THROMBOSIS RESEARCH 65; 677-686,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
SAFETY OF VIRUS INACTIVATED ANTITHROMBIN III ANTITHROMBIN III IMMUNO (AT III)
CONCENTRATE
Dieter Ulrich Preiss*, Delawer Abdullah*, Bruno Eberspiiche?, Karlheinz Wilhelm*
and
* Department of Anesthesia (D.U.P.), the Department of Cardiovascular Surgery (D.A.), Benedikt Kreutz Rehabilitation Center, Siidring 15, D-7812 Bad Krozingen, FRG and # IMMUNO GmbH, D-6900 Heidelberg, FRG. (Received 2.12.1991;
accepted in revised form 1512.1991
by H.A. Vinazzer)
ABSTRACT
In a prospective clinical trial the risk of infection after application of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III) was investigated in patients undergoing cardiovascular surgery. The study was conducted according to the recommendations of the International Committee on Thrombosis and Hemostasis (ICTH), with the exception that most patients required additional blood products as well as AT III. Twenty-seven patients were eligible to test for the risk of acquiring hepatitis B. Twenty-six patients could be evaluated in terms of hepatitis NANB transmission considering ALT-levels whereas 20 patients could be tested for anti-HCV one year after surgery. Samples from 78 patients could be monitored for antiHIV-l. None of these patients showed any signs of infection. AT III IMMUNO seems to be an antithrombin III concentrate with low or absent infectivity.
Key Words: virus safety, AT III concentrate, cardiovascular surgery Address for reprints: Dr. D.U. Preiss, Department of Anesthesia, Benedikt Kreutz Rehabilitation Center, Siidring 15, D-7812 Bad Krozingen, FRG
677
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SAFETY
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Vol. 65, No. 6
Introduction Screening of plasma donors for HBsAg drastically reduced the risk of transmission of hepatitis B virus (HBV) via plasma derivatives. But at present only about 7 to 17% of all forms of posttransfusion hepatitis cases are caused by HBV (8), whith the majority being caused by hepatitis C virus (HCV) and other hepatitis NANB viruses. One major problem with hepatitis NANB viruses is the relatively high rate of progression towards chronicity. Sixty to 85% of all patients with posttransfusion hepatitis NANB test positive for HCV antibodies. These can be detected predominantly in patients with chronic hepatitis NANB (60 to 80%), whereas only 15 to 25% of those with acute, resolving infections are anti HCV positive (5,12,15,16,17). Therefore, a major goal during production of plasma derivatives is the prevention of transmission of hepatitis NANB viruses and HIV. In a prospective clinical trial, a virus inactivated antithrombin III concentrate, ANTITHROMBIN AT III IMMUNO (AT III), has been investigated for its safety with regard to transmission of hepatitis viruses and HIV-1 in cardiac surgical patients.
The study followed the recommendations made by the International Committee on Thrombosis and Hemostasis (ICTH) in November 1984 (13) with a slightly modified protocol: most patients required other blood products in addition to AT III. Only patients lacking markers of hepatitis B, testing negative for anti-HIV-l, and having normal preoperative values of alanine aminotransferase (ALT) were studied. The antithrombin III concentrate under investigation (ANTITHROMBIN III IMMUNO) was manufactured from pooled plasma by IMMUNO AG, Vienna. Only plasma from donors seronegative for both HBsAg and for anti-HIV-l, and who had ALT levels less than twice the upper limit of normal, was used. During the production process, each lot was heated in aqueous solution for 10 hours at 60°C at pH 7.5 in the presence of 0.5 M citrate, in order to inactivate residual bloodborne viruses. AT III was given to patients with extracorporeal circulation to improve heparin activity in a
state of hypercoagulation, in order to establish a hemostatic equilibrium and to reduce postoperative platelet consumption (4,lO). There have been two groups of patients with regard to their availability for regular blood sampling and the tests that have been performed. Group I: Blood samples were drawn immediately after induction of anesthesia and biweekly for 4 months. Additional samples were taken at 6 and at 12 months post surgery. ALT was measured in every sample (BOEHRINGER- MANNHBIM, upper limit of normal range: male 23 u/l, female 19 u/l). HBsAg (RIAQUICK HBsAg, IMMUNO), anti-HBs
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(RIAQUICK Anti-HBs, IMMUNO) and anti-HBc (CORAB, ABBOTT) were determined in samples taken immediately after induction of anesthesia, and also 3, 6, and 12 months postoperatively. Anti-HCV (ORTHO HCV ANTIBODY EUSA TEST SYTEM) was tested retrospectively at various time intervals in samples as far as they were still available. Anti-HIV-l (ETI-HTLV-III-K, SORIN BIOMEDICA) was determined in samples taken preoperatively, and also 3,6, and 12 months postoperatively. Group II: Anti-HIV-l (ETI-HTLV-III-K, SORIN BIOMEDICA) was measured in samples taken preoperatively and 12 months after surgery or later. Criteria used in defining transmission of infectious diseases were determined as follows: Hepatitis NANB is defined by an increase in ALT beyond 2.5 times the upper limit of the normal range in two consecutive samples within the four month period if other causes can be excluded; hepatitis B is diagnosed if hepatitis B-markers (HBsAg, anti-HBs, anti-HBc) occur within 6 months after surgery; HIV-l-transmission is assumed if patients develop anti-HIV-l antibodies within 12 months postoperatively. HCV-transmission is assumed if patients develop anti-HCV antibodies within 12 months after surgery.
Results Group I: Twenty-seven out of 28 patients entered the study and could be continually monitored according to ICIH-criteria for up to 12 months postoperatively. One patient (patient 7) died after having completed the initial 6 months period. Patients received an average of 1.230 I.U. of ANTITHROMFJIN III IMMUNO from 12 different lots. Surgical procedures are listed in table 1; transfusion requirements are given in table 2. During the observation period, patient 8 tested anti-HBs positive 6 months postoperatively but she was negative in terms of HBsAg and anti-HBc. Three months after surgery all of her HBV markers had been negative and ALT-levels stayed normal throughout the initial 4 months period. At month 6, patient 8 had a common bile duct obstruction due to gallstones. A peak AUT-level of 465 was observed that normalized quickly after the gallstones had been removed. Anti-HIV-l was negative whenever tested. The occurrence of isolated anti-HBs is somewhat disturbing. One would expect anti-HBc to precede anti-HBs and that the patient should have tested anti-HBc positive either 12 weeks or 6 months postoperatively. The patient lived alone; there were no family members who could be checked for HBV markers. Patient 8 had received blood and blood derivatives of 11 different donors in addition to AT III concentrate. When these donors were tested one year later, two of them showed signs of a recently acquired hepatitis B infection: they were positive in terms of anti-HBs, anti-HBc and anti-HBe.
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Table 1 Surgical procedures in group I patients
TypeofSWPY
Number of patients
2xCABG 3xCABG 4xCABG SxCAElG
n= Il= n= n=
Closure of aorto-atrial fistula
n= 1
Aortic valve replacement
n= 3
+ 3xCABG + 4xcABG
n= 2 n=l
Mitral valve replacement
n= 1
7 4 5 4
For this reason, the data of patient 8 had to be omitted from the evaluation of the risk of transmitting hepatitis viruses via AT III, but they could be used to calculate the risk of HIV-infection. ALT-data of patient 18 could also not be considered because of two consecutive samples missing (table 3). Results showed 26 patients whose ALT-values never reached or exceeded 2,5 times the upper limit of normal, and 27 patients testing negative whenever tested for hepatitis Bmarkers, respectively. Only a limited number of samples were available to investigate the risk of HCV-transmission (table 5). Twenty patients could be checked for anti-HCV one year after surgery, and all were found to be negative. Twenty-seven patients were monitored for anti-HIV-l for one year (as mentioned, patient 7 could be followed for 6 months only). None showed any signs of HIV-transmission. Group II: Fifty-One patients who were not available for regular screening of infection markers could be tested for anti-HIV-l twelve months postoperatively or later. These patients also stayed negative. This gives a total of 78 patients without HIV-l-infection according to ICTH criteria (table 4).
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VIRUS SAFETY AND AT III CONCENTRATE
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Table 3 Serial levels of Serum Alanlne Aminotmnsferase (U/I) in group I patients treated with ANTllHROMBlN Ill IMMUNO (2.5 times Upper Limit of Normal: male 57,5 U/I, female 47,s U/I) Patient
1
sex
Week
0
1
2 3 4 5 6 7 6 9
m m m m m m m
2
16
18
20
22
Month 6 12
4
6
8
10
12
14
12
15
13
14
10
8
10
11
14
t 10 9
40
16
17
12
16
16
14
12
12
12
10
6
12
6
10
10
10
10
2
6
8
27
31
30
20 6 7 43
10
13
12
10
8
9
10
8
6
5
10
6
15
30
10
23
11
12
13
18
13
19
7
9
4
11
4
465
6
6
6
6
11
6
10
6
7
6
8
5
10
12 8 8
19
f
18
20
20
m
15
22
26
22 22
m
9
12
10
10
9
10
10
9
9
11
13
11
m
4
6
7
6
26
10
7
9
8
9
16
12
f
12
10
9
9
11
9
10
9
10
15
13
f
13
15
5
5
8
6
7
8
6
17
14
m
3
8
6
8
8
10
8
8
9
11
12
6
10
17
10
20
22 10
9
15
m
16
12
10
10
5
6
5
16
f
26
24
18
18
24
29
19
17
f
6
13
12
12
8
13
14
12
16
m
8
10
8
8
6
6
4
6
4
5
5
6
5
6
11
14
12
12
11
10
10
14
19 3
5
19
m
10
20
m
8
10
21
f
10
8
4
8
8
8
5
8
10
5
10
22
m
10
10
15
9
6
13
7
10
11
11
12
23
m
5
8
8
6
8
8
10
12
9
13
9
24
m
16
16
10
15
12
10
10
8
8
10
13
25
m
14
10
10
24
12
12
10
11
12
26
m
13
10
8
8
6
9
15
15
18
14
16
27
m
6
10
6
10
10
8
8
10
10
12
13
10
10
7
13
10
12 10
12
9
10
m
9
5
11
12
26
6
7 16 14
20 20
1120
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VIRUS SAFETY AND AT III CONCENTRATE
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Table 4 Results of safety study with virus inactivated antitluombii III concentrate (ANTITHROMBINIII IMMUNO) I 1 Type
I
I
1 Observation period months)
of infection
1 Number of
eliglble patients
Cases of infection O/26 o/20 O/27 O/78
26 20 27 78
4 12 6- 12 2 12
Hepatitis NANB Hepatitis C Hepatitis B HIV-l-infection
I 1
Table 5: Samples tested for anti-HCV in group I patients Patients 1 2 3 4 5 5 7 3 3 LO 11 12 13 14 15 16 17 18 19 m 21 22 23 24 25 26 27 28
&
gw
low
l2w
14w 15w 16w 17w l&v
26w 27w 3&v
ly
>ly
neg *et3
*eg neg
*eg
neg
*eg
neg
neg
neg
neg
neg neg neg
neg
netI
neg neg
*eg
w
*eg
=-g neg
*eg
nei3 *et3 -3
*eg
*eg *eg
w3
*eg w
*eg
neg nei3
*et3 w3
*eg neg
ne.3
*eg
*ei3
*ef3
w neg w
I 1
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VIRUS SAFETY AND AT III CONCENTRATE
Vol. 65, No. 6
Blood derivatives in general carry the risk of virus transmission. While different screening tests have been established (tests for HBsAg, antSIBs, anti-HIV and ALT) in order to reduce the viral burden of the plasma pool used in the production process, additional virus inactivation procedures are required to yield a noninfectious product. The efficiency of such a procedure can be shown by virus inactivation experiments, chimpanzee studies and clinical trials, the latter of which are mandatory to assess the clinical safety. Thirteen volunteers who received a virus inactivated AT III concentrate and were followed according to ICTH criteria stayed free from infections (2). However, no data have been published concerning the risk of infection after AT III-substitution in patients. The risk of acquiring hepatitis while undergoing cardiac surgery is 1.8 - 6.3 % in patients not given large-pool preparations of blood derivatives (1,9,11,17). When non virus inactivated products derived from large plasma pools are given additionally, hepatitis develops in 42-70% of the patients (6,11,14). This prospective clinical trial to evaluate the safety of ANTITHROMBIN III IMMUNO in terms of transmission of viral diseases is the first study in patients receiving AT III concentrate and being thoroughly controlled according to ICIH-criteria. Six months after surgery, patient 8 was antSIBs positive; but no other HBV-markers such as HBsAg or anti-HBc could be found. Patient 8 had been treated with blood and blood derivatives from donors who showed signs of a hepatitis B infection when tested one year after. The period of 3 months for the occurrence of anti-HBs seems to be rather short (3). One could argue that this time interval is sufficient for HBsAg to arise and disappear again, but then one would at least expect to detect anti-HBc. ALT-levels stayed normal up to six months postoperatively when a transient increase in relation with a common bile duct obstruction was observed. After gallstone removal, ALT-values returned to normal levels very quickly. Whether patient 8 developed a hepatitis B infection or not could not be clarified. Therefore, her data could not be used for evaluation of the risk of hepatitis-transmission, but were considered for assessment of the risk of HIV-transmission. The “Rule of Three” (7,13) is a biostatistical approach for quantitative evaluation of clinical safety data when no cases of infection occured in the study group. Accordingly, the 95% confidence interval of the true risk of acquiring an infection after substitution with ANTITHROMBIN III IMMUNO can be calculated as O-12% for hepatitis NANB, O-15% for hepatitis C, and O-11% for hepatitis B. For HIV-l infection, the true risk range is 04%. The data from this study suggest ANTITHROMBIN III IMMUNO to be an antithrombin III concentrate with low to absent infectivity which could be demonstrated in a group of patients particularly prone to virus infection.
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685
We wish to thank Mrs. G. Ullmann for her valuable organisational assistance.
References
1. AYMARD J., JANOT C., GAYET S., GUILLEMJN C., CANTON P., GAUCHER P., STREIFF F. Post-transfusion Non-A, Non-B hepatitis after Cardiac Surgery. VOXSung, 51: 236-238.1986 R, WEIDMANN E, MERKLE W, BOCK H, MAULER R, HILFENHAUS J. Virus Safety of a Pasteurized Antithrombin III Concentrate. Anneim.-Forsch./Dnzg Res., 37 (II), Nr. 7: 759-762, 1987
2. CLEMENS
3. DEINHARDT F, GUST I. Viral hepatitis Bulletin WHO, 60: 661-691, 1982
4. DIETRICH W, SCHROLL A, GijB E, BARANKA Y A, RICHTER J. AntithrombinIII-Substitution zur Optimierung der Heparinwirkung w&rend extrakoporaler Zirkulation in der Herzchirurgie. Arzczesthakt, 33: 422-427,1984 5. ESTEBAN J, ESTEBAN R, VILAMIU L, LOPEZ-TALAVERA J, GONZALEZ A, HERNANDEZ J, ROGET M, VARGAS V, GENESCA J, BUTI M, GUARDIA J, HOUGHTON M, CHOO Q, KU0 G. Hepatitis C Virus Antibodies Among Risk Groups in Spain. Lancet, II: 294-296,1989 6. GERBER A, ENGLENDER S, SELVEY D, CARLSON I, MATTHEWS D, WEBSTER H, CALDWELL G. An Outbreak of Non-A, Non-B Hepatitis Associated with the Infusion of a Commercial Factor IX Complex during Cardiovascular Surgery. VOXSang., 58: 270-275,199O 7. HANLEY J, LIPPMAN-HAND A. If Nothing Goes Wrong, IS Everything All Right? JAMA ,249: 1743-1745,1983 8. HOOFNAGLE J. Posttransfusion hepati
8. HOOFNAGLE J. Posttransfusion hep
9. HOPPE I. Hepatitis Safety of a New Prothrombin Complex Preparation, Sterilized According to the Method of LoGrippo. Results of a Prospective Clinical Trial. Blut, 50: 363-368,1985 10. KESTEVEN P, AHMED A, WILLIAMS B, SAVIDGE G. Antithrombin III Concentrates in Cardiopulmonary Bypass. Thmmb. Haemost. 54: 89,1984
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11. KUNKEL B, SPITZER E, SATTER P, KALTENBACH M. Hepatitis bei kardiochirurgischen Patienten. In: Plasmaprokne und Viihepatitk G FRijSNER, H LASCH, E LECHLER (Eds), Springer-Verlag 1982, pp 39 - 48 12. KU0 G, CHOO Q, ALTER H, GITNICK G, REDEKER A, PURCELL R, M-IYAMURA T, DIENSTAG J, ALTER M, STEVENS C, TEGTMEIER G, BONINO F, COLOMBO M, LEE W, KU0 C, BERGER K, SHUSTER J, OVERBY L, BRADLEY D, HOUGHTON M. An Assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A, Non-B Hepatitis Science 244: 362-364, 1989 13. MANNUCCI P, COLOMBO M. Revision of the Protocol Recommended for Studies of Safety from Hepatitis of Clotting Factor Concentrates. Thromb. Huemost. 61: 532-534, 1989 14. MATTHEWS D, HARMON R, ENGLENDER S, NOVICK L, CALDWELL G. NonA, Non-B Hepatitis Associated with a Factor IX Complex Infused During Cardiovascular Surgery - Arizona. M4WZ? 35: 391-399,1986 15. MOSLEY J, AACH R, HOLLINGER F, STEVENS C, BARBOSA L, NEMO G, HOLLAND P, BANCROFT W, ZIMMERMANN H, KU0 G, CHOO Q, HOUGHTON M. Non-A, Non-B Hepatitis and Antibody to Hepatitis C Virus JAM4 263: 77-78, 1990 16. ROGGENDORF M, DEINHARDT F, RASSHOFER R, EBERLE J, HOPF U, MdLLER B, ZACHOVAL R, PAPE G, SCHRAMM W, ROMMEL F. Antibodies to Hepatitis C-Virus. Lancet II: 324-325, 1989 17. VAN DER POEL C, REESINK H, LELJE P, LEENTVAAR-KUYPERS A, CHOO Q-L, KU0 G, HOUGHTON M. Anti-Hepatitis C Antibodies and Non-A, Non-B PostTransfusion Hepatitis in the Netherlands. Lancet II: 297-298,1989
SAFETY OF VIRUS INACTIVATED ANTITHROMBIN III ANTITHROMBIN III IMMUNO (AT III)
CONCENTRATE
Dieter Ulrich Preiss*, Delawer Abdullah*, Bruno Eberspiiche?, Karlheinz Wilhelm*
and
* Department of Anesthesia (D.U.P.), the Department of Cardiovascular Surgery (D.A.), Benedikt Kreutz Rehabilitation Center, Siidring 15, D-7812 Bad Krozingen, FRG and # IMMUNO GmbH, D-6900 Heidelberg, FRG. (Received 2.12.1991;
accepted in revised form 1512.1991
by H.A. Vinazzer)
ABSTRACT
In a prospective clinical trial the risk of infection after application of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III) was investigated in patients undergoing cardiovascular surgery. The study was conducted according to the recommendations of the International Committee on Thrombosis and Hemostasis (ICTH), with the exception that most patients required additional blood products as well as AT III. Twenty-seven patients were eligible to test for the risk of acquiring hepatitis B. Twenty-six patients could be evaluated in terms of hepatitis NANB transmission considering ALT-levels whereas 20 patients could be tested for anti-HCV one year after surgery. Samples from 78 patients could be monitored for antiHIV-l. None of these patients showed any signs of infection. AT III IMMUNO seems to be an antithrombin III concentrate with low or absent infectivity.
Key Words: virus safety, AT III concentrate, cardiovascular surgery Address for reprints: Dr. D.U. Preiss, Department of Anesthesia, Benedikt Kreutz Rehabilitation Center, Siidring 15, D-7812 Bad Krozingen, FRG
677
678
VIRUS
SAFETY
AND AT III CONCENTRATE
Vol. 65, No. 6
Introduction Screening of plasma donors for HBsAg drastically reduced the risk of transmission of hepatitis B virus (HBV) via plasma derivatives. But at present only about 7 to 17% of all forms of posttransfusion hepatitis cases are caused by HBV (8), whith the majority being caused by hepatitis C virus (HCV) and other hepatitis NANB viruses. One major problem with hepatitis NANB viruses is the relatively high rate of progression towards chronicity. Sixty to 85% of all patients with posttransfusion hepatitis NANB test positive for HCV antibodies. These can be detected predominantly in patients with chronic hepatitis NANB (60 to 80%), whereas only 15 to 25% of those with acute, resolving infections are anti HCV positive (5,12,15,16,17). Therefore, a major goal during production of plasma derivatives is the prevention of transmission of hepatitis NANB viruses and HIV. In a prospective clinical trial, a virus inactivated antithrombin III concentrate, ANTITHROMBIN AT III IMMUNO (AT III), has been investigated for its safety with regard to transmission of hepatitis viruses and HIV-1 in cardiac surgical patients.
The study followed the recommendations made by the International Committee on Thrombosis and Hemostasis (ICTH) in November 1984 (13) with a slightly modified protocol: most patients required other blood products in addition to AT III. Only patients lacking markers of hepatitis B, testing negative for anti-HIV-l, and having normal preoperative values of alanine aminotransferase (ALT) were studied. The antithrombin III concentrate under investigation (ANTITHROMBIN III IMMUNO) was manufactured from pooled plasma by IMMUNO AG, Vienna. Only plasma from donors seronegative for both HBsAg and for anti-HIV-l, and who had ALT levels less than twice the upper limit of normal, was used. During the production process, each lot was heated in aqueous solution for 10 hours at 60°C at pH 7.5 in the presence of 0.5 M citrate, in order to inactivate residual bloodborne viruses. AT III was given to patients with extracorporeal circulation to improve heparin activity in a
state of hypercoagulation, in order to establish a hemostatic equilibrium and to reduce postoperative platelet consumption (4,lO). There have been two groups of patients with regard to their availability for regular blood sampling and the tests that have been performed. Group I: Blood samples were drawn immediately after induction of anesthesia and biweekly for 4 months. Additional samples were taken at 6 and at 12 months post surgery. ALT was measured in every sample (BOEHRINGER- MANNHBIM, upper limit of normal range: male 23 u/l, female 19 u/l). HBsAg (RIAQUICK HBsAg, IMMUNO), anti-HBs
Vol. 65, No. 6
VIRUS SAFETY AND AT III CONCENTRATE
679
(RIAQUICK Anti-HBs, IMMUNO) and anti-HBc (CORAB, ABBOTT) were determined in samples taken immediately after induction of anesthesia, and also 3, 6, and 12 months postoperatively. Anti-HCV (ORTHO HCV ANTIBODY EUSA TEST SYTEM) was tested retrospectively at various time intervals in samples as far as they were still available. Anti-HIV-l (ETI-HTLV-III-K, SORIN BIOMEDICA) was determined in samples taken preoperatively, and also 3,6, and 12 months postoperatively. Group II: Anti-HIV-l (ETI-HTLV-III-K, SORIN BIOMEDICA) was measured in samples taken preoperatively and 12 months after surgery or later. Criteria used in defining transmission of infectious diseases were determined as follows: Hepatitis NANB is defined by an increase in ALT beyond 2.5 times the upper limit of the normal range in two consecutive samples within the four month period if other causes can be excluded; hepatitis B is diagnosed if hepatitis B-markers (HBsAg, anti-HBs, anti-HBc) occur within 6 months after surgery; HIV-l-transmission is assumed if patients develop anti-HIV-l antibodies within 12 months postoperatively. HCV-transmission is assumed if patients develop anti-HCV antibodies within 12 months after surgery.
Results Group I: Twenty-seven out of 28 patients entered the study and could be continually monitored according to ICIH-criteria for up to 12 months postoperatively. One patient (patient 7) died after having completed the initial 6 months period. Patients received an average of 1.230 I.U. of ANTITHROMFJIN III IMMUNO from 12 different lots. Surgical procedures are listed in table 1; transfusion requirements are given in table 2. During the observation period, patient 8 tested anti-HBs positive 6 months postoperatively but she was negative in terms of HBsAg and anti-HBc. Three months after surgery all of her HBV markers had been negative and ALT-levels stayed normal throughout the initial 4 months period. At month 6, patient 8 had a common bile duct obstruction due to gallstones. A peak AUT-level of 465 was observed that normalized quickly after the gallstones had been removed. Anti-HIV-l was negative whenever tested. The occurrence of isolated anti-HBs is somewhat disturbing. One would expect anti-HBc to precede anti-HBs and that the patient should have tested anti-HBc positive either 12 weeks or 6 months postoperatively. The patient lived alone; there were no family members who could be checked for HBV markers. Patient 8 had received blood and blood derivatives of 11 different donors in addition to AT III concentrate. When these donors were tested one year later, two of them showed signs of a recently acquired hepatitis B infection: they were positive in terms of anti-HBs, anti-HBc and anti-HBe.
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Table 1 Surgical procedures in group I patients
TypeofSWPY
Number of patients
2xCABG 3xCABG 4xCABG SxCAElG
n= Il= n= n=
Closure of aorto-atrial fistula
n= 1
Aortic valve replacement
n= 3
+ 3xCABG + 4xcABG
n= 2 n=l
Mitral valve replacement
n= 1
7 4 5 4
For this reason, the data of patient 8 had to be omitted from the evaluation of the risk of transmitting hepatitis viruses via AT III, but they could be used to calculate the risk of HIV-infection. ALT-data of patient 18 could also not be considered because of two consecutive samples missing (table 3). Results showed 26 patients whose ALT-values never reached or exceeded 2,5 times the upper limit of normal, and 27 patients testing negative whenever tested for hepatitis Bmarkers, respectively. Only a limited number of samples were available to investigate the risk of HCV-transmission (table 5). Twenty patients could be checked for anti-HCV one year after surgery, and all were found to be negative. Twenty-seven patients were monitored for anti-HIV-l for one year (as mentioned, patient 7 could be followed for 6 months only). None showed any signs of HIV-transmission. Group II: Fifty-One patients who were not available for regular screening of infection markers could be tested for anti-HIV-l twelve months postoperatively or later. These patients also stayed negative. This gives a total of 78 patients without HIV-l-infection according to ICTH criteria (table 4).
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VIRUS SAFETY AND AT III CONCENTRATE
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VIRUS SAFETY AND AT III CONCENTRATE
Vol. 65, No. 6
Table 3 Serial levels of Serum Alanlne Aminotmnsferase (U/I) in group I patients treated with ANTllHROMBlN Ill IMMUNO (2.5 times Upper Limit of Normal: male 57,5 U/I, female 47,s U/I) Patient
1
sex
Week
0
1
2 3 4 5 6 7 6 9
m m m m m m m
2
16
18
20
22
Month 6 12
4
6
8
10
12
14
12
15
13
14
10
8
10
11
14
t 10 9
40
16
17
12
16
16
14
12
12
12
10
6
12
6
10
10
10
10
2
6
8
27
31
30
20 6 7 43
10
13
12
10
8
9
10
8
6
5
10
6
15
30
10
23
11
12
13
18
13
19
7
9
4
11
4
465
6
6
6
6
11
6
10
6
7
6
8
5
10
12 8 8
19
f
18
20
20
m
15
22
26
22 22
m
9
12
10
10
9
10
10
9
9
11
13
11
m
4
6
7
6
26
10
7
9
8
9
16
12
f
12
10
9
9
11
9
10
9
10
15
13
f
13
15
5
5
8
6
7
8
6
17
14
m
3
8
6
8
8
10
8
8
9
11
12
6
10
17
10
20
22 10
9
15
m
16
12
10
10
5
6
5
16
f
26
24
18
18
24
29
19
17
f
6
13
12
12
8
13
14
12
16
m
8
10
8
8
6
6
4
6
4
5
5
6
5
6
11
14
12
12
11
10
10
14
19 3
5
19
m
10
20
m
8
10
21
f
10
8
4
8
8
8
5
8
10
5
10
22
m
10
10
15
9
6
13
7
10
11
11
12
23
m
5
8
8
6
8
8
10
12
9
13
9
24
m
16
16
10
15
12
10
10
8
8
10
13
25
m
14
10
10
24
12
12
10
11
12
26
m
13
10
8
8
6
9
15
15
18
14
16
27
m
6
10
6
10
10
8
8
10
10
12
13
10
10
7
13
10
12 10
12
9
10
m
9
5
11
12
26
6
7 16 14
20 20
1120
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VIRUS SAFETY AND AT III CONCENTRATE
Vol. 65, No. 6
Table 4 Results of safety study with virus inactivated antitluombii III concentrate (ANTITHROMBINIII IMMUNO) I 1 Type
I
I
1 Observation period months)
of infection
1 Number of
eliglble patients
Cases of infection O/26 o/20 O/27 O/78
26 20 27 78
4 12 6- 12 2 12
Hepatitis NANB Hepatitis C Hepatitis B HIV-l-infection
I 1
Table 5: Samples tested for anti-HCV in group I patients Patients 1 2 3 4 5 5 7 3 3 LO 11 12 13 14 15 16 17 18 19 m 21 22 23 24 25 26 27 28
&
gw
low
l2w
14w 15w 16w 17w l&v
26w 27w 3&v
ly
>ly
neg *et3
*eg neg
*eg
neg
*eg
neg
neg
neg
neg
neg neg neg
neg
netI
neg neg
*eg
w
*eg
=-g neg
*eg
nei3 *et3 -3
*eg
*eg *eg
w3
*eg w
*eg
neg nei3
*et3 w3
*eg neg
ne.3
*eg
*ei3
*ef3
w neg w
I 1
684
VIRUS SAFETY AND AT III CONCENTRATE
Vol. 65, No. 6
Blood derivatives in general carry the risk of virus transmission. While different screening tests have been established (tests for HBsAg, antSIBs, anti-HIV and ALT) in order to reduce the viral burden of the plasma pool used in the production process, additional virus inactivation procedures are required to yield a noninfectious product. The efficiency of such a procedure can be shown by virus inactivation experiments, chimpanzee studies and clinical trials, the latter of which are mandatory to assess the clinical safety. Thirteen volunteers who received a virus inactivated AT III concentrate and were followed according to ICTH criteria stayed free from infections (2). However, no data have been published concerning the risk of infection after AT III-substitution in patients. The risk of acquiring hepatitis while undergoing cardiac surgery is 1.8 - 6.3 % in patients not given large-pool preparations of blood derivatives (1,9,11,17). When non virus inactivated products derived from large plasma pools are given additionally, hepatitis develops in 42-70% of the patients (6,11,14). This prospective clinical trial to evaluate the safety of ANTITHROMBIN III IMMUNO in terms of transmission of viral diseases is the first study in patients receiving AT III concentrate and being thoroughly controlled according to ICIH-criteria. Six months after surgery, patient 8 was antSIBs positive; but no other HBV-markers such as HBsAg or anti-HBc could be found. Patient 8 had been treated with blood and blood derivatives from donors who showed signs of a hepatitis B infection when tested one year after. The period of 3 months for the occurrence of anti-HBs seems to be rather short (3). One could argue that this time interval is sufficient for HBsAg to arise and disappear again, but then one would at least expect to detect anti-HBc. ALT-levels stayed normal up to six months postoperatively when a transient increase in relation with a common bile duct obstruction was observed. After gallstone removal, ALT-values returned to normal levels very quickly. Whether patient 8 developed a hepatitis B infection or not could not be clarified. Therefore, her data could not be used for evaluation of the risk of hepatitis-transmission, but were considered for assessment of the risk of HIV-transmission. The “Rule of Three” (7,13) is a biostatistical approach for quantitative evaluation of clinical safety data when no cases of infection occured in the study group. Accordingly, the 95% confidence interval of the true risk of acquiring an infection after substitution with ANTITHROMBIN III IMMUNO can be calculated as O-12% for hepatitis NANB, O-15% for hepatitis C, and O-11% for hepatitis B. For HIV-l infection, the true risk range is 04%. The data from this study suggest ANTITHROMBIN III IMMUNO to be an antithrombin III concentrate with low to absent infectivity which could be demonstrated in a group of patients particularly prone to virus infection.
Vol. 65, No. 6
VIRUS SAFETY AND AT III CONCENTRATE
685
We wish to thank Mrs. G. Ullmann for her valuable organisational assistance.
References
1. AYMARD J., JANOT C., GAYET S., GUILLEMJN C., CANTON P., GAUCHER P., STREIFF F. Post-transfusion Non-A, Non-B hepatitis after Cardiac Surgery. VOXSung, 51: 236-238.1986 R, WEIDMANN E, MERKLE W, BOCK H, MAULER R, HILFENHAUS J. Virus Safety of a Pasteurized Antithrombin III Concentrate. Anneim.-Forsch./Dnzg Res., 37 (II), Nr. 7: 759-762, 1987
2. CLEMENS
3. DEINHARDT F, GUST I. Viral hepatitis Bulletin WHO, 60: 661-691, 1982
4. DIETRICH W, SCHROLL A, GijB E, BARANKA Y A, RICHTER J. AntithrombinIII-Substitution zur Optimierung der Heparinwirkung w&rend extrakoporaler Zirkulation in der Herzchirurgie. Arzczesthakt, 33: 422-427,1984 5. ESTEBAN J, ESTEBAN R, VILAMIU L, LOPEZ-TALAVERA J, GONZALEZ A, HERNANDEZ J, ROGET M, VARGAS V, GENESCA J, BUTI M, GUARDIA J, HOUGHTON M, CHOO Q, KU0 G. Hepatitis C Virus Antibodies Among Risk Groups in Spain. Lancet, II: 294-296,1989 6. GERBER A, ENGLENDER S, SELVEY D, CARLSON I, MATTHEWS D, WEBSTER H, CALDWELL G. An Outbreak of Non-A, Non-B Hepatitis Associated with the Infusion of a Commercial Factor IX Complex during Cardiovascular Surgery. VOXSang., 58: 270-275,199O 7. HANLEY J, LIPPMAN-HAND A. If Nothing Goes Wrong, IS Everything All Right? JAMA ,249: 1743-1745,1983 8. HOOFNAGLE J. Posttransfusion hepati
8. HOOFNAGLE J. Posttransfusion hep
9. HOPPE I. Hepatitis Safety of a New Prothrombin Complex Preparation, Sterilized According to the Method of LoGrippo. Results of a Prospective Clinical Trial. Blut, 50: 363-368,1985 10. KESTEVEN P, AHMED A, WILLIAMS B, SAVIDGE G. Antithrombin III Concentrates in Cardiopulmonary Bypass. Thmmb. Haemost. 54: 89,1984
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11. KUNKEL B, SPITZER E, SATTER P, KALTENBACH M. Hepatitis bei kardiochirurgischen Patienten. In: Plasmaprokne und Viihepatitk G FRijSNER, H LASCH, E LECHLER (Eds), Springer-Verlag 1982, pp 39 - 48 12. KU0 G, CHOO Q, ALTER H, GITNICK G, REDEKER A, PURCELL R, M-IYAMURA T, DIENSTAG J, ALTER M, STEVENS C, TEGTMEIER G, BONINO F, COLOMBO M, LEE W, KU0 C, BERGER K, SHUSTER J, OVERBY L, BRADLEY D, HOUGHTON M. An Assay for Circulating Antibodies to a Major Etiologic Virus of Human Non-A, Non-B Hepatitis Science 244: 362-364, 1989 13. MANNUCCI P, COLOMBO M. Revision of the Protocol Recommended for Studies of Safety from Hepatitis of Clotting Factor Concentrates. Thromb. Huemost. 61: 532-534, 1989 14. MATTHEWS D, HARMON R, ENGLENDER S, NOVICK L, CALDWELL G. NonA, Non-B Hepatitis Associated with a Factor IX Complex Infused During Cardiovascular Surgery - Arizona. M4WZ? 35: 391-399,1986 15. MOSLEY J, AACH R, HOLLINGER F, STEVENS C, BARBOSA L, NEMO G, HOLLAND P, BANCROFT W, ZIMMERMANN H, KU0 G, CHOO Q, HOUGHTON M. Non-A, Non-B Hepatitis and Antibody to Hepatitis C Virus JAM4 263: 77-78, 1990 16. ROGGENDORF M, DEINHARDT F, RASSHOFER R, EBERLE J, HOPF U, MdLLER B, ZACHOVAL R, PAPE G, SCHRAMM W, ROMMEL F. Antibodies to Hepatitis C-Virus. Lancet II: 324-325, 1989 17. VAN DER POEL C, REESINK H, LELJE P, LEENTVAAR-KUYPERS A, CHOO Q-L, KU0 G, HOUGHTON M. Anti-Hepatitis C Antibodies and Non-A, Non-B PostTransfusion Hepatitis in the Netherlands. Lancet II: 297-298,1989
