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Donation of factor VIII by plasma exchange after DDAVP administration
Transfus. Sci. 1989; 10:253-256
09X-3886/89
Donation
$3.00+0.00
Copyright @ 1989 Pergamon Press plc
Printed in Great Britain. All rights reserved
of Factor VIII by Plasma Exchange After DDAVP Administration B. Noel E. Coudurier G. Brochier
n We report our experience with a new factor VIII product, the potency and
might be of special interest for countries that lack industrial fractionation capabilities. n
safety of which are at least equivalent to industrial products. Carefully screened, highly motivated donors are informed about requirements for the treatment of hemophilia and committed to give donations for factor VIII monthly. 2.5 L of plasma is obtained by plasma exchange after i.v. infusion of DDAVP, which triples the level of factor VIII and doubles the level of Von Willebrand Factor. Plasma replacement is with autologous supernatant from previous donations or from manual plasmapheresis. An exchange can usually be completed in 2 h. Cryoprecipitate is obtained in minibags after freezing and fast thawing. The factor VIII yield has averaged 2764 + 922 IU per donation. The concentration of factor VIII in cryoprecipitate has averaged 23.8 + 7.7IU/mL and the specific activity was mean 1.22 + 0.48 IU/mg protein. Cost of production was estimated at 1.3 Fr per IU. After 6 months quarantine, during which the donors are checked repeatedly for infectious diseases, the concentrate may be released. Thus, plasma exchange and use of DDAVP in a small number of donors allow us to obtain a “clean” factor VIII concentrate with a lower cost than industrial concentrates. This strategy
INTRODUCTION Replacement therapy in hemophilia A is characterized by high cost and an important risk of viral transmission. l The viral risk is related to the very large number of individual donors involved in the preparation of anti-hemophilic products. Wet heat or solvent/detergent treatment of products derived from plasma fractionation may make them more reliable as regards non-transmission of the human immunodeficiency virus (HIV) and the non-A non-B hepatitis virus2 However, the complex technology used for industrial fractionation and the decreased production yield following viral inactivation have result in a high cost for these products. We have explored an alternative to these products; factor VIII obtained by cryoprecipitation of plasma collected by plasma exchange (PE) from a small number of highly motivated and dedicated donors. We follow a procedure similar to that described by McLeod et d3 including injection of DDAVP just prior to donation. PE allows the collection of a large volume of plasma, and the use of DDAVP, a structural analogue of vasopressin which has been previously used in Sweden,4 gives an increased factor VIII content. The reduction in the number of
From the Centre Departement de Transfusion Sanguine de la Savoie, Faubourg Mache, B.P. 1125, 73011 Chambery Cedex, France. Received and Accepted 5/89.
253
254
Transfus. Sci.
Vol. 10, No. 3
donors lowers the risk of viral transmission. The safety of the product is further enhanced by holding it in quarantine until the donors give negative tests for hepatitis and HIV at 6 months after donation.
METHODS Donors 35 volunteer donors gave informed consent for this investigation as approved by the Ethics Committee of Claude Bernard University and Lyon’s Civil Hospitals.
Plasma Exchange Plasma exchange was carried out with a COBE 2997 blood cell separator (centrifugation) or a COBE CENTRY TPE [filtration]. Fresh plasma was collected and replaced by the autologous cryoprecipitate-supernatant of plasma removed at a previous exchange donation and preserved frozen after separation of the precipitate. Three initial plasmaphereses of 600mL provide the replacement plasma for the first exchange. The volume of plasma exchanged varied from 2 to 2.5 L. This was accomplished within a time of about 2h. Each PE was preceded by the intravenous injection of DDAVP (20 yg in 50mL of saline solution administered over 2&30 min). Donors were requested to refrain from excessive fluid intake for 8 h preceding and 24 h following a donation. Cryoprecipitate Plasma from a donation was distributed into 6 double bags of about 400mL and immediately frozen in an alcohol bath at -50°C. Cryoprecipitate was separated by centrifugation at 0°C after thawing plasma at 4°C for 1.5-2 h in a circulating water bath. The cryoprecipitate was immediately diluted with 15 mL citrate saline solution per bag. The product was then frozen again at -40°C and stored at this temperature for 6 months before use.
Dosage for individual infusions could be adjusted by combining 2 or more bags. Donor Monitoring The frequency of donation was adjusted for donor convenience, but averaged once per month. Pulse and blood pressure were recorded before and after DDAVP administration. A complete blood count, electrolyte panel, protein electrophoresis, and electrocardiogram were performed at the inception of the program and then at 6 month intervals. A transaminase assay, an assay for anti-HBc, and testing for Ag P 24 (HIV protein) and corresponding antibodies were carried out at each donation. Product was not released for transfusion until negative results had been obtained in all re-testings for a period of 6 months following the donation. Product Testing The product was assayed for factor VIII by the 2-stage method derived from the thromboplastin generation test,5 for Van Willebrand Factor antigen (VWF:Ag) by the method of Laurell,6 for fibrinogen by the chronometric method,’ and for total protein by the Biuret reaction.* RESULTS 106 exchanges have been performed. All were well tolerated by the donors, both clinically and biologically. The only symptoms reported were facial flushing linked to DDAVP injection and hypocalcemia due to citrate. The average volume of plasma collected was 2301+ 154 mL. The average factor VIII content of the plasma was 5388 f 13801U. The characteristics of cryoprecipitate from plasma collected by filtration and centrifugal apheresis were similar. The average factor VIII content was 2764 + 9221U per donation for a of separation of mean efficiency 51.3 f 13%. In the cryoprecipitate, the of factor VIII was concentration 23.8 + 7.7 IU/mL, the VWF:Ag concentration was 18.2 +3.7IU/mL, and the
Factor VIII Donation Using PE and DDAVP 255
fibrinogen concentration was 6.01+ 2.2 mg/mL. The total protein content of the cryoprecipitate was 19.2 f 57mg/mL, so that the average specific activity for factor VIII was 1.22 + 0.48 IU/mg protein (Table 1). The cost of one IU of factor VIII prepared in this way was estimated to be 1.3 Fr. DISCUSSION The prevention of viral transmission is a natural goal of transfusion in general and hemophilia therapy in particular. While we hope that products of a very high degreee of purity (proteins and viruses) will be obtained by genetic engineering in the future, replacement therapy in hemophilia is currently based upon plasma-derived anti-hemophilic factor concentrates treated with heat, or with solvents and detergents (SD), to inactivate viruses. This is a pilot study based on McLeod’s work. However it differs in many aspects, including, for instance, the way the donors are selected and the repetitive screening and testing during a prolonged quarantine. This delay in release means that the product is probably very safe both in regards to HIV and also chronic hepatitis. The donor in our study is an anonymous volunteer, according to the French ethical standards. This status is desired by our patients as the hemophiliacs do not like the psychological ties which in some cases reveal parental problems. Generally the donor determines the frequency of his donation, but our volunteers are very keen to give factor VIII every month. We did not observe any
serious adverse effects of DDAVP. No unrelated volunteer donors have rejected the infusion of DDAVP after signing an informed consent. Several ethics committees, including those in Sweden, Chicago9 and Lyon, have approved the procedure. In fact, there is no restriction for the use of DDAVP in mild hemophilia and Von Willebrand disease except the decreasing response. In this study, the joint use of plasma exchange and DDAVP allows us to obtain a cryoprecipitate product, the composition of which is quite comparable to that of factor VIII SD from the fractionation industrylO as shown in Table 1. The use of DDAVP is not a disadvantage. Its action on the liberation of plasminogen activators was checked on a restricted number of donors. The results showed highly variable individual sensitivity, rapid inhibition of these activators, and absence of these materials in the final product. The duration of plasma exchange does not appear to be an impediment, since no donor cancelled a scheduled donation. Furthermore plasma exchange has had no adverse clinical or biological consequences. Immediate invivo recovery was measured in several recipients and found to be about 2% per IU infused per kg, in agreement with the results of McLeod et ~1.~The only significant drawback is the need to keep the cryoprecipitate frozen, and a study of freeze-drying has been implemented. This procedure is efficient at two levels: (1) it recovers about 50% of the factor VIII in the raw material, as opposed to only 10-15% for industrial fractionation, and (2) the sale price is markedly lower than that of the industrial pro-
Table 1. Protein Composition and Degree of Purity of Different Preparations Cryoprecipitate
Components Factor VIII:C VWF:Ag Fibrinogen Total proteins Specific activity Purity (approx.) 1s 10:3-G
Plasma 200 ng 20 lJg 3mg 70 mg O.O14U/mg 0.003%
from PE 200 ng 16.5 pg
0.3 mg 0.82 mg 1.22 U/mg 0.024%
SD Concentrate Intermediate Purity 200 ng 32Yg
0.2mg 1.8mg
0.6-0.8 U/mg 0.012%
256
Transfus. Sci. Vol. 10, No. 3
ducts. This strategy could be adapted to a transfusion organization lacking a fractionation capacity. The viral safety of the product is afforded by the limited number of highly motivated and responsible donors allowed by plasma exchange and DDAVP. Also, the 6-month re-testing interval between donation and release of product will allow detection of seroconversion in an individual who donates in the early stages of HIV or hepatitis infection. The product complies with the recommendations of the National Hemophilia Foundation in the United States to attending physicians of hemophilic subjects. This foundation has indeed proposed in its bulletin dated February 13, 198811that the use of cryoprecipitate prepared from a single, repetitively tested, selected donor or from a small number of such donors as an alternative to industrial products.
3.
4.
5.
6.
7. 8. 9.
10. REFERENCES 1. Ratnoff OD: Some complications of the therapy of classic hemophilia. I Lab Clin Med 1984; 103:653-659. 2. Horowitz MS, Rooks C, Horowitz B,
11.
Hilgartner MW: Virus safety of solvent detergent treated antihemophilic factor concentrate. Lancet 1988; July:186-189. McLeod BC, Sassetti RJ, Cole ER, Scott JP: A high potency, single donor cryoprecipitate of known Factor VIII content dispensed in vials. Ann Intern Med 1987; 106:35-40. Nilson IM, Walter H, Mikaelsonn M, Vilhardt H: Factor VIII concentrate prepared from DDAVP stimulated blood donor plasma. Stand 1 Haematol 1979; 22 ~42-46. Biggs RM, Eveling J, Richards G: The assay of antihemophilic globulin activity. Br / Haematol 1955; 1:20. Laurel1 CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966; 15:45-52. Clauss A: Acta Haematol 1957; 17:237. Kingsley GR: IBiol Chem 1939; 131: 197200. McLeod BC, Sassetti RJ, Cole ER, Scott JP: Long term frequent plasma exchange donation of cryoprecipitate. Transfusion 1988; 28(4):307-310. In pamphlet distributed by Biotransfusion, B.P. 99, 91943 Les Uelis Cedex, France. The National Hemophilia Foundation, Med. Bulletin, Ref. 66, Chap. 72. February 19, 1988.
09X-3886/89
Donation
$3.00+0.00
Copyright @ 1989 Pergamon Press plc
Printed in Great Britain. All rights reserved
of Factor VIII by Plasma Exchange After DDAVP Administration B. Noel E. Coudurier G. Brochier
n We report our experience with a new factor VIII product, the potency and
might be of special interest for countries that lack industrial fractionation capabilities. n
safety of which are at least equivalent to industrial products. Carefully screened, highly motivated donors are informed about requirements for the treatment of hemophilia and committed to give donations for factor VIII monthly. 2.5 L of plasma is obtained by plasma exchange after i.v. infusion of DDAVP, which triples the level of factor VIII and doubles the level of Von Willebrand Factor. Plasma replacement is with autologous supernatant from previous donations or from manual plasmapheresis. An exchange can usually be completed in 2 h. Cryoprecipitate is obtained in minibags after freezing and fast thawing. The factor VIII yield has averaged 2764 + 922 IU per donation. The concentration of factor VIII in cryoprecipitate has averaged 23.8 + 7.7IU/mL and the specific activity was mean 1.22 + 0.48 IU/mg protein. Cost of production was estimated at 1.3 Fr per IU. After 6 months quarantine, during which the donors are checked repeatedly for infectious diseases, the concentrate may be released. Thus, plasma exchange and use of DDAVP in a small number of donors allow us to obtain a “clean” factor VIII concentrate with a lower cost than industrial concentrates. This strategy
INTRODUCTION Replacement therapy in hemophilia A is characterized by high cost and an important risk of viral transmission. l The viral risk is related to the very large number of individual donors involved in the preparation of anti-hemophilic products. Wet heat or solvent/detergent treatment of products derived from plasma fractionation may make them more reliable as regards non-transmission of the human immunodeficiency virus (HIV) and the non-A non-B hepatitis virus2 However, the complex technology used for industrial fractionation and the decreased production yield following viral inactivation have result in a high cost for these products. We have explored an alternative to these products; factor VIII obtained by cryoprecipitation of plasma collected by plasma exchange (PE) from a small number of highly motivated and dedicated donors. We follow a procedure similar to that described by McLeod et d3 including injection of DDAVP just prior to donation. PE allows the collection of a large volume of plasma, and the use of DDAVP, a structural analogue of vasopressin which has been previously used in Sweden,4 gives an increased factor VIII content. The reduction in the number of
From the Centre Departement de Transfusion Sanguine de la Savoie, Faubourg Mache, B.P. 1125, 73011 Chambery Cedex, France. Received and Accepted 5/89.
253
254
Transfus. Sci.
Vol. 10, No. 3
donors lowers the risk of viral transmission. The safety of the product is further enhanced by holding it in quarantine until the donors give negative tests for hepatitis and HIV at 6 months after donation.
METHODS Donors 35 volunteer donors gave informed consent for this investigation as approved by the Ethics Committee of Claude Bernard University and Lyon’s Civil Hospitals.
Plasma Exchange Plasma exchange was carried out with a COBE 2997 blood cell separator (centrifugation) or a COBE CENTRY TPE [filtration]. Fresh plasma was collected and replaced by the autologous cryoprecipitate-supernatant of plasma removed at a previous exchange donation and preserved frozen after separation of the precipitate. Three initial plasmaphereses of 600mL provide the replacement plasma for the first exchange. The volume of plasma exchanged varied from 2 to 2.5 L. This was accomplished within a time of about 2h. Each PE was preceded by the intravenous injection of DDAVP (20 yg in 50mL of saline solution administered over 2&30 min). Donors were requested to refrain from excessive fluid intake for 8 h preceding and 24 h following a donation. Cryoprecipitate Plasma from a donation was distributed into 6 double bags of about 400mL and immediately frozen in an alcohol bath at -50°C. Cryoprecipitate was separated by centrifugation at 0°C after thawing plasma at 4°C for 1.5-2 h in a circulating water bath. The cryoprecipitate was immediately diluted with 15 mL citrate saline solution per bag. The product was then frozen again at -40°C and stored at this temperature for 6 months before use.
Dosage for individual infusions could be adjusted by combining 2 or more bags. Donor Monitoring The frequency of donation was adjusted for donor convenience, but averaged once per month. Pulse and blood pressure were recorded before and after DDAVP administration. A complete blood count, electrolyte panel, protein electrophoresis, and electrocardiogram were performed at the inception of the program and then at 6 month intervals. A transaminase assay, an assay for anti-HBc, and testing for Ag P 24 (HIV protein) and corresponding antibodies were carried out at each donation. Product was not released for transfusion until negative results had been obtained in all re-testings for a period of 6 months following the donation. Product Testing The product was assayed for factor VIII by the 2-stage method derived from the thromboplastin generation test,5 for Van Willebrand Factor antigen (VWF:Ag) by the method of Laurell,6 for fibrinogen by the chronometric method,’ and for total protein by the Biuret reaction.* RESULTS 106 exchanges have been performed. All were well tolerated by the donors, both clinically and biologically. The only symptoms reported were facial flushing linked to DDAVP injection and hypocalcemia due to citrate. The average volume of plasma collected was 2301+ 154 mL. The average factor VIII content of the plasma was 5388 f 13801U. The characteristics of cryoprecipitate from plasma collected by filtration and centrifugal apheresis were similar. The average factor VIII content was 2764 + 9221U per donation for a of separation of mean efficiency 51.3 f 13%. In the cryoprecipitate, the of factor VIII was concentration 23.8 + 7.7 IU/mL, the VWF:Ag concentration was 18.2 +3.7IU/mL, and the
Factor VIII Donation Using PE and DDAVP 255
fibrinogen concentration was 6.01+ 2.2 mg/mL. The total protein content of the cryoprecipitate was 19.2 f 57mg/mL, so that the average specific activity for factor VIII was 1.22 + 0.48 IU/mg protein (Table 1). The cost of one IU of factor VIII prepared in this way was estimated to be 1.3 Fr. DISCUSSION The prevention of viral transmission is a natural goal of transfusion in general and hemophilia therapy in particular. While we hope that products of a very high degreee of purity (proteins and viruses) will be obtained by genetic engineering in the future, replacement therapy in hemophilia is currently based upon plasma-derived anti-hemophilic factor concentrates treated with heat, or with solvents and detergents (SD), to inactivate viruses. This is a pilot study based on McLeod’s work. However it differs in many aspects, including, for instance, the way the donors are selected and the repetitive screening and testing during a prolonged quarantine. This delay in release means that the product is probably very safe both in regards to HIV and also chronic hepatitis. The donor in our study is an anonymous volunteer, according to the French ethical standards. This status is desired by our patients as the hemophiliacs do not like the psychological ties which in some cases reveal parental problems. Generally the donor determines the frequency of his donation, but our volunteers are very keen to give factor VIII every month. We did not observe any
serious adverse effects of DDAVP. No unrelated volunteer donors have rejected the infusion of DDAVP after signing an informed consent. Several ethics committees, including those in Sweden, Chicago9 and Lyon, have approved the procedure. In fact, there is no restriction for the use of DDAVP in mild hemophilia and Von Willebrand disease except the decreasing response. In this study, the joint use of plasma exchange and DDAVP allows us to obtain a cryoprecipitate product, the composition of which is quite comparable to that of factor VIII SD from the fractionation industrylO as shown in Table 1. The use of DDAVP is not a disadvantage. Its action on the liberation of plasminogen activators was checked on a restricted number of donors. The results showed highly variable individual sensitivity, rapid inhibition of these activators, and absence of these materials in the final product. The duration of plasma exchange does not appear to be an impediment, since no donor cancelled a scheduled donation. Furthermore plasma exchange has had no adverse clinical or biological consequences. Immediate invivo recovery was measured in several recipients and found to be about 2% per IU infused per kg, in agreement with the results of McLeod et ~1.~The only significant drawback is the need to keep the cryoprecipitate frozen, and a study of freeze-drying has been implemented. This procedure is efficient at two levels: (1) it recovers about 50% of the factor VIII in the raw material, as opposed to only 10-15% for industrial fractionation, and (2) the sale price is markedly lower than that of the industrial pro-
Table 1. Protein Composition and Degree of Purity of Different Preparations Cryoprecipitate
Components Factor VIII:C VWF:Ag Fibrinogen Total proteins Specific activity Purity (approx.) 1s 10:3-G
Plasma 200 ng 20 lJg 3mg 70 mg O.O14U/mg 0.003%
from PE 200 ng 16.5 pg
0.3 mg 0.82 mg 1.22 U/mg 0.024%
SD Concentrate Intermediate Purity 200 ng 32Yg
0.2mg 1.8mg
0.6-0.8 U/mg 0.012%
256
Transfus. Sci. Vol. 10, No. 3
ducts. This strategy could be adapted to a transfusion organization lacking a fractionation capacity. The viral safety of the product is afforded by the limited number of highly motivated and responsible donors allowed by plasma exchange and DDAVP. Also, the 6-month re-testing interval between donation and release of product will allow detection of seroconversion in an individual who donates in the early stages of HIV or hepatitis infection. The product complies with the recommendations of the National Hemophilia Foundation in the United States to attending physicians of hemophilic subjects. This foundation has indeed proposed in its bulletin dated February 13, 198811that the use of cryoprecipitate prepared from a single, repetitively tested, selected donor or from a small number of such donors as an alternative to industrial products.
3.
4.
5.
6.
7. 8. 9.
10. REFERENCES 1. Ratnoff OD: Some complications of the therapy of classic hemophilia. I Lab Clin Med 1984; 103:653-659. 2. Horowitz MS, Rooks C, Horowitz B,
11.
Hilgartner MW: Virus safety of solvent detergent treated antihemophilic factor concentrate. Lancet 1988; July:186-189. McLeod BC, Sassetti RJ, Cole ER, Scott JP: A high potency, single donor cryoprecipitate of known Factor VIII content dispensed in vials. Ann Intern Med 1987; 106:35-40. Nilson IM, Walter H, Mikaelsonn M, Vilhardt H: Factor VIII concentrate prepared from DDAVP stimulated blood donor plasma. Stand 1 Haematol 1979; 22 ~42-46. Biggs RM, Eveling J, Richards G: The assay of antihemophilic globulin activity. Br / Haematol 1955; 1:20. Laurel1 CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 1966; 15:45-52. Clauss A: Acta Haematol 1957; 17:237. Kingsley GR: IBiol Chem 1939; 131: 197200. McLeod BC, Sassetti RJ, Cole ER, Scott JP: Long term frequent plasma exchange donation of cryoprecipitate. Transfusion 1988; 28(4):307-310. In pamphlet distributed by Biotransfusion, B.P. 99, 91943 Les Uelis Cedex, France. The National Hemophilia Foundation, Med. Bulletin, Ref. 66, Chap. 72. February 19, 1988.