- Email: [email protected]
Polymerase chain reaction amplification from dried blood spots on Guthrie cards
639
1
2
3
4
5
Pedigree of family with autosomal dominant vWD. Results are shown of coagulation factor assays (U/dl) and vWF.VNTR analysis. Mutant vWF allele cosegregates with vWF.VNTR(6*). Insert: vWF.VNTR bands obtained by PCR for 11-1 and 111-1. Oligonucleotide primers detect (ATCT)n VNTR in intron 40 of the vWF gene such that 11 tandem repeats givesa 119 bp amphfied fragment and 6 repeats a 99 bp fragment The marker track (M) contains plasmid pBR322 DNA digested with MspI.
Analysis of cord blood samples from the infant showed a borderline raised vWFAg of 203 U/dl (normal range 50-200) and a vWFRicof level of 56 U/dl which was in the normal range (50-200). However, PCR amplification of leucocyte DNA in the cord blood showed that the infant was homozygous for the vWF.VNTR(6) marker (figure, insert), providing unequivocal confirmation that the mutant maternal allele had been inherited. In families with mild vWD in whom prenatal diagnosis is inappropriate, rapid diagnosis in the baby may nevertheless be important if, for example, there is significant birth injury or surgery is required. The technique described offers a simple and reliable means of obtaining this information providing appropriate family studies have been done. Department of Haematology, University of Wales College of Medicine, Cardiff
P. BIGNELL
Department of Haematology, Bristol Royal Infirmary, Bristol BS2 8HW, UK
G. R. STANDEN
D. J. BOWEN Department of Haematology, University of Wales College of Medicine 1.
Ruggeri ZM, Zimmerman TS.
von
Willebrand factor and
I. R. PEAKE A. L. BLOOM
von
Willebrand disease.
Blood 1987: 70: 895-904.
Johnson SS, Montgomery RR, Hathaway WE. Newborn factor VIII complex: elevated activities in term infants and alterations in electrophoretic mobility related 1981: 47: 597-606. to illness and activated coagulation. Br Haematol J 3. Sadler JE, Shelton-Inloes BB, Sorace JM, Harlan JM, Titani K, Davie EW. Cloning and characterisation of two cDNAs coding for human von Willebrand factor. Proc 2
Fig 1-Agarose-gel electrophoresis of
the
PCR-amplified
product. Lane 1 = molecular weight marker obtained from digestion of pBR322 with MspI; lanes 2-5 = 1/lOth of the product obtained after amplification from Guthrie cards. In each case, amplification from Guthrie cards resulted in the specific amplification of the expected 245 bp fragment.
mutations that can be examined from each patient. With the continuing characterisation of new mutations within the human genome, there will be a demand to simultaneously identify carriers of the most prevalent mutations. Thus, we have developed a method for detecting specific mutations from Guthrie cards which does not require extraction of genomic DNA. This method permits the genetic analysis of several hundred mutations from a single blood spot. Briefly, 0-5 jjmol/1 of each suitable oligonucleotide amplification primer was added to a tube containing 10 nimol/I "tris" (pH 8-3), 50 mmol/1 KCI, 1 -5 mmol/1 MgCI2, 0-0% gelatin, and 200 umol/1 of each deoxynucleoside triphosphate in a volume of 100 1. A small piece (1-2 mmz) cut from the Guthrie card was added to the reaction mixture. The contents of the tube were denatured at 97°C for 7 min and annealed at 50°C for 1 min before addition of approximately 2 units of thermostable DNA polymerase from Thermus thermophilus.4 After addition of the enzyme, one extension cycle was done at 72°C for 1 min, followed by an additional 35 amplification cycles under the following conditions: 92°C for 10 s, 50°C for 10 s, and 72°C for 40 s.
Normal
probe
Mutant
probe
Natl Acad Sci USA 1985. 82: 6394-98. 4. Verweij CL, de Vries CJM, Distel B, et al. Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene. Nucleic Acids Res 1985: 13: 4699-717. 5. Peake IR, Bowen D, Bignell P, et al. Family studies and prenatal diagnosis in severe von Willebrand’s disease by polymerase chain reaction amplification of a variable number tandem repeat (VNTR) region of the von Willebrand factor gene. Blood
(in press). GR, Bignell P, Bowen DJ, Peake IR, Bloom AL. Family studies in von Willebrand’s disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence. Br J Haematol (in press).
6. Standen
Polymerase chain reaction amplification from dried blood spots on Guthrie cards SIR,-Polymerase chain reaction (PCR) amplification and allelespecific oligonucleotide hybridisation methods have greatly facilitated the identification and screening of specific mutations within genomic DNA. Together, these methods form the basis of reports describing the detection of specific mutations in genomic DNA extracted from Guthrie cards. 1-3 Unfortunately, the relatively low recovery of genomic DNA from each card limits the number of
Fig 2-Slot-blot hybridisation analysis of PCR-amplified product using allele-specific oligonucleotide probes. Aliquots of amplified DNA were applied to duplicate zeta-probe membranes and hybridised with either normal or R408W mutant probes. Patient AS is homozygous normal at this position; patient B is heterozygous, bearing one normal allele and one mutant allele ; patient C is homozygous for the R408W mutation.
640
A
typical result of this procedure is shown in fig 1. In this experiment, the exon-12-containing region of the human phenylalanine hydroxylase gene was amplified from the Guthrie cards of four phenylketonuria patients from Leningrad. This region of the phenylalanine hydroxylase gene contains the most prevalent caucasian phenylketonuria mutation (R408W).5 The primers used in this amplification were identical to those described by DiLella et al. This experiment demonstrates amplification of a single band of appropriate size (245 bp). The sequence identity of the amplification product was confirmed by slot-blot hybridisation analysis using allele-specific oligonucleotides as described by DiLella et al (fig 2).6 The primary advantage of this technique over other methods is the significant increase in the number of potential mutations which can be determined from a single card. Based on the size of the average blood spot on a Guthrie card, all the predominant mutant alleles of many common genetic diseases could be screened from a single blood spot. Thus, Guthrie cards represent an important repository of genetic information on the frequency and distribution of specific mutant alleles within a given population.
Supported in part by the Human Genome Project, Academy of Science of the USSR (E. I. S.), and by National Institutes of Health grant HD-17711 to S. L. C. W., who is an investigator with the Howard Hughes Medical Institute. B. P Konstantinov Institute of Nuclear
Academy of Sciences of the USSR, Gatchina, Leningrad Region, USSR
Department of Cell Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA
Physics,
E. I. SCHWARTZ S. E. KHALCHITSKY
R. C. EISENSMITH S. L. C. Woo
1. McCabe ERB, Huang S-Z, Seltzer WK, Law ML. DNA microextraction from dried blood spots on filter paper blotters: poential applications to newborn screening Hum Genet 1987; 75: 213-16 2. Jinks DC, Minter M, Tarver DA, Vanderford M, Hejtmancik JF, McCabe ERB. Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening. Hum Genet 1989; 81: 363-66. 3. McCabe ERB, Zhang Y-H, Descartes M, Therrell BL Jr, Erlich HA. Rapid detection of &bgr;s DNA from Guthrie cards by chromogenic probes. Lancet 1989; ii: 741. 4. Schwartz EI, Kaboev OK, Gol’tsov AA, Vinogradov SV, Lebedenko EN, Berlin YuA. Amplification of two segments of the human &bgr;-globin gene by means of polymerase chain reaction. Bioorg Khim 1988; 14: 1577-79. 5. DiLella AG, Marvit J, Brayton K, Woo SLC. An amino-acid substitution in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature 1987; 327: 333-36. 6. DiLella AG, Huang W-M, Woo SLC. Screening for phenylketonuria mutations by DNA amplification with the polymerase chain reaction Lancet 1988; i: 497-99.
of NBBS 300 flgjkg induced a syndrome of motor dysfunction identical to that of the intracisternally inoculated rabbits. Neuropathological findings in rabbits injected intraperitoneally were indistinguishable from those in rabbits injected intracistemally, which indicated that NBBS crossed the bloodbrain barrier. Scattered axonal spheroids, present in the brainstem and spinal cord, and fusiform thickening of the intramedullary portion of the spinal motor neurons were immunoreactive with monoclonal antibodies against phosphorylated epitopes of the 200 kD and 160 kD neurofilament polypeptides. Abnormalities in the dendritic processes of the nucleus motoris lateralis were evident on routine histopathological staining, as well as on immunostaining with monoclonal antibodies specific for microtubule-associated protein 2. Distended dendritic processes were seen during early stages (up to 4 months) of either intracistemal or intraperitoneal inoculation. After 12 months of NBBS exposure, the same dendritic processes were absent or greatly reduced in number. Teased fibre preparations, toluidine-blue stained semi-thin sections, and ’Epon’embedded ultrathin sections of proximal and distal sciatic nerves were normal. NBBS, a sulphonamide plasticiser, is used for the polymerisation of polyamide compounds in the production of plastic resins and as a starting agent in the synthesis of an agricultural herbicide. Human exposure to NBBS may also occur from agricultural fungicides and waste water effluents.1 Although it has been demonstrated that workers engaged in the production of plastics seem to be at an increased risk for the development of amyotrophic lateral sclerosis,2 the levels of NBBS required to induce human neurodegenerative disease, the conditions most likely to result in leaching of NBBS, and the human bioavailability of NBBS have, to our knowledge, not been determined. Leaching of plasticisers from plastic products depends not only on pH, temperature, and duration of storage but also on the chemical composition of foods or biological products stored in them.3-5 Our demonstration that NBBS produces a dose-dependent, progressive motor dysfunction in rabbits demands a careful assessment of the safety, bioavailabilty, and conditions of leaching of this group of plasticisers.
Bethesda, Maryland 20892, USA
MICHAEL J. STRONG RALPH M. GARRUTO AXEL V. WOLFF RICHARD YANAGIHARA
Department of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio
SAMUEL M. CHOU
Laboratory of CNS Studies, National Institutes of Health,
Chemical Synthesis and Analysis Laboratory,
N-butylbenzenesulphonamide, a novel neurotoxic plasticising agent SIR,-We have found, by chance, that a container-derived contaminant from plastic ware, identified as N-butylbenzenesulphonamide (NBBS), induces a progressive spastic myelopathy in New Zealand white rabbits. Following the neurological deterioration of rabbits inoculated intracisternally with 0-9% saline solution we identifed NBBS as the sole contaminant in the saline and as a component of the plastic ware. In a series of three experiments in young adult NZ white rabbits, we examined: (1) the dose-response toxicity of NBBS (monthly intracisternal inoculations with 0.1,1,10,50, or 100 ug for 4 months with monthly necropsies; (2) the effects of long-term exposure of the central nervous system to NBBS (monthly intracisternal inoculations with 10, 50, or 100 µg NBBS with rabbits allowed to survive for 12 months); and (3) the permeability of the blood-brain barrier to NBBS (intraperitoneal inoculations thrice weekly for 4 months with NBBS suspended in olive oil). Rabbits inoculated intracisternally with 10,50, or 100 ug NBBS developed external rotation and splaying of the hind limbs, reflex spreading, hypertonia, focal paralysis, and impaired back-pedalling during the initial 4 months of the study. Thereafter, fore-limb splaying with hyperreflexia, focal muscle weakness and wasting, exaggerated hind-limb extensor tremors during weight-bearing, and impaired righting reflexes developed. Intraperitoneal injections
Program Resources, Inc, Frederick Cancer Research and Development Center,
Frederick, Maryland
STEPHEN D. FOX
LS, Hites RA. Sources and movement of organic chemicals in the Delaware River. Environ Sci Technol 1979; 13: 574-79. 2 Deapen DM, Henderson BE A case-control study of amyotrophic lateral sclerosis Am J Epidemiol 1986, 123: 790-97. 3. Hollifield HC, Breder CV, Dennison JL, Roach JAG, Adams WS. Container-derived contamination of maple syrup with methyl methacrylate, toluene, and styrene as determined by headspace gas-liquid chromatography. J Assoc Off Anal Chem 1980, 63: 173-77. 4 Bieber WD, Freytag W, Figge K, vom Bruck CG. Transfer of additives from plastics materials into foodstuffs and into food stimulants: a comparison. Food Chem Toxicol 1984; 22: 737-42. 5. Rubin RJ, Ness PM. What price progress? an update on vinyl blood bags. Transfusion 1989; 29: 358-61. 1. Sheldon
CORRECTION Antibody to hepatitis C virus in Hungary. In this letter by Dr A. Par (July 14, p 123) the last sentence should have read "We would emphasise that donors at high risk should be identified by both anti-HIV testing and questioning about risk behaviour, but we also recommend anti-HBc and anti-HCV testing for the prevention of parenterally-transmitted HBV and HCV infections ...".
1
2
3
4
5
Pedigree of family with autosomal dominant vWD. Results are shown of coagulation factor assays (U/dl) and vWF.VNTR analysis. Mutant vWF allele cosegregates with vWF.VNTR(6*). Insert: vWF.VNTR bands obtained by PCR for 11-1 and 111-1. Oligonucleotide primers detect (ATCT)n VNTR in intron 40 of the vWF gene such that 11 tandem repeats givesa 119 bp amphfied fragment and 6 repeats a 99 bp fragment The marker track (M) contains plasmid pBR322 DNA digested with MspI.
Analysis of cord blood samples from the infant showed a borderline raised vWFAg of 203 U/dl (normal range 50-200) and a vWFRicof level of 56 U/dl which was in the normal range (50-200). However, PCR amplification of leucocyte DNA in the cord blood showed that the infant was homozygous for the vWF.VNTR(6) marker (figure, insert), providing unequivocal confirmation that the mutant maternal allele had been inherited. In families with mild vWD in whom prenatal diagnosis is inappropriate, rapid diagnosis in the baby may nevertheless be important if, for example, there is significant birth injury or surgery is required. The technique described offers a simple and reliable means of obtaining this information providing appropriate family studies have been done. Department of Haematology, University of Wales College of Medicine, Cardiff
P. BIGNELL
Department of Haematology, Bristol Royal Infirmary, Bristol BS2 8HW, UK
G. R. STANDEN
D. J. BOWEN Department of Haematology, University of Wales College of Medicine 1.
Ruggeri ZM, Zimmerman TS.
von
Willebrand factor and
I. R. PEAKE A. L. BLOOM
von
Willebrand disease.
Blood 1987: 70: 895-904.
Johnson SS, Montgomery RR, Hathaway WE. Newborn factor VIII complex: elevated activities in term infants and alterations in electrophoretic mobility related 1981: 47: 597-606. to illness and activated coagulation. Br Haematol J 3. Sadler JE, Shelton-Inloes BB, Sorace JM, Harlan JM, Titani K, Davie EW. Cloning and characterisation of two cDNAs coding for human von Willebrand factor. Proc 2
Fig 1-Agarose-gel electrophoresis of
the
PCR-amplified
product. Lane 1 = molecular weight marker obtained from digestion of pBR322 with MspI; lanes 2-5 = 1/lOth of the product obtained after amplification from Guthrie cards. In each case, amplification from Guthrie cards resulted in the specific amplification of the expected 245 bp fragment.
mutations that can be examined from each patient. With the continuing characterisation of new mutations within the human genome, there will be a demand to simultaneously identify carriers of the most prevalent mutations. Thus, we have developed a method for detecting specific mutations from Guthrie cards which does not require extraction of genomic DNA. This method permits the genetic analysis of several hundred mutations from a single blood spot. Briefly, 0-5 jjmol/1 of each suitable oligonucleotide amplification primer was added to a tube containing 10 nimol/I "tris" (pH 8-3), 50 mmol/1 KCI, 1 -5 mmol/1 MgCI2, 0-0% gelatin, and 200 umol/1 of each deoxynucleoside triphosphate in a volume of 100 1. A small piece (1-2 mmz) cut from the Guthrie card was added to the reaction mixture. The contents of the tube were denatured at 97°C for 7 min and annealed at 50°C for 1 min before addition of approximately 2 units of thermostable DNA polymerase from Thermus thermophilus.4 After addition of the enzyme, one extension cycle was done at 72°C for 1 min, followed by an additional 35 amplification cycles under the following conditions: 92°C for 10 s, 50°C for 10 s, and 72°C for 40 s.
Normal
probe
Mutant
probe
Natl Acad Sci USA 1985. 82: 6394-98. 4. Verweij CL, de Vries CJM, Distel B, et al. Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene. Nucleic Acids Res 1985: 13: 4699-717. 5. Peake IR, Bowen D, Bignell P, et al. Family studies and prenatal diagnosis in severe von Willebrand’s disease by polymerase chain reaction amplification of a variable number tandem repeat (VNTR) region of the von Willebrand factor gene. Blood
(in press). GR, Bignell P, Bowen DJ, Peake IR, Bloom AL. Family studies in von Willebrand’s disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence. Br J Haematol (in press).
6. Standen
Polymerase chain reaction amplification from dried blood spots on Guthrie cards SIR,-Polymerase chain reaction (PCR) amplification and allelespecific oligonucleotide hybridisation methods have greatly facilitated the identification and screening of specific mutations within genomic DNA. Together, these methods form the basis of reports describing the detection of specific mutations in genomic DNA extracted from Guthrie cards. 1-3 Unfortunately, the relatively low recovery of genomic DNA from each card limits the number of
Fig 2-Slot-blot hybridisation analysis of PCR-amplified product using allele-specific oligonucleotide probes. Aliquots of amplified DNA were applied to duplicate zeta-probe membranes and hybridised with either normal or R408W mutant probes. Patient AS is homozygous normal at this position; patient B is heterozygous, bearing one normal allele and one mutant allele ; patient C is homozygous for the R408W mutation.
640
A
typical result of this procedure is shown in fig 1. In this experiment, the exon-12-containing region of the human phenylalanine hydroxylase gene was amplified from the Guthrie cards of four phenylketonuria patients from Leningrad. This region of the phenylalanine hydroxylase gene contains the most prevalent caucasian phenylketonuria mutation (R408W).5 The primers used in this amplification were identical to those described by DiLella et al. This experiment demonstrates amplification of a single band of appropriate size (245 bp). The sequence identity of the amplification product was confirmed by slot-blot hybridisation analysis using allele-specific oligonucleotides as described by DiLella et al (fig 2).6 The primary advantage of this technique over other methods is the significant increase in the number of potential mutations which can be determined from a single card. Based on the size of the average blood spot on a Guthrie card, all the predominant mutant alleles of many common genetic diseases could be screened from a single blood spot. Thus, Guthrie cards represent an important repository of genetic information on the frequency and distribution of specific mutant alleles within a given population.
Supported in part by the Human Genome Project, Academy of Science of the USSR (E. I. S.), and by National Institutes of Health grant HD-17711 to S. L. C. W., who is an investigator with the Howard Hughes Medical Institute. B. P Konstantinov Institute of Nuclear
Academy of Sciences of the USSR, Gatchina, Leningrad Region, USSR
Department of Cell Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA
Physics,
E. I. SCHWARTZ S. E. KHALCHITSKY
R. C. EISENSMITH S. L. C. Woo
1. McCabe ERB, Huang S-Z, Seltzer WK, Law ML. DNA microextraction from dried blood spots on filter paper blotters: poential applications to newborn screening Hum Genet 1987; 75: 213-16 2. Jinks DC, Minter M, Tarver DA, Vanderford M, Hejtmancik JF, McCabe ERB. Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening. Hum Genet 1989; 81: 363-66. 3. McCabe ERB, Zhang Y-H, Descartes M, Therrell BL Jr, Erlich HA. Rapid detection of &bgr;s DNA from Guthrie cards by chromogenic probes. Lancet 1989; ii: 741. 4. Schwartz EI, Kaboev OK, Gol’tsov AA, Vinogradov SV, Lebedenko EN, Berlin YuA. Amplification of two segments of the human &bgr;-globin gene by means of polymerase chain reaction. Bioorg Khim 1988; 14: 1577-79. 5. DiLella AG, Marvit J, Brayton K, Woo SLC. An amino-acid substitution in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature 1987; 327: 333-36. 6. DiLella AG, Huang W-M, Woo SLC. Screening for phenylketonuria mutations by DNA amplification with the polymerase chain reaction Lancet 1988; i: 497-99.
of NBBS 300 flgjkg induced a syndrome of motor dysfunction identical to that of the intracisternally inoculated rabbits. Neuropathological findings in rabbits injected intraperitoneally were indistinguishable from those in rabbits injected intracistemally, which indicated that NBBS crossed the bloodbrain barrier. Scattered axonal spheroids, present in the brainstem and spinal cord, and fusiform thickening of the intramedullary portion of the spinal motor neurons were immunoreactive with monoclonal antibodies against phosphorylated epitopes of the 200 kD and 160 kD neurofilament polypeptides. Abnormalities in the dendritic processes of the nucleus motoris lateralis were evident on routine histopathological staining, as well as on immunostaining with monoclonal antibodies specific for microtubule-associated protein 2. Distended dendritic processes were seen during early stages (up to 4 months) of either intracistemal or intraperitoneal inoculation. After 12 months of NBBS exposure, the same dendritic processes were absent or greatly reduced in number. Teased fibre preparations, toluidine-blue stained semi-thin sections, and ’Epon’embedded ultrathin sections of proximal and distal sciatic nerves were normal. NBBS, a sulphonamide plasticiser, is used for the polymerisation of polyamide compounds in the production of plastic resins and as a starting agent in the synthesis of an agricultural herbicide. Human exposure to NBBS may also occur from agricultural fungicides and waste water effluents.1 Although it has been demonstrated that workers engaged in the production of plastics seem to be at an increased risk for the development of amyotrophic lateral sclerosis,2 the levels of NBBS required to induce human neurodegenerative disease, the conditions most likely to result in leaching of NBBS, and the human bioavailability of NBBS have, to our knowledge, not been determined. Leaching of plasticisers from plastic products depends not only on pH, temperature, and duration of storage but also on the chemical composition of foods or biological products stored in them.3-5 Our demonstration that NBBS produces a dose-dependent, progressive motor dysfunction in rabbits demands a careful assessment of the safety, bioavailabilty, and conditions of leaching of this group of plasticisers.
Bethesda, Maryland 20892, USA
MICHAEL J. STRONG RALPH M. GARRUTO AXEL V. WOLFF RICHARD YANAGIHARA
Department of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio
SAMUEL M. CHOU
Laboratory of CNS Studies, National Institutes of Health,
Chemical Synthesis and Analysis Laboratory,
N-butylbenzenesulphonamide, a novel neurotoxic plasticising agent SIR,-We have found, by chance, that a container-derived contaminant from plastic ware, identified as N-butylbenzenesulphonamide (NBBS), induces a progressive spastic myelopathy in New Zealand white rabbits. Following the neurological deterioration of rabbits inoculated intracisternally with 0-9% saline solution we identifed NBBS as the sole contaminant in the saline and as a component of the plastic ware. In a series of three experiments in young adult NZ white rabbits, we examined: (1) the dose-response toxicity of NBBS (monthly intracisternal inoculations with 0.1,1,10,50, or 100 ug for 4 months with monthly necropsies; (2) the effects of long-term exposure of the central nervous system to NBBS (monthly intracisternal inoculations with 10, 50, or 100 µg NBBS with rabbits allowed to survive for 12 months); and (3) the permeability of the blood-brain barrier to NBBS (intraperitoneal inoculations thrice weekly for 4 months with NBBS suspended in olive oil). Rabbits inoculated intracisternally with 10,50, or 100 ug NBBS developed external rotation and splaying of the hind limbs, reflex spreading, hypertonia, focal paralysis, and impaired back-pedalling during the initial 4 months of the study. Thereafter, fore-limb splaying with hyperreflexia, focal muscle weakness and wasting, exaggerated hind-limb extensor tremors during weight-bearing, and impaired righting reflexes developed. Intraperitoneal injections
Program Resources, Inc, Frederick Cancer Research and Development Center,
Frederick, Maryland
STEPHEN D. FOX
LS, Hites RA. Sources and movement of organic chemicals in the Delaware River. Environ Sci Technol 1979; 13: 574-79. 2 Deapen DM, Henderson BE A case-control study of amyotrophic lateral sclerosis Am J Epidemiol 1986, 123: 790-97. 3. Hollifield HC, Breder CV, Dennison JL, Roach JAG, Adams WS. Container-derived contamination of maple syrup with methyl methacrylate, toluene, and styrene as determined by headspace gas-liquid chromatography. J Assoc Off Anal Chem 1980, 63: 173-77. 4 Bieber WD, Freytag W, Figge K, vom Bruck CG. Transfer of additives from plastics materials into foodstuffs and into food stimulants: a comparison. Food Chem Toxicol 1984; 22: 737-42. 5. Rubin RJ, Ness PM. What price progress? an update on vinyl blood bags. Transfusion 1989; 29: 358-61. 1. Sheldon
CORRECTION Antibody to hepatitis C virus in Hungary. In this letter by Dr A. Par (July 14, p 123) the last sentence should have read "We would emphasise that donors at high risk should be identified by both anti-HIV testing and questioning about risk behaviour, but we also recommend anti-HBc and anti-HCV testing for the prevention of parenterally-transmitted HBV and HCV infections ...".