On the Way to Virus-Safe Blood Coagulation Factor Concentrates

In 1972, I took over the Kurpfalzkrankenhaus of the ``Stiftung Rehabilitation'' in Heidelberg, a hospital for internal medicine and neurology that was then newly constructed. At that time it was still called ``Rehabilitationsklinik Heidelberg.'' The university hospital for internal medicine offered me the opportunity to take a part of the hemophilia treatment with me to the new hospital. So, I founded the supraregional Hemophilia Center Heidelberg and soon had more than 100 persons with hemophilia as my patients. Already in 1972, a factor (F) VIII concentrate of higher purity was available. However, this concentrate was still in somewhat short supply. It could be infused in shorter time and did not have to be infused as slowly as usual. One of my patients, who underwent his vocational training nearby, never came to see me for treatment between his classes when he had an acute joint bleeding but only after, and then it was virtually too late. He did not want to miss a lecture. Once we had the new concentrate, which could be infused more quickly, he came for treatment during his 15-minute break between classes. That was the beginning of early treatment. Moreover, this concentrate enabled us to give our patients sufficient replacement therapy, what at least we considered to be sufficient, and also allowed us to begin to introduce self-treatment. However, this also meant that we stopped using cryoprecipitate from only a few donations but began using concentrates that were manufactured from larger plasma pools from many donors. To translate ``pool,'' the German dictionary also allows us to use ``Tümpel,'' which is a very small and dirty pond, for it came to light that these ``Tümpels'' were contaminated by viruses. In this context, I must express myself correctly, for the cryoprecipitates were also ``Tümpel.'' Therefore, we had to warn all patients about hepatitis before infusing the concentrate. After using concentrates for 1 year I looked into the clinical records of 114 patients and found that during the year, 17 of them (15%) had developed infectious jaundice. This corresponded with findings in the international literature (Table [1]).[1] [2] [3] [4] [5] [6] [7] [8] The average incubation period after beginning intensive replacement therapy was 46 days (Table [2]).[8] It was believed that this was an indication of non-A, non-B (NANB) hepatitis. Until the onset of jaundice, the average amount of concentrate that had been infused was 10,827 units, with considerable variations in amounts (Table [3]).[8]

Although only 15% of the 114 patients developed jaundice, the infection rate was nearly 100%, showing serological markers of hepatitis B (Table [4]).[8] [10] What did this mean? Looking for an explanation, I came across an article published by Creutzfeldt et al,[9] who in 1966 had done a prospective study and observed posttransfusion hepatitis in nonhemophilic patients who had received blood transfusions. Of those patients who had received more than 10 units of whole blood, 14.3% had developed icteric posttransfusion hepatitis (Table [5]).[9] This corresponded with our findings.

However, Creutzfeldt et al were not satisfied with only diagnosing the clinical picture of jaundice but observed and monitored the transaminase levels and performed biopsies on all patients who had received transfusions. They found anicteric hepatitis in a further 14.3%. Looking at Creutzfeldt's patient cohort (Table [5]), one will note that those patients who had received fewer transfusions had developed less jaundice but had developed anicteric hepatitis approximately as frequently as those patients who had received a greater number of transfusions. What did this mean for our patients with hemophilia?

To find an answer to this question, we decided to perform liver biopsies on our patients with hemophilia.[8] [10] [11] [12] [13] In the medical literature, there were only a few reports published on liver biopsies in patients with hemophilia.[14] If liver biopsies had been tried at all, also later,[15] [16] [17] [18] they were performed only in patients with elevated transaminase levels. To get an insight into the real situation, we decided not to do it in the same manner.

We asked all patients with hemophilia who had to undergo surgery for their consent to perform a biopsy after starting anesthesia. We also informed them that the histological findings would not have any therapeutic consequences; this corresponded to what was then the treatment standard. The histological examinations were conducted by Uwe Bleyl, M.D., from the Institute for Pathology of the Faculty of Medicine in Mannheim of the Heidelberg University. Table [6] shows that, in general, single values of the transaminases do not give any clue concerning the histological state of the patient's liver.[8] [11] [12] [19] However, the more dramatic the histological findings were, the more elevated were the average values of the transaminases.

Until the early 1980s, 51 biopsies had been performed on 43 patients.[13] [19] Tables [7] [8] [10] [11] [12] and [8] [8] [10] [11] [12] [15] [16] [17] [18] [19] [32] show the distribution of liver damage. In the 1970s, however, when the number of our biopsy findings was smaller,[8] [10] the distribution was similar. In 66% of diagnoses, chronic hepatitis was found, and in these 66% are included 9% of the total number of patients in whom liver cirrhosis was present, although patients had not been selected by the criteria of elevated transaminase levels. Later, all biopsy samples were sent to Dr. Aledort in New York for a multicenter study.

At a seminar held at the Medizinische Klinik of the University in Giessen in the mid-1970s, I reported on the problems of hepatitis caused by replacement therapy in hemophilia. After the discussion that followed, Dr. Heimburger approached me. He was looking very upset when he said to me, ``Dr. Schimpf, I am shocked that my nice little factor VIII concentrate Haemate causes such damage.'' We asked ourselves, ``Which side effect of the FVIII therapy must be considered to be the most hazardous?'' and we agreed on hepatitis. Consequently, Dr. Heimburger said, ``Dr. Schimpf, I promise that I will do something.'' What followed? In 1979, the pasteurized Haemate HS^℗ was clinically tested, and in 1981, it was available on the market. It was manufactured by 80% from plasma of United States origin. We all were anticipating an increase of FVIII inhibitors from infusion of this concentrate, but this did not happen. The first supply of Haemate HS was insufficient, and the hemophilia centers in Germany, therefore, agreed informally to give Haemate HS only to those patients with hemophilia who were treated for the first time and then also continue treatment with Haemate HS. For obvious reasons, no control group of new and untreated patients treated with old preparations could be created, for we were hoping for a decrease of posttransfusion hepatitis. At a hearing at the German Federal Health Office in 1983, I speculated that the reduction of infections would be approximately 50%.

Now let me mention human immunodeficiency virus (HIV), the most dangerous virus transmitted through blood products. It seemed after some time that HIV in the contaminated plasma would be more susceptible to virus inactivation than hepatitis viruses would be. This also holds true for pasteurization. To prove this clinically, all patients in Germany and Austria who had been treated exclusively with Haemate HS and had not received any other plasma products up to December 1986 were enrolled in a study; there were 155 patients (Fig. [1]).[20] Their data were collected until September 1988. The entire patient cohort was given a total of 15,916,260 units of Haemate HS. Table [9] [20] shows how the varying total amounts per patient were distributed among the individual patients of the cohort. By September 1988, 657 anti-HIV tests had been done in this cohort, and all patients had remained seronegative (Table [1]).[20] The longest preceding treatment period with Haemate HS was 110 months (Table [1]).[20] Most surprising was the finding that, in 68 of the 155 enrolled patients, therapy had already been started prior to April 1983 (Table [1]).[20] April 1983 was the date when the Centers for Disease Control (CDC) in the United States introduced criteria for eliminating high-risk blood or plasma donors. All those patients of the study cohort who had received replacement therapy before April 1983 remained seronegative; these were the patients who had been given Haemate HS that had been manufactured from, presumably, plasma that was highly contaminated with HIV (Table [1]). The statistical numbers we had, that is, the 68 patients with a high risk for HIV infection and the total of 155 noninfected patients, meant that the findings were of statistical significance, although there was no control group.[21] Thus, pasteurization had obviously safely inactivated HIV in the source plasma, even in times when plasma pools were contaminated with HIV to a higher degree than later. Our publication was the first study that proved with statistical significance the HIV safety of a concentrate. Other studies with smaller numbers of patients that were published later substantiated our findings.

We were able to present the results of a similar retrospective multicenter study, in which 60 German, Austrian, and Italian patients with hemophilia were enrolled, in the form of an abstract at the International Society on Thrombosis and Haemostasis (ISTH) Congress held in Brussels in 1987.[22] These patients had been given a vapor-treated FVIII concentrate and also remained seronegative. However, the patient data were less detailed than in the study with Haemate HS. Also, the period of observation did not go back that far; the longest observation period was 24 months. Moreover, several of these patients had been pretreated with other concentrates but were HIV-seronegative when treatment was switched to the vapor-treated concentrate (Tables [12] and [13]).[22]

Together with H.-H. Brackmann and T. Mandalaki, we prepared another retrospective study for publication in which 56 patients with hemophilia B were enrolled who had been given prothrombin complex concentrate (PCC) that had been virus inactivated by means of ultraviolet light and β-propiolactone. Then the notorious incidence occurred, namely, that 8 patients who were treated with this concentrate became infected with HIV.

We also investigated the hepatitis safety of Heimburger's Haemate HS in a study with participation from Germany, Austria, and Italy, in which previously untreated patients who had been given Haemate HS were prospectively studied (Fig. [2]).[23] We were unable to find more than 26 suitable young patients within an acceptable period of time. In this study, the transaminases were monitored biweekly over 4 months and then monthly for another 2 months. Testing for hepatitis B markers was done after 4, 6, and 12 months. All parameters were within normal ranges, and when the study was completed, there was no indication of NANB hepatitis or hepatitis B virus (HBV) infection. This study, which was finally published in 1987, was long regarded as the gold standard. The graphic presentation of the transaminase levels and dosages of FVIII concentrate was later adopted by other study groups (Fig. [3]).[23] We collaborated in other prospective multicenter studies of ``hepatitis safety,'' namely a study on Beriate P<sup>℗</sup>, which is the more purified version of Haemate HS that contains less von Willebrand factor (Fig. [4])[24]; a study on vapor-treated FVIII concentrate (Fig. [5])[25]; and a study on vapor-treated FVII and FIX concentrates (Fig. [6]).[26] Because the patient cohorts of these studies did not contain more than 25 to 29 patients either, the statistical significance, as requested by established rules, was not proved, although there was no clue for infection with hepatitis viruses. The titles of the published studies were, therefore, not phrased as ``absence of'' but as ``low risk for viral infection'' or ``low risk for hepatitis C after administration of,'' and so on. There is also the prospective multicenter study published in 1985. Previously untreated patients with hemophilia A showed NANB hepatitis in 84% of cases after having been treated with a FVIII concentrate (Fig. [7]) that had been heat treated at 60°C over 72 hours in a lyophilized state for virus inactivation.[27]

In summary, the history of Haemate HS is a wonderful example of how essential it is that communication is ongoing between the treating physicians and the scientists working for the pharmaceutical industry. Dear Dr. Heimburger, thank you for the development of Haemate HS!

REFERENCES

• 1 Iwarson S, Kjellmann H, Teger-Nilsson A C. Incidence of viral hepatitis after administration of factor IX concentrates.  Vox Sang . 1976;  31 136-140
• 2 Mannucci P M, Capitano A, del Nino E. Asymptomatic liver disease in haemophiliacs.  J Clin Pathol . 1975;  28 620-624
• 3 Schwartz B, Landbeck G. Australia-SH-Antigen und Antikörper nach Mehrfachtransfusionen bei Kindern und Jugendlichen bei schwerer und mittelschwerer Hämophilie.  Dtsch Med Wochenschr . 1973;  98 2016-2019
• 4 Stampfli K. Nebenwirkungen der Substitutionstherapie. In: Landbeck G, Marx R, eds. 8 Hamburger Hämophilie-Symposium 1977 Heidelberg: Global 1978: 185-186
• 5 Sultan Y, Maisonneneuve P, Simeon J, Intratar L. Modifications of factor VIII related antigen in hemophiliacs with acute hepatitis and subclinical liver disease.  Scand J Haematol . 1977;  30(Suppl) 16-26
• 6 Tilsner V. Häufigkeit von klinisch manifester Hepatitis B bei 38 Hämophilie-Patienten unter Substitutionstherapie. In: Landbeck G, Marx R, eds. 6 Hämophilie-Symposium Hamburg 1975 Heidelberg: Immuno GmbH 1976: 65-66
• 7 Yannitsiosis A, Bossinakov I, Louizou C, Panayotopoulou R, Mandalaki T. Jaundice and hepatitis B surface antigen and antibody in Greek haemophiliacs.  Scand J Haematol . 1977;  30(Suppl) 11-15
• 8 Schimpf K, Zimmermann K. Hepatitishäufigkeit, serologische Befunde und Leberhistologie nach Therapie schwerer hämorrhagischer Diathesen mit Gerinnungsfaktorenkonzentraten. In: Schimpf K, ed. Fibrinogen, Fibrin und Fibrinkleber-Nebenwirkungen der Therapie mit Gerinnungsfaktoren-Konzentraten Verhandlungsbericht der Deutschen Arbeitsgemeinschaft für Blutgerinnungsforschung über die 23 Tagung in Heidelberg, Februar 1979. Stuttgart: Schattauer Verlag 1980: 299-308
• 9 Creutzfeldt W, Severidt H J, Schmitt H. Untersuchungen über Häufigkeit und Verlauf der ikterischen und anikterischen Transfusionshepatitis.  Dtsch Med Wochenschr . 1966;  91 1813-1820
• 10 Schimpf K, Zimmermann K, Rüdel J, Thamer G, Zeltsch P. Results of liver biopsies, rate of icteric hepatitis, and frequency of anti-HBs and HBs-antigen in patients of the Heidelberg haemophilia centre.  Thromb Haemost (Abst) . 1977;  38 340
• 11 Schimpf K, Zimmermann K, Bleyl U, Döhnert G. Liver biopsy findings in haemophilia. In: Seligsohn U, Rimon A, Horoszowski H, eds.: Haemophilia-Based on Symposia Held during the XIIIth Congress of the World Federation of Hemophilia, Tel-Aviv, Israel, 1979 Castle House Publications; 1981: 149-153
• 12 Schimpf K, Zimmermann K, Zeltsch P. Leberhistologie bei 43 Patienten mit Hämophilie A, B und Faktor VII-Mangel. Ergebnisse von 51 Biopsien In: Kommerell B, Hahn P, Kübler W, Mörl H, Weber E, eds. Fortschritte in der Inneren Medizin, Berlin: Springer-Verlag; 1982: 350-353
• 13 Schimpf K. Liver disease in hemophilia.  Transfus Sci . 1990;  11 15S-22S
• 14 Lesnesne H R, Morgan J E, Blatt P M, Webster W B, Roberts H R. Liver biopsy in hemophilia A.  Ann Intern Med . 1977;  86 703-707
• 15 Hay C RM, Preston F E, Triger D R, Underwood J CE. Progressive liver disease in haemophilia: an understated problem?.  Lancet . 1985;  II 1495-1498
• 16 Mannucci P M, Ronchi G, Rota L, Colombo M. A clinicopathological study of liver disease in haemophiliacs.  J Clin Pathol . 1978;  31 779-783
• 17 Preston F E, Underwood J CE, Mitchell V E. Percutaneous liver biopsy and chronic liver disease in haemophiliacs.  Lancet . 1978;  II 592-594
• 18 Spero J A, Lewis J H, van Thiel H D, Hasiba U, Rabin B S. Asymptomatic structural liver disease in hemophilia.  N Engl J Med . 1978;  298 1373-1378
• 19 Schimpf, K. Liver disease in haemophilia (and correction p.  572). Lancet . 1986;  II 373
• 20 Schimpf K, Brackmann H H, Kreuz W. Absence of anti- human immundeficiency virus types 1 and 2 seroconversion after the treatment of hemophilia or von Willebrand's disease with pasteurized factor VIII concentrate.  N Engl J Med . 1989;  321 1148-1152
• 21 Hanley J A, Lippman-Hand A. If nothing goes wrong, is everything all right?.  <~>Interpreting zero numerators. JAMA . 1983;  249 1743-1745
• 22 Schimpf K, Brackmann H H, Bock D. No anti-HIV seroconversion after replacement therapy with steam-treated factor VIII concentrate-a study of 60 patients with hemophilia A and von Willebrand's disease.  Thromb Haemost (Abst) . 1987;  58 346
• 23 Schimpf K, Mannucci P M, Kreuz W. Absence of hepatitis after treatment with a pasteurized factor VIII concentrate in patients with hemophilia and no previous transfusions.  N Engl J Med . 1987;  316 918-922
• 24 Mannucci P M, Schimpf K, Brettler D B. Low risk for hepatitis C in hemophiliacs given a high-purity, pasteurized factor VIII concentrate.  Ann Intern Med . 1990;  113 27-32
• 25 Mannucci P M, Schimpf K, Abe T. Low risk of viral infection after administration of vapor-heated factor VIII concentrate.  Transfusion . 1992;  32 134-138
• 26 Shapiro A, Abe T, Aledort L M. Low risk of viral infection after administration of vapor-heated factor VII concentrate or factor IX complex in first-time recipients of blood components.  Transfusion . 1995;  35 204-208
• 27 Colombo M, Carnelli V, Gazengel C. Transmission of non-A, non-B hepatitis by heat-treated factor VIII concentrate.  Lancet . 1985;  II 1-4
• 28 Schimpf K, Zimmermann K, Thamer G, Rüdel J. Hepatitishäufigkeit, HBs-Antigen- und Anti-HBs-Frequenz bei Patienten des Hämophiliezentrums Heidelberg.  Verh Dtsch Ges Inn Med . 1976;  82 414-417
• 29 Cederbaum H I, Levine P H, Blatt P M. Abnormal serum transaminase levels in patients with factor VIII deficiency. XVII Congress of the International Society of Hematology, Paris, July 24-26, 1978. 211 (Abst)
• 30 Hasiba U, Spero J A, Lewis J H. Chronic hepatitis in hemophilia.  Scand J Haematol . 1977;  30(Suppl) 27-32
• 31 Hilgartner M W, Giardina P. Liver dysfunction in patients with hemophilia.  Scand J Haematol . 1977;  30(Suppl) 6-10
• 32 Spero J A, Lewis J H, van Thiel H D, Ragni M V, Bontempo F A. Chronic liver disease in hemophilia: an update.  Blood (Abst) . 1983;  62(Suppl) 239