1473
could be taken and a definitive hollow-box obturator constructed. We believe that this technique has considerable advantages over the use of conventional split-thickness skin grafts in the oral cavity, and in allowing the defects to epithelialise spontaneously. Infection and scar contracture have not been observed.
impressions
Kings College Hospital Medical School, London SE5, UK
J. D.
Department of Oral Pathology, London Hospital Medical College, London E1 2AD, UK
I. M. LEIGH H. A. NAVSARIA D. M. WILLIAMS
LANGDON
JG, Green G. Serial cultivation of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975; 6: 331-44.
1. Rheinwald
Detection by PCR of hepatitis C virus in factor VIII concentrates treated haemophilia patients have chronic abnormalities of their liver enzymes and at least 20% of these patients have histological evidence of chronic active hepatitis or cirrhosis.l,2 The most important cause of this liver disease is the transmission by factor VIII concentrates of hepatitis C virus (HCV).3 The sterilisation of factor VIII concentrates by heating or by chemical methods,’ together with the self-exclusion of donors at high risk of HIV infection and donor screening for surrogate markers, has considerably reduced but not eliminated the risk of post-infusion non-A, non-B hepatitis (NANBH). Nor will screening for anti-HCV detect all potentially infectious donors.S We have found that detection by the polymerase chain reaction (PCR) of HCV RNA sequences in blood donations predicts infectivity in man (see p 1419)6 and report here the application of PCR to the detection of HCV in clotting factor concentrates. In our "nested" PCR technique6 RNA extracted from 500 III reconstituted concentrate is reverse transcribed and the cDNA is amplified with HCV-specific oligonucleotide The of the outer primers. sequence primers was 5’-ATGGGGCAAAGGACGTCCG and 5’-TACCTAGTCATAGCCTCCGTGAAG, and of the inner 5’-GAGGTTTTCTGCGTCCA and primers 5’-GCGATAGCCGCAGTTCT. 18 samples were tested, 13 from European or American paid-donor pools and 5 manufactured in the UK from volunteerdonor pools (table). A striking correlation was observed between the presence of HCV RNA in a sample and the likelihood of that product transmitting NANBH. Except for sample 14, an NHS intermediate purity product derived from fewer than 3000 donations, all the unheated concentrates contained HCV RNA. Unheated concentrates have almost invariably resulted in NANBH when administered to previously untreated patients with
SIR,-Most regularly
HCV RNA SEQUENCES IN CLOTTING FACTOR CONCENTRATES*
haemophilia.7 HCV sequences were not detected in any of the three batches of "superheated" material (80°C for 72 h) whereas they were found in both batches of a concentrate heated less intensively (60°C for 32 h). Whilst the superheated material has not been implicated in NANBH,8 hepatitis has occurred in most patients receiving the less intensively heated products.4 Samples 9 and 11, both from "wet" (heptane) treated concentrates, provide further evidence for a correlation between PCR positivity and infectivity: sample 9 (PCR positive) was from a batch of high infectivity9 whereas sample 11 (PCR negative) was apparently non-infectious.9 Recipients of concentrates represented by PCR-positive samples 7, 9, and 10 are known to have developed post-infusion HCV viraemia (unpublished). It is hoped that clotting factors generated by recombinant DNA technology will eventually replace plasma derived-material. Until then stringent virological monitoring will be essential. The PCR technique described here represents a significant step towards the hitherto elusive goal of zero-risk concentrates. We thank J. A. Glazebrook of Wellcome Diagnostics for sequence information used to derive the PCR primers.
Virology Section, Department of Medica Microbiology and Department of Haematology, University College and Middlesex School of Medicine, London W1 P 7PN, UK, and Department of Haematology, Royal Hallamshire Hospital, Sheffield
generating the
J. A. GARSON F. E. PRESTON M. MAKRIS P. TUKE C. RING S. J. MACHIN R. S. TEDDER
1. Aledort LM, Levine
PH, Hilgartner M, et al. A study of liver biopsies and liver disease hemophiliacs. Blood 1985; 66: 367-72. 2. Hay CRM, Preston FE, Triger DR, Underwood JCE. Progressive liver disease in haemophilia: an understated problem? Lancet 1985; i: 1495-98. 3. Makris M, Preston FE, Triger DR, et al. Hepatitis C antibody and chronic liver disease in haemophilia. Lancet 1990; 335: 1117-19. 4. Brettler DB, Levine PH. Factor concentrates for treatment of hemophilia Which one among
choose? Blood 1989; 73: 2067-73. HJ, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively follow ed transfusion recipients with acute and chronic non-A, non-B hepatitis N Engl J Med 1989; 321: 1494-500. Garson JA, Tedder RS, Briggs M, et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. Fletcher ML, Trowell JM, Craske J, Pavier K, Rizza CR. Non-A, non-B hepatitis after transfusion of factor VIII in infrequently treated patients. Br Med J 1983; 287: 1754-57. Study Group of the UK Haemophilia Centre Directors. Effect of dry-heating of coagulation factor concentrates at 80°C for 72 hours on transmission of non-A, non-B hepatitis. Lancet 1988; ii: 814. Kemoff PBA, Miller EJ, Savidge GF, et al. Reduced risk of non-A, non-B hepatitis after a first exposure to "wet heated" factor VIII concentrate. Br J Haematol 1987; 67: 207-11. to
5. Alter
6.
7.
8.
9.
Prevention of hepatitis C virus infection in
haemophiliacs SiR,—We agree with Dr Makris and colleagues (May 12, p 1117) that hepatitis C virus (HCV) seems to be a major cause of liver disease in haemophilia and that there is an urgent need to prevent transmission by elimination of HCV from large-pool clotting factor concentrates. Until now, in contrast to hepatitis B and HIV where donor screening is available, the lack of a diagnostic test for non-A, non-B hepatitis (NANBH) has prevented the use of such screening to reduce NANBH transmission; as an alternative, various virucidal methods have been introduced into the manufacture of factor concentrates.
the new generation of virally inactivated factor VIII is BPL 8Y (Blood Products Laboratory, Elstree) which is manufactured from plasma from volunteers. Each unit of this product has been screened and proved negative for HBsAg and HIV antibody, but not screened for HCV antibody or surrogate markers of NANBH. The lyophilised concentrate is virus inactivated by protracted heating in the freeze-dried state at 800C for 72 h. We have investigated 17 patients with haemophilia A and 1 with type III von Willebrand’s disease, all complying with International Committee on Thrombosis and Haemostasis [Miami, Florida, 1984] criteria for the evaluation of new clotting factor concentrates
Among
concentrates
*All factor VIII except sample 18
(factor IX)
could be taken and a definitive hollow-box obturator constructed. We believe that this technique has considerable advantages over the use of conventional split-thickness skin grafts in the oral cavity, and in allowing the defects to epithelialise spontaneously. Infection and scar contracture have not been observed.
impressions
Kings College Hospital Medical School, London SE5, UK
J. D.
Department of Oral Pathology, London Hospital Medical College, London E1 2AD, UK
I. M. LEIGH H. A. NAVSARIA D. M. WILLIAMS
LANGDON
JG, Green G. Serial cultivation of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975; 6: 331-44.
1. Rheinwald
Detection by PCR of hepatitis C virus in factor VIII concentrates treated haemophilia patients have chronic abnormalities of their liver enzymes and at least 20% of these patients have histological evidence of chronic active hepatitis or cirrhosis.l,2 The most important cause of this liver disease is the transmission by factor VIII concentrates of hepatitis C virus (HCV).3 The sterilisation of factor VIII concentrates by heating or by chemical methods,’ together with the self-exclusion of donors at high risk of HIV infection and donor screening for surrogate markers, has considerably reduced but not eliminated the risk of post-infusion non-A, non-B hepatitis (NANBH). Nor will screening for anti-HCV detect all potentially infectious donors.S We have found that detection by the polymerase chain reaction (PCR) of HCV RNA sequences in blood donations predicts infectivity in man (see p 1419)6 and report here the application of PCR to the detection of HCV in clotting factor concentrates. In our "nested" PCR technique6 RNA extracted from 500 III reconstituted concentrate is reverse transcribed and the cDNA is amplified with HCV-specific oligonucleotide The of the outer primers. sequence primers was 5’-ATGGGGCAAAGGACGTCCG and 5’-TACCTAGTCATAGCCTCCGTGAAG, and of the inner 5’-GAGGTTTTCTGCGTCCA and primers 5’-GCGATAGCCGCAGTTCT. 18 samples were tested, 13 from European or American paid-donor pools and 5 manufactured in the UK from volunteerdonor pools (table). A striking correlation was observed between the presence of HCV RNA in a sample and the likelihood of that product transmitting NANBH. Except for sample 14, an NHS intermediate purity product derived from fewer than 3000 donations, all the unheated concentrates contained HCV RNA. Unheated concentrates have almost invariably resulted in NANBH when administered to previously untreated patients with
SIR,-Most regularly
HCV RNA SEQUENCES IN CLOTTING FACTOR CONCENTRATES*
haemophilia.7 HCV sequences were not detected in any of the three batches of "superheated" material (80°C for 72 h) whereas they were found in both batches of a concentrate heated less intensively (60°C for 32 h). Whilst the superheated material has not been implicated in NANBH,8 hepatitis has occurred in most patients receiving the less intensively heated products.4 Samples 9 and 11, both from "wet" (heptane) treated concentrates, provide further evidence for a correlation between PCR positivity and infectivity: sample 9 (PCR positive) was from a batch of high infectivity9 whereas sample 11 (PCR negative) was apparently non-infectious.9 Recipients of concentrates represented by PCR-positive samples 7, 9, and 10 are known to have developed post-infusion HCV viraemia (unpublished). It is hoped that clotting factors generated by recombinant DNA technology will eventually replace plasma derived-material. Until then stringent virological monitoring will be essential. The PCR technique described here represents a significant step towards the hitherto elusive goal of zero-risk concentrates. We thank J. A. Glazebrook of Wellcome Diagnostics for sequence information used to derive the PCR primers.
Virology Section, Department of Medica Microbiology and Department of Haematology, University College and Middlesex School of Medicine, London W1 P 7PN, UK, and Department of Haematology, Royal Hallamshire Hospital, Sheffield
generating the
J. A. GARSON F. E. PRESTON M. MAKRIS P. TUKE C. RING S. J. MACHIN R. S. TEDDER
1. Aledort LM, Levine
PH, Hilgartner M, et al. A study of liver biopsies and liver disease hemophiliacs. Blood 1985; 66: 367-72. 2. Hay CRM, Preston FE, Triger DR, Underwood JCE. Progressive liver disease in haemophilia: an understated problem? Lancet 1985; i: 1495-98. 3. Makris M, Preston FE, Triger DR, et al. Hepatitis C antibody and chronic liver disease in haemophilia. Lancet 1990; 335: 1117-19. 4. Brettler DB, Levine PH. Factor concentrates for treatment of hemophilia Which one among
choose? Blood 1989; 73: 2067-73. HJ, Purcell RH, Shih JW, et al. Detection of antibody to hepatitis C virus in prospectively follow ed transfusion recipients with acute and chronic non-A, non-B hepatitis N Engl J Med 1989; 321: 1494-500. Garson JA, Tedder RS, Briggs M, et al. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. Fletcher ML, Trowell JM, Craske J, Pavier K, Rizza CR. Non-A, non-B hepatitis after transfusion of factor VIII in infrequently treated patients. Br Med J 1983; 287: 1754-57. Study Group of the UK Haemophilia Centre Directors. Effect of dry-heating of coagulation factor concentrates at 80°C for 72 hours on transmission of non-A, non-B hepatitis. Lancet 1988; ii: 814. Kemoff PBA, Miller EJ, Savidge GF, et al. Reduced risk of non-A, non-B hepatitis after a first exposure to "wet heated" factor VIII concentrate. Br J Haematol 1987; 67: 207-11. to
5. Alter
6.
7.
8.
9.
Prevention of hepatitis C virus infection in
haemophiliacs SiR,—We agree with Dr Makris and colleagues (May 12, p 1117) that hepatitis C virus (HCV) seems to be a major cause of liver disease in haemophilia and that there is an urgent need to prevent transmission by elimination of HCV from large-pool clotting factor concentrates. Until now, in contrast to hepatitis B and HIV where donor screening is available, the lack of a diagnostic test for non-A, non-B hepatitis (NANBH) has prevented the use of such screening to reduce NANBH transmission; as an alternative, various virucidal methods have been introduced into the manufacture of factor concentrates.
the new generation of virally inactivated factor VIII is BPL 8Y (Blood Products Laboratory, Elstree) which is manufactured from plasma from volunteers. Each unit of this product has been screened and proved negative for HBsAg and HIV antibody, but not screened for HCV antibody or surrogate markers of NANBH. The lyophilised concentrate is virus inactivated by protracted heating in the freeze-dried state at 800C for 72 h. We have investigated 17 patients with haemophilia A and 1 with type III von Willebrand’s disease, all complying with International Committee on Thrombosis and Haemostasis [Miami, Florida, 1984] criteria for the evaluation of new clotting factor concentrates
Among
concentrates
*All factor VIII except sample 18
(factor IX)
