal is completed, the blood iscooled to 4", if the plasma is to be stored frozen it is separated from the cellular components by

as the blank solution I ml of water treated in the same manner. Prepare a calibration curve by measuring the absorbance of solutions prepared by treating in the same manner t-ml quantities of suitable dilutions of a solution in water containing 2 .5 mg per ml of C6 H8O7. prepared by using anhydrous citric acid, previously dned for 3 hours at 90* Calculate the total citrate content, as C6 H8 O7, in mg per ml of the solution being examined from the expression0.2 Cwhere C is the concentration in ug per ml of C6H8O7, read from the curve. Calculate the quantity, in mg. of C6H5Na3O7, 2H2O in 1 ml of the solution being examined from tfie expression 1.53 (A - B)where A ts the concentration in mg per ml of total citrate as C6H8O7 and B is the concentration in mg per ml of free citnc acid in the solution For free crtric acid - Carry out the Assay for free citric acid descnbed under. Anticoagulant Citrate Dextrose Solution From the volume of 0.1M sodium hydrxide required subtract a valumeH in ml,, equal to 1.28 times the number of mg of NaH2PO4 ,2H2O present, as determined in the Assay for sodium acid phosphate Each ml of the remainder is equivalent to 0.007005 g of C6,H8,O7,H2O. For sodium acid phosphate -Dilute 5 0 ml to 100 0 ml wiih water Transfer 5.0 ml to a25-ml graduated flask and add 10. 0 ml of a 2 8% w/v solution of sulphunc acid followed by 2.0 ml of a 2.5% w/v solution of ammonium motybdate. mixing after each addition. Add 1.0 ml of aminohydroxynaphthale in sulphonic acid solution and sufficient water to produce 25 0 ml. mix and keep aside at 25* for 10 minutes. Measure the absorbance (A,) of the resutong solution at about 660 run, Appendix 5.5, using as the blank 5 ml of water treated in the same manner. Calculate the content of NaH2PO4H2O in each ml of the solution being examined from the absorbance (A2) obtained by simultaneously carrying out the operation using 5 0 ml of a solution of potassium dihydrogen phosphate containing 0.11 mg of KH2PO4 per ml (1) and from the expression22.92 COMAFor dextrose - Weigh a clean, medium porosity smterefl-glass crucible containing a few glass beads. To 50 ml. of potassium cupn-tartrate solution add the glass beads from the weighed crucible, 45 ml of water and 5.0 ml of the, solution being examined. Hear the solution at such a rate that it begins to boil in 3.5 to 4 minutes, boil the solution for exactly 2 minutes and filter immediately through the weighed crucible, taking care to transfer all the glass beads to the crucible, along with the precipitate Wash the precipitate with hot water and then with 10 ml of ethanol (95%) and dry it to constant weight at 110*. Perform a blank determination and make any necessary correction Each mg of the precipitate is equivalent to 0000496 g of C6H12O6.H2O DRIED HUMAN ANT1HAEMOPHILIC FRACTlONDned Factor VIII Fraction, Freeze-dned Human Coagulation Factor VIIDned Human Antihaemophilic Fraction is a preparation of antihaemophihc factor which is obtained from human plasma. It is rich in clotting factor VIII The plasma to be used for preparing Dried Human Antihaernophilic Fraction is obtained from blood of healthy human donors who are, as far as can be ascertained after clinical examination, laboratory tests on their blood and consideration of their medical history, free from detectable agents of infection transmtssible by blood transfusion. The examinations and tests to be carned out are decided by the appropriate national authority In particular, the blood must be tested with negative results for (a) evidence of syphilitic infection, (b) hepatitis B surface antigen and (c) HIV antibodies by suitably sensitive methods. The haemoglobin value of the donor's blood is not less than 12.5% w/v. The blood is withdrawn asepucaiiy through a closed system of stenie tubing into a sterile container in which a suitable anticoagulant ,solution has been placed before sterilisation. During tne withdrawal there is no interruption in the flow fram the donor, and the container is gently agitated Immediately after the withdrawal is completed, the blood iscooled to 4", if the plasma is to be stored frozen it is separated from the cellular components by centrifugation and frozen to -30* or below, preferably within 12 hours of collection, if the plasma is not to be frozen it is separated from the celluter components by centrifugallon as soon as possible and not later than 1B hours after collection, and fracaonation begun without delay, Dried Human Antihaemophilic Fraction may be prepared from human plasma so obtained by precipitation under controlled conditions of pH, ionic strength and temperature with organic solvents, or by freezing and thawing. The precipitate may be washed by extraction with suitable solvents, dissolved in a solution of Sodium Citrate adjusted to a pH of B to 7.2, which may also contain Sodium Chloride. The solution is steilised by filtration through a membrane filter, distnbuted in sterile containers and