Preparation of Factor VIII Concentrates by Cryoprecipitate Sedimentation D. J. BOUDARTA N D M. F. NAUD From the Blood Transfusion Center Laboratory, Hotel-Dieu, Nantes Cedex, France
A new method for factor VIII concentrate preparation is p r o m . The method is the coliectlon of cry+ precipitate afkr sedimentationat 4 C in a jacketed glass vessel. The Rnd product is standardized (4.0 2 0.3 factor VIII units per ml) and CM be infused in hemophllic patients regardless of their ABO blood type. Factor VIII recovery (52.2 2 6.4%) is comparable to other processes.
Materials and Methods
Blood was obtained from healthy donors, and collected in Fenwal double bag system containing 56 ml of citrate-phosphate-dextrose (CPD). Blood was twice centrifuged at 1,200 x g for 15 minutes at 10 C, no more than six hours after collection from the donor, and the plasma of donors of the same ABO blood group was CRYOPRECIPITATION has been widely pooled. Bottles containing approximately 400 ml of two plasma donors were spin-frozen by adopted for preparation of factor VIII conrotation in vertical position in alcohol at -40 C centrate. Two methods can be achieved, one and stored at -60 C until use. for large scale production (LSP)6 and one The apparatus (Fig. 1) used for decantation is for small scale production (SSP) which is composed of a glass vessel of about 12 liters represented by the closed bag ~ y s t e mEach .~ whose top and bottom present a blood bottle neck with rubber cap. The top neck is used for the process has advantages and inconveniences. LSP classicaly requires sterile fi1tration6*l3 filling with thawed plasma and the botton neck for the collection of cryoprecipitate rich plasma as a final step and the factor VIII yield is and cryoprecipitate poor plasma. The glass less than in SSP.7The LSP method provides vessel is inserted in a plastic support that is able factor VIII concentrates which can be into contain cold water and to maintain the temperature lower than 8 C, as recommended by fused in hemophilic patients regardless of Vermeer ef al. 'I The glass vessel is washed and their ABO blood type, reduces the number sterilized before each preparation. of biological controls and gives reproductible For each preparation 20 bottles of plasma (nine factor VIII level in the product.'O In contrast, group A, nine group 0, one group B and one SSP gives higher yields than LSP, but the group AB) were taken from storage at -60 C and kept at -27 C for 48 hours. They were then factor VIII recovery for the final product is maintained at 4 C for 48 hours. The glass vessel variable, closely related to the variation of the apparatus was filled and after thirty factor VIII concentration in normal plasma.8 of minutes the cryoprecipitate sedimented. The Moreover, that process gives factor VIII cryoprecipitate rich plasma was then collected in concentrate of each ABO blood group three or four 500 ml tared bottles containing a magnetic mixing bar. The bottles were censpecificity and thus complicates the storage trifuged at 1,200 x g for 15 minutes at 4 C and the management. Finally, SSP is not adapted supernatant plasma was discarded. The cryoprefor large scale production and requires cipitate was weighed (for each bottle) and diluted numerous biological controls. The difficulty by addition of sodium citrate 0.055 M, pH 6.8. to collect homogenous samples for biological The volume of citrate solution was determined from a graphic curve chosen in order to give four controls must be noted. In order to avoid factor VIII unitshl in the final solution (i.e., 7.9 such disadvantages, we proposed a method ml per gram of cryoprecipitate). The contents based on cryoprecipitate sedimentation. were mixed for 30 minutes at 25 C with a magnetic stirrer. The contents of the three or four bottles were pooled and then redistributed in Received for publication August 1 , 1978; accepted aliquots of 50 or 100 ml. The resulting October 21. 1978. 0041-1132/79/0900/0594$00.65 0 J. B. Lippincott Co. Transfusion September-October 1979
594
Volume 19 Number 5
Volume 19 Number 5
SEDIMENTED CRYOPRECIPITATE
-*.
595
Table 1. Yields of Cryoprecipitate and Factor VIII Activity ~
Factor Vlll Activity Gram of Cryoprecipitate per Liter of Starting Nondiluted Plasma
Units per Gram of Cry* precipitate
Units per Milllllter of Final Solution
Recovery Per h n t
17.7 T 1.7
35.4 r 3.2
4.0 T 0.3
52.2 e 6.4
to 0.80 ml of “nondiluted” plasma. Yields and composition of the cryoprecipitate extracted from 88 liters of CPD plasma (1 1 batches) are shown in Tables 1 and 2. The yield of cryoprecipitate was 17.7 2 1.7 g per liter of nondiluted plasma and the factor VIII activity was 35.4 2 3.2 units per gram of the cryoprecipitate and 4.0 0.3 units per ml of the final solution. Fibrinogen (4.1 -C 0.6 mg per ml of the final solution) represented about 50 per cent of the total proteins (8.6 2 1.5 mg per ml). The anti-A and anti-B hemagglutinin titers did not exceed 8. Factor VIII recovery was 52.2 f 6.4 per cent.
*
Discussion
FIG.I . Apparatus with sedimented cryoprecipitate. cryoprecipitate units were frozen at -60 C. Each lot was assayed for factor Vlll activity by the two stage method of Biggs et al.’ The presence of hepatitis B antigen (HBsAg) was checked by radioimmunoassay (AusRIA 11, Abbot Laboratones). Protein determination was made by the Biuret method using purified albumin as a standard. Anti-A and anti-B hemagglutinin were determined by saline and indirect antiglobulin tests. Fibrinogen content was measured and tests for sterility were performed as recommended by Arnaud et al. I
Results Factor VIII recovery was calculated after corrections of plasma volumes were done as re. ~ VIII potency ported by Paleirac et ~ 1 Factor was expressed in units equivalent to one ml of fresh plasma obtained from nine ml of normal blood collected mixed with one ml of citrate solution. Citrate and CPD plasma were expressed in terms of “nondiluted” plasma. one factor VIII unit is equivalent to 0.83 ml of “nondiluted” plasma and one ml of CPD plasma is equivalent
The closed bag system’ is now the most commonly used method by a large number of Blood Transfusion Centers for factor VIII concentrate production. However, this method is not easily adaptable for a large scale production and it is not possible to standardized the final p r ~ d u c t . ~ *The ’~ sedimentation method avoids these two inconveniences. The final product is standardized to provide 4.0 k 0.3 U/ml. The method could easily be altered to provide larger volumes of cryoprecipitate by increasing the capacity of each sedimentation apparatus or having several available. The factor VIII recovery is better than the LSP7*l0and satisfactory, compared with Table 2. Composition of the Final Cryoprecipitate Solution Anti-A
Factor Vlll Units per Milliter
Proteins (mglml)
4.0 2 0.3
8.6 2 1.5
Fibrinogen (mglml) 4.1
?
0.6
and Anti-B Hemagglutinin Titer s8
BOUDART AND NAUD
596
SSP.4,e,11However, the yield can be imThis proved by thawing in 4 C water is a time and money saving method since one can process eight liters of plasma in 3.5 hours using only one centrifuge. Finally, it presents good guaranties of sterility. We think that this preparation method of factor VIII concentrate by cryoprecipitate sedimentation can be applied for a small price to any blood transfusion center. Acknowledgment The authors wish to thank Dr. M. Steinbuch from the National Blood Transfusion Center of Paris for his courtesy and Mrs. Le Hecho for invaluable assistance in the preparation of this manuscript.
References 1. Arnaud, R., E. Borderon, J. C. Borderon, and J.
2.
3.
4.
5.
Loulergue: Etudes sur les contrbles de sterilite du plasma. Rev. Fr. Transf. 16:289, 1973. Biggs, R. M., J. Eveling, and G. Richards: The assay ofantihaemophilic-globulinactivity. Br. J. Haematol. 1:20, 1955. Burka, E. R., T. Puffer, and J. Martinez: The influence of donor characteristics and preparation methods on the potency of human cryoprecipitate. Transfusion 15323, 1975. Kasper, C. K., B. A. Myhre, J. D. MacDonald, Y.Nakasako. and D. I. Feinstein: Determinants of factor VIII recovery in cryoprecipitate. Transfusion 15312, 1975. Paleirac, G., E. Lete, R. Graafland, P. Izarn, and P. Cazal: Preparation des cryoprecipites lyo-
Transfusion September-October 1979
philists (facteur VIII concentre). Roc. 9th Congr. Nat. Blood Transf. Nancy 1973. 6. Pool, J. G., and E. J. Hershgold: High-potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate. Nature 2033 12, 1%4. 7.
-, and
A. E. Shannon: Production of highpotency concentrates of antihemophilic globulin in a closed-bag system. N. Engl. J. Med. 273: 1443, 1%5.
8. Prest0n.A. E.,andA. Barr: Theplasmaconcentration of factor VIII in the normal population. Br. J. Haematol. 10238, 1964. 9. Slichter, S. J., R. B. Counts, R. Henderson, and L. A. Harker: Preparation of cryoprecipitated factor VIII concentrates. Transfusion 16:616, 1976. 10. Steinbuck, M.: Personal communication. 11. Vermeer, C., B. A. M. Soute, G. Ates, and H. G. J.
Brummelhuis: Contributions to the optimal use of human blood. Vox Sang. %1, 1976. 12. Verstraete, M., P. Olislaegers, H. Van Itterbeek, P. Waumans, and A. Lust: Human plasma and plasma fractions as sources of factor VIII (Antihaemophilic factor). Vox Sang. 16:382, 1969. 13. Wickerhauser, M.: Preparation of antihemophilic factor from indated plasma. Transfusion 16:345, 1976.
D. J. Boudart, Ph.D., Chief, Cryobiologic Service, Blood Transfusion Center, BD 349, 44011, Nantes. Cedex, France. M. F. Naud, Blood Bank Technician, Blood Transfusion Center.
A new method for factor VIII concentrate preparation is p r o m . The method is the coliectlon of cry+ precipitate afkr sedimentationat 4 C in a jacketed glass vessel. The Rnd product is standardized (4.0 2 0.3 factor VIII units per ml) and CM be infused in hemophllic patients regardless of their ABO blood type. Factor VIII recovery (52.2 2 6.4%) is comparable to other processes.
Materials and Methods
Blood was obtained from healthy donors, and collected in Fenwal double bag system containing 56 ml of citrate-phosphate-dextrose (CPD). Blood was twice centrifuged at 1,200 x g for 15 minutes at 10 C, no more than six hours after collection from the donor, and the plasma of donors of the same ABO blood group was CRYOPRECIPITATION has been widely pooled. Bottles containing approximately 400 ml of two plasma donors were spin-frozen by adopted for preparation of factor VIII conrotation in vertical position in alcohol at -40 C centrate. Two methods can be achieved, one and stored at -60 C until use. for large scale production (LSP)6 and one The apparatus (Fig. 1) used for decantation is for small scale production (SSP) which is composed of a glass vessel of about 12 liters represented by the closed bag ~ y s t e mEach .~ whose top and bottom present a blood bottle neck with rubber cap. The top neck is used for the process has advantages and inconveniences. LSP classicaly requires sterile fi1tration6*l3 filling with thawed plasma and the botton neck for the collection of cryoprecipitate rich plasma as a final step and the factor VIII yield is and cryoprecipitate poor plasma. The glass less than in SSP.7The LSP method provides vessel is inserted in a plastic support that is able factor VIII concentrates which can be into contain cold water and to maintain the temperature lower than 8 C, as recommended by fused in hemophilic patients regardless of Vermeer ef al. 'I The glass vessel is washed and their ABO blood type, reduces the number sterilized before each preparation. of biological controls and gives reproductible For each preparation 20 bottles of plasma (nine factor VIII level in the product.'O In contrast, group A, nine group 0, one group B and one SSP gives higher yields than LSP, but the group AB) were taken from storage at -60 C and kept at -27 C for 48 hours. They were then factor VIII recovery for the final product is maintained at 4 C for 48 hours. The glass vessel variable, closely related to the variation of the apparatus was filled and after thirty factor VIII concentration in normal plasma.8 of minutes the cryoprecipitate sedimented. The Moreover, that process gives factor VIII cryoprecipitate rich plasma was then collected in concentrate of each ABO blood group three or four 500 ml tared bottles containing a magnetic mixing bar. The bottles were censpecificity and thus complicates the storage trifuged at 1,200 x g for 15 minutes at 4 C and the management. Finally, SSP is not adapted supernatant plasma was discarded. The cryoprefor large scale production and requires cipitate was weighed (for each bottle) and diluted numerous biological controls. The difficulty by addition of sodium citrate 0.055 M, pH 6.8. to collect homogenous samples for biological The volume of citrate solution was determined from a graphic curve chosen in order to give four controls must be noted. In order to avoid factor VIII unitshl in the final solution (i.e., 7.9 such disadvantages, we proposed a method ml per gram of cryoprecipitate). The contents based on cryoprecipitate sedimentation. were mixed for 30 minutes at 25 C with a magnetic stirrer. The contents of the three or four bottles were pooled and then redistributed in Received for publication August 1 , 1978; accepted aliquots of 50 or 100 ml. The resulting October 21. 1978. 0041-1132/79/0900/0594$00.65 0 J. B. Lippincott Co. Transfusion September-October 1979
594
Volume 19 Number 5
Volume 19 Number 5
SEDIMENTED CRYOPRECIPITATE
-*.
595
Table 1. Yields of Cryoprecipitate and Factor VIII Activity ~
Factor Vlll Activity Gram of Cryoprecipitate per Liter of Starting Nondiluted Plasma
Units per Gram of Cry* precipitate
Units per Milllllter of Final Solution
Recovery Per h n t
17.7 T 1.7
35.4 r 3.2
4.0 T 0.3
52.2 e 6.4
to 0.80 ml of “nondiluted” plasma. Yields and composition of the cryoprecipitate extracted from 88 liters of CPD plasma (1 1 batches) are shown in Tables 1 and 2. The yield of cryoprecipitate was 17.7 2 1.7 g per liter of nondiluted plasma and the factor VIII activity was 35.4 2 3.2 units per gram of the cryoprecipitate and 4.0 0.3 units per ml of the final solution. Fibrinogen (4.1 -C 0.6 mg per ml of the final solution) represented about 50 per cent of the total proteins (8.6 2 1.5 mg per ml). The anti-A and anti-B hemagglutinin titers did not exceed 8. Factor VIII recovery was 52.2 f 6.4 per cent.
*
Discussion
FIG.I . Apparatus with sedimented cryoprecipitate. cryoprecipitate units were frozen at -60 C. Each lot was assayed for factor Vlll activity by the two stage method of Biggs et al.’ The presence of hepatitis B antigen (HBsAg) was checked by radioimmunoassay (AusRIA 11, Abbot Laboratones). Protein determination was made by the Biuret method using purified albumin as a standard. Anti-A and anti-B hemagglutinin were determined by saline and indirect antiglobulin tests. Fibrinogen content was measured and tests for sterility were performed as recommended by Arnaud et al. I
Results Factor VIII recovery was calculated after corrections of plasma volumes were done as re. ~ VIII potency ported by Paleirac et ~ 1 Factor was expressed in units equivalent to one ml of fresh plasma obtained from nine ml of normal blood collected mixed with one ml of citrate solution. Citrate and CPD plasma were expressed in terms of “nondiluted” plasma. one factor VIII unit is equivalent to 0.83 ml of “nondiluted” plasma and one ml of CPD plasma is equivalent
The closed bag system’ is now the most commonly used method by a large number of Blood Transfusion Centers for factor VIII concentrate production. However, this method is not easily adaptable for a large scale production and it is not possible to standardized the final p r ~ d u c t . ~ *The ’~ sedimentation method avoids these two inconveniences. The final product is standardized to provide 4.0 k 0.3 U/ml. The method could easily be altered to provide larger volumes of cryoprecipitate by increasing the capacity of each sedimentation apparatus or having several available. The factor VIII recovery is better than the LSP7*l0and satisfactory, compared with Table 2. Composition of the Final Cryoprecipitate Solution Anti-A
Factor Vlll Units per Milliter
Proteins (mglml)
4.0 2 0.3
8.6 2 1.5
Fibrinogen (mglml) 4.1
?
0.6
and Anti-B Hemagglutinin Titer s8
BOUDART AND NAUD
596
SSP.4,e,11However, the yield can be imThis proved by thawing in 4 C water is a time and money saving method since one can process eight liters of plasma in 3.5 hours using only one centrifuge. Finally, it presents good guaranties of sterility. We think that this preparation method of factor VIII concentrate by cryoprecipitate sedimentation can be applied for a small price to any blood transfusion center. Acknowledgment The authors wish to thank Dr. M. Steinbuch from the National Blood Transfusion Center of Paris for his courtesy and Mrs. Le Hecho for invaluable assistance in the preparation of this manuscript.
References 1. Arnaud, R., E. Borderon, J. C. Borderon, and J.
2.
3.
4.
5.
Loulergue: Etudes sur les contrbles de sterilite du plasma. Rev. Fr. Transf. 16:289, 1973. Biggs, R. M., J. Eveling, and G. Richards: The assay ofantihaemophilic-globulinactivity. Br. J. Haematol. 1:20, 1955. Burka, E. R., T. Puffer, and J. Martinez: The influence of donor characteristics and preparation methods on the potency of human cryoprecipitate. Transfusion 15323, 1975. Kasper, C. K., B. A. Myhre, J. D. MacDonald, Y.Nakasako. and D. I. Feinstein: Determinants of factor VIII recovery in cryoprecipitate. Transfusion 15312, 1975. Paleirac, G., E. Lete, R. Graafland, P. Izarn, and P. Cazal: Preparation des cryoprecipites lyo-
Transfusion September-October 1979
philists (facteur VIII concentre). Roc. 9th Congr. Nat. Blood Transf. Nancy 1973. 6. Pool, J. G., and E. J. Hershgold: High-potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate. Nature 2033 12, 1%4. 7.
-, and
A. E. Shannon: Production of highpotency concentrates of antihemophilic globulin in a closed-bag system. N. Engl. J. Med. 273: 1443, 1%5.
8. Prest0n.A. E.,andA. Barr: Theplasmaconcentration of factor VIII in the normal population. Br. J. Haematol. 10238, 1964. 9. Slichter, S. J., R. B. Counts, R. Henderson, and L. A. Harker: Preparation of cryoprecipitated factor VIII concentrates. Transfusion 16:616, 1976. 10. Steinbuck, M.: Personal communication. 11. Vermeer, C., B. A. M. Soute, G. Ates, and H. G. J.
Brummelhuis: Contributions to the optimal use of human blood. Vox Sang. %1, 1976. 12. Verstraete, M., P. Olislaegers, H. Van Itterbeek, P. Waumans, and A. Lust: Human plasma and plasma fractions as sources of factor VIII (Antihaemophilic factor). Vox Sang. 16:382, 1969. 13. Wickerhauser, M.: Preparation of antihemophilic factor from indated plasma. Transfusion 16:345, 1976.
D. J. Boudart, Ph.D., Chief, Cryobiologic Service, Blood Transfusion Center, BD 349, 44011, Nantes. Cedex, France. M. F. Naud, Blood Bank Technician, Blood Transfusion Center.
