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         Human Plasma for Fractionation is the liquid part of human blood remaining after separation of the cellular elements from blood collected in a receptacle containing an anticoagulant, or separated by continuous filtration or centrifugation of anticoagulated blood in an apheresis procedure; it is intended for the manufacture of plasma-derived products.

         The plasma or serum obtained from counselled healthy donors who must, as far as can be ascertained after medical examination, laboratory blood tests and a study of their medical history, be free from detectable agents of infection transmissible by plasmaderived products, may be used in the collection of plasma for fractionation. The examinations and tests to be carried out are decided by the National Blood Centre and the Ministry of Public Health; in particular, tests for hepatitis B surface antigen (HBsAg), for hepatitis C antibodies (anti-HCV) and for HIV antibodies such as anti-HIV-1 and anti-HIV-2 are carried out by suitable sensitive methods. The nucleic acid test for HBV, HCV and HIV is also recommended and must give non-reactive results in all three cases.

         For individual plasma units, the plasma is prepared by a method that removes cells and cell debris as completely as possible. Whether prepared from whole blood or by plasmapheresis, the plasma is separated from the cells by a method designed to prevent the introduction of micro-organisms. No antimicrobial agent is added to the plasma. Requirements and advice concerning the containers are given in Appendix 11.3. The containers are closed so as to prevent contamination.

         If two or more units are pooled prior to freezing, the operations are carried out using sterile connecting devices or under aseptic conditions and using containers that have not previously been used.

         Plasma intended for the manufacture of coagulation factors and other labile derivatives is either processed shortly after separation or collection or frozen by cooling rapidly at –18º or below. Plasma obtained from whole blood and intended for the manufacture of coagulation factors and other labile derivatives is separated from cellular elements and frozen as soon as possible and at the latest within 24 hours of donation. Plasma intended for the manufacture of non-labile derivatives is separated within 5 days of the expiration date of the whole blood.

         It is not intended that the determination of total protein and factor VIII shown below be carried out on each unit of plasma. They are rather given as guidelines for good manufacturing practice, the test for factor VIII being relevant for plasma intended for use in the preparation of concentrates of labile components.

         The total protein content of a unit of plasma depends on the serum protein content of the donor and the degree of dilution inherent in the donation procedure. When plasma is obtained from a suitable donor and using the intended proportion of anticoagulant solution, a total protein content complying with the limit of 50 g per litre is obtained. If a volume of blood or plasma smaller than intended is collected into the anticoagulant solution, the resulting plasma is not necessarily unsuitable for pooling for fractionation. The aim of good manufacturing practice must be to achieve the prescribed limit for all normal donations.

         Preservation of factor VIII in the donation depends on the collection procedure and the subsequent handling of the blood and plasma. With good practice, 0.7 IU per ml can usually be achieved, but units of plasma with a lower content may still be suitable for use in the production of blood coagulation factor concentrates. The aim of good manufacturing practice is to conserve labile components as much as possible.

         For pooled plasma, during the manufacture of plasma products, the first homogeneous pool of plasma (for example, after removal of cryoprecipitate) is tested for hepatitis B surface antigen and for HIV antibodies such as anti-HIV-1 and anti-HIV-2 using test methods of suitable sensitivity and specificity; the pool must give non-reactive results in these tests.

         The pool is also tested for hepatitis C virus RNA using a validated nucleic acid amplification technique (Appendix 14.6). A positive control with 100 IU per ml of hepatitis C virus RNA and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control is non-reactive or if the result obtained with the internal control indicates the presence of inhibitors. The plasma pool complies with the test if it is found nonreactive for hepatitis C virus RNA.

Description Before freezing, a clear or slightly turbid liquid without visible signs of hemolysis; it may vary in colour from light yellow to green.

Packaging and storage Store frozen plasma at or below –18º; the plasma may still be used for fractionation if a temperature of –18º is exceeded on at most one occasion for not more than 72 hours and if the plasma is at all times maintained at a temperature of –5º or lower.

Labelling The label enables each individual unit to be traced to a specific donor.

Total protein Not less than 5 per cent w/v of protein. Carry out the test using a pool of not less than 10 individual plasma units. Dilute the pool with saline TS to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of this solution in a round-bottomed centrifuge tube add 2 ml of a 7.5 per cent w/v solution of sodium molybdate, 2 ml of a mixture of 1 volume of nitrogen-free sulfuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid, and allow the inverted tube to drain on filter paper. 

Using the residue thus obtained, carry out the “Determination of Nitrogen” (Method II, Appendix 6.7) and calculate the content of protein by multiplying by 6.25.

Factor VIII Thaw the samples of a pool of not less than 10 individual plasma units, if necessary, at a temperature not exceeding 37º. Carry out the “Biological Assay of Human Coagulation Factor VIII”(Appendix 15.1.5), using a reference plasma calibrated against the International Standard for blood coagulation factor VIII in plasma. The activity is not less than 0.7 IU per ml.