1077 IN VITRO ASSAY AND IN VIVO RECOVERY OF FACTOR-VIII
CONCENTRATES
i
i
iI
been a heavy drinker for 16 years and the mother’s drinking had been intermittent, but often heavy, for a similar period. Although thev had given up drinking 1 z years before conception, their child has multiple congenital malformations which are associated with fetal alcohol syndrome. The child was born to a 27-year-old gravida vii, para n, abortus v woman. Delivery was by cacsarean section at term; the baby weighed 2215gand was 48 cm in length: head circumference was not recorded. Ftosis of the left lid was recorded. At 48 h of age, congestive heart-failure developed. Emergency cardiac catheterisation was done, and a coarctation of the aorta just distal to the subclavian artery was resected; this was associated with a small ventricular septal defect.
i
i
age of 2 Fe.
higher estimate of the F VIII concentrates was obtained by the two-stage than by the one-stage assay. For all concentrates, when the injected dose was calculated on the basis of the one-stage factor vm assay, the recovery was close to 100% of the expected value. When the dose was calculated according to the two-stage assay, the recovery was significantly lower for five products and higher for only one (product 6). This discrepancy was not found when the recovery of F VIII from cryoprecipitate was measured; recovery was close to 100%, whether a one-stage or two-stage assay was used (table). Purification procedures used in the preparation of concentrates may modify the factor-vm molecule so that it behaves differently in one and two stage assays. Since the values obtained by one-stage assay seem to correlate more closely with the expected in-vivo recovery, we suggest that, in practice, F vm activity in concentrates should be estimated either by onestage assay using a reference plasma or by a two-stage assay against a concentrate whose activity has been established by a one-stage assay.
Patient at age 4 months.
Blepharophimosis, relative ocular hypertelorism, incompletely formed ear helices, long and poorly demarcated philtrum, small upper lip with thin vermilion, upturned nose.
-
National Blood Transfusion Centre,
P. ALLAIN F. VERROUST J. P. SOULIER
J.
75739 Paris, France, and Centre for Hæmophilic Children, La Queue Lez Yvelines
FETAL ALCOHOL SYNDROME IN CHILD WHOSE PARENTS HAD STOPPED DRINKING
SIR,-We have
seen a
child with features of the fetal alcohol
syndrome, born to alcoholic parents who had stopped drinking before conception. Moderate to heavy alcohol consumption during pregnancy is a major risk to fetal morphological development and to intellectual function. 1-4 The mechanism for the teratogenesis is not understood. In most reported cases, the mother ’had drunk moderate to large amounts of alcohol during the first trimester. The presence or absence of an alcoholic father, the possible effects of alcohol on spermatogenesis, and the possible pregestational effects on the sperm or ovum have all been considered. It has been assumed that the fetal alcohol syndrome results from the direct effects of alcohol, its metabolites, or associated nutritional factors upon embryogenesis, and that the mother should refrain from drinking only during pregnancy. The child we describe was born to parents both of whom are members of Alcoholics Anonymous. The father had formerly 1 2.
A P., Herman, C. S., Smith, D. W. J Pediat. 1978, 92, 363. Hanson, J.W.,Streissguth, A. P., Smith, D. W. ibid p. 457. 3 Ouellette, P.M., Rosette, H. L., Rossman, N. P., Weiner, L. New Engl. J.
Streissguth, Med.
She did well medically but was developmentally delayed in language and gross motor areas. At 18 and 32 months her weight was in the 10th percentile, and her length and head circumference were below the 5th percentile. She had a left mild ptosis with relative ocular hypertelorism, blepharophimosis, incompletely formed ear helices, long and poorly demarcated philtrum, a small upper lip with a thin vermilion, and an upturned nose (see figure). She had no limitation of movement at her elbows but had mild pes planus bilaterally with dysplasia of the fourth and fifth toenails. She made substantial progress, and at 32 months had normal gross motor skills with a Stanford-Binet i.Q. of 106. Chromosomal analysis showed a normal female karyotype. The fetal alcohol syndrome is variable in phenotype.’ Hanson et al.1 found that 2 of 62 children examined were born to non-drinking women and had features of the syndrome. Our patient was small for gestational age, had blepharophimosis, hypertelorism, a long philtrum, and a cardiac lesion, and had shown catch-up growth in weight as well as intellectual development. Children with fetal alcohol syndrome generally do not catch Up,I,2 but Streissguth et al.’ described a 9-year-old child, reported to have the syndrome, with an i.Q. of 105. we feel that the substantial congenital malformations are suggestive of fetal alcohol syndrome. Explanations for this child’s malformations include an adverse effect of alcoholism and its complications on the male or female germ cells before conception; congenital malformations might be secondary to such alterations. Alternatively, alcoholism could be a genotype or phenotype which is terato-
1977, 297, 528.
4 Clarren, S. K, Smith, D. W. ibid. 5 FDA Drug Bull. 1977, 7, 18.
1978, 298, 1063.
6. -
Friedler, G., Cochin, J Serer Hanson, J. W. and others J.
1972, 175, 654. Pearal 1976, 89, 662.
1078
genic, even in the absence of actual intake of the agent during pregnancy. A parallel suggestion has been made for maternal phenytoin,7 epilepsy, and congenital malformations .8,1 BX’e cannot rule out the possibility that the mother did, in fact, drink while pregnant, or the possibility that the child has, not fetal alcohol syndrome, but a suggestive phenotype with some other aetiology. Child
Development Service, University of Massachusetts Medical Center, Worcester, Massachusetts, U.S.A.
ALBERT P. SCHEINER CAROL M. DONOVAN
Center for Genetic Counseling and Birth Defects Evaluation, Tufts—New England Medical Center,
LOUIS E. BARTOSHESKY
Boston, Massachusetts
MONKEY SCOLIOSIS
SiR,-Your editorial on scoliosis (March 10, p. 537) cites the important observations of Lloyd-Roberts et al.1 who showed that idiopathic scoliosis in humans may be related to failure of the spinal cord to rotate m company with the rotation of the vertebral column, causing nerve-root traction and cell degeneration in the dorsal root ganglion. This prompts us to - report the development of scoliosis in cynomolgus monkeys (Macaca fascicularis) following the routine safety testing of live, attenuated, oral poliomyelitis vaccines in the neurovirulence monkey test2 with the standard technique of
intraspinal inoculation.3 Shapiro, S., and others. Lancet, 1976, i, 272 Fraser, F. C., Metrakos, J D. Zletkin, M. ibid. 1978, i, 884. 1. Llovd-Roberts, G. C., Pincott, J. R., McMeniman, P., Bayley, dall, B. J Bone Jt Surg. 1978, 60B, 451. 2. Boulder, L. R.J. biol Standard. 1973, 1, 119. 3 Beswick, T. S. L., Cord, C. R J. Hyg., Camb. 1961, 59, 395.
2-Four sections of upper lumbar cord of scoliotic shown in fig. 1.
Fig.
monkey
Note needle track trauma in right posterior horn. Nerve tissue was mechanically destroyed on same side i.e., right) as convex side of spinal curve. Little or no histological poliomyelitis (viral activity observed in this monkey. (Einarson-gallocyamn.)
8. 9
I.
J. L., Ken-
Of 1370
monkeys
inoculated
intraspinally,
under
general
anxsthesia, with oral poliovaccines, 23 (1.7"() acquired scoliosis (14 left, 2 with marked curvature; 9 right, 6 with marked curvature) and 1 monkey acquired marked
kyphosis. Scoliosis detected between the first and twenty-second day after injection. In 16 cases scoliosis was first observed within 7 days. Three cases were first noticed between the 9th and 16th days, and in4 monkeys scoliosis was detected only on day 22, the final day of observation before routine cardiac perfusion and removal of the brain and spinal cord for histology. The kyphosis was first seen on day 22. In only 3 instances did leg paralysis (normal in some monkeys after vaccine inoculation) accompany the scoliosis. There seems to be no correlation between scoliosis and vaccine manufacturer, poliovirus type, cell substrate, or dilution of vaccine inoculated. Radiographs of five spines examined by Prof. L. C. Vaughan (Royal Veterinary College), Dr P. A. Zorab (Brompton and St. Mary’s Hospitals), and Dr Min Mehta (Royal National Orthopxdic Hospital) showed no bone abnormality, thus the condition might be associated with muscle and/or neuronal damage. As might be expected after the inoculation of live virus, histological examination of the grey-matter of the lumbar cord showed varying degrees of cellular infiltration and destruction of nerve cells through needle trauma and viral activity. Perhaps, however, the most interesting findings were that 2 monkeys, 1 (fig. 1) with marked scoliosis, had few or no nerve cells destroyed through viral activity at the site of inoculation in the grey-matter of the lumbar cord (fig. 2); and that the needle track (trauma) occurred in every case on the same side of the cord as the convex side of the spinal curve. It is therefore possible that the scoliosis seen infrequently in monkeys following intraspinal inoculation may be due to mechanical damage of the dorsal nerve root and/or posterior horn caused by the needle as it passes into the spinal cord, or to the failure of the spinal cord to rotate in company with the vertebrx during the violent muscular contractions of one or both legs which on occasion accompany the injection. This possibility is noBB being
was
Fig. I-Radiograph of cynomolgus monkey. Severe right lateral curvature and rotation of spine, first observed 2 davs after intraspinal inoculation with 0.1 ml of a type m live, attenuated poliomyelitis vaccine. Sedated and X-rayed 22 days after inoculation.
explored. Whatever the
wause
and mechanism of monkey section.
CONCENTRATES
i
i
iI
been a heavy drinker for 16 years and the mother’s drinking had been intermittent, but often heavy, for a similar period. Although thev had given up drinking 1 z years before conception, their child has multiple congenital malformations which are associated with fetal alcohol syndrome. The child was born to a 27-year-old gravida vii, para n, abortus v woman. Delivery was by cacsarean section at term; the baby weighed 2215gand was 48 cm in length: head circumference was not recorded. Ftosis of the left lid was recorded. At 48 h of age, congestive heart-failure developed. Emergency cardiac catheterisation was done, and a coarctation of the aorta just distal to the subclavian artery was resected; this was associated with a small ventricular septal defect.
i
i
age of 2 Fe.
higher estimate of the F VIII concentrates was obtained by the two-stage than by the one-stage assay. For all concentrates, when the injected dose was calculated on the basis of the one-stage factor vm assay, the recovery was close to 100% of the expected value. When the dose was calculated according to the two-stage assay, the recovery was significantly lower for five products and higher for only one (product 6). This discrepancy was not found when the recovery of F VIII from cryoprecipitate was measured; recovery was close to 100%, whether a one-stage or two-stage assay was used (table). Purification procedures used in the preparation of concentrates may modify the factor-vm molecule so that it behaves differently in one and two stage assays. Since the values obtained by one-stage assay seem to correlate more closely with the expected in-vivo recovery, we suggest that, in practice, F vm activity in concentrates should be estimated either by onestage assay using a reference plasma or by a two-stage assay against a concentrate whose activity has been established by a one-stage assay.
Patient at age 4 months.
Blepharophimosis, relative ocular hypertelorism, incompletely formed ear helices, long and poorly demarcated philtrum, small upper lip with thin vermilion, upturned nose.
-
National Blood Transfusion Centre,
P. ALLAIN F. VERROUST J. P. SOULIER
J.
75739 Paris, France, and Centre for Hæmophilic Children, La Queue Lez Yvelines
FETAL ALCOHOL SYNDROME IN CHILD WHOSE PARENTS HAD STOPPED DRINKING
SIR,-We have
seen a
child with features of the fetal alcohol
syndrome, born to alcoholic parents who had stopped drinking before conception. Moderate to heavy alcohol consumption during pregnancy is a major risk to fetal morphological development and to intellectual function. 1-4 The mechanism for the teratogenesis is not understood. In most reported cases, the mother ’had drunk moderate to large amounts of alcohol during the first trimester. The presence or absence of an alcoholic father, the possible effects of alcohol on spermatogenesis, and the possible pregestational effects on the sperm or ovum have all been considered. It has been assumed that the fetal alcohol syndrome results from the direct effects of alcohol, its metabolites, or associated nutritional factors upon embryogenesis, and that the mother should refrain from drinking only during pregnancy. The child we describe was born to parents both of whom are members of Alcoholics Anonymous. The father had formerly 1 2.
A P., Herman, C. S., Smith, D. W. J Pediat. 1978, 92, 363. Hanson, J.W.,Streissguth, A. P., Smith, D. W. ibid p. 457. 3 Ouellette, P.M., Rosette, H. L., Rossman, N. P., Weiner, L. New Engl. J.
Streissguth, Med.
She did well medically but was developmentally delayed in language and gross motor areas. At 18 and 32 months her weight was in the 10th percentile, and her length and head circumference were below the 5th percentile. She had a left mild ptosis with relative ocular hypertelorism, blepharophimosis, incompletely formed ear helices, long and poorly demarcated philtrum, a small upper lip with a thin vermilion, and an upturned nose (see figure). She had no limitation of movement at her elbows but had mild pes planus bilaterally with dysplasia of the fourth and fifth toenails. She made substantial progress, and at 32 months had normal gross motor skills with a Stanford-Binet i.Q. of 106. Chromosomal analysis showed a normal female karyotype. The fetal alcohol syndrome is variable in phenotype.’ Hanson et al.1 found that 2 of 62 children examined were born to non-drinking women and had features of the syndrome. Our patient was small for gestational age, had blepharophimosis, hypertelorism, a long philtrum, and a cardiac lesion, and had shown catch-up growth in weight as well as intellectual development. Children with fetal alcohol syndrome generally do not catch Up,I,2 but Streissguth et al.’ described a 9-year-old child, reported to have the syndrome, with an i.Q. of 105. we feel that the substantial congenital malformations are suggestive of fetal alcohol syndrome. Explanations for this child’s malformations include an adverse effect of alcoholism and its complications on the male or female germ cells before conception; congenital malformations might be secondary to such alterations. Alternatively, alcoholism could be a genotype or phenotype which is terato-
1977, 297, 528.
4 Clarren, S. K, Smith, D. W. ibid. 5 FDA Drug Bull. 1977, 7, 18.
1978, 298, 1063.
6. -
Friedler, G., Cochin, J Serer Hanson, J. W. and others J.
1972, 175, 654. Pearal 1976, 89, 662.
1078
genic, even in the absence of actual intake of the agent during pregnancy. A parallel suggestion has been made for maternal phenytoin,7 epilepsy, and congenital malformations .8,1 BX’e cannot rule out the possibility that the mother did, in fact, drink while pregnant, or the possibility that the child has, not fetal alcohol syndrome, but a suggestive phenotype with some other aetiology. Child
Development Service, University of Massachusetts Medical Center, Worcester, Massachusetts, U.S.A.
ALBERT P. SCHEINER CAROL M. DONOVAN
Center for Genetic Counseling and Birth Defects Evaluation, Tufts—New England Medical Center,
LOUIS E. BARTOSHESKY
Boston, Massachusetts
MONKEY SCOLIOSIS
SiR,-Your editorial on scoliosis (March 10, p. 537) cites the important observations of Lloyd-Roberts et al.1 who showed that idiopathic scoliosis in humans may be related to failure of the spinal cord to rotate m company with the rotation of the vertebral column, causing nerve-root traction and cell degeneration in the dorsal root ganglion. This prompts us to - report the development of scoliosis in cynomolgus monkeys (Macaca fascicularis) following the routine safety testing of live, attenuated, oral poliomyelitis vaccines in the neurovirulence monkey test2 with the standard technique of
intraspinal inoculation.3 Shapiro, S., and others. Lancet, 1976, i, 272 Fraser, F. C., Metrakos, J D. Zletkin, M. ibid. 1978, i, 884. 1. Llovd-Roberts, G. C., Pincott, J. R., McMeniman, P., Bayley, dall, B. J Bone Jt Surg. 1978, 60B, 451. 2. Boulder, L. R.J. biol Standard. 1973, 1, 119. 3 Beswick, T. S. L., Cord, C. R J. Hyg., Camb. 1961, 59, 395.
2-Four sections of upper lumbar cord of scoliotic shown in fig. 1.
Fig.
monkey
Note needle track trauma in right posterior horn. Nerve tissue was mechanically destroyed on same side i.e., right) as convex side of spinal curve. Little or no histological poliomyelitis (viral activity observed in this monkey. (Einarson-gallocyamn.)
8. 9
I.
J. L., Ken-
Of 1370
monkeys
inoculated
intraspinally,
under
general
anxsthesia, with oral poliovaccines, 23 (1.7"() acquired scoliosis (14 left, 2 with marked curvature; 9 right, 6 with marked curvature) and 1 monkey acquired marked
kyphosis. Scoliosis detected between the first and twenty-second day after injection. In 16 cases scoliosis was first observed within 7 days. Three cases were first noticed between the 9th and 16th days, and in4 monkeys scoliosis was detected only on day 22, the final day of observation before routine cardiac perfusion and removal of the brain and spinal cord for histology. The kyphosis was first seen on day 22. In only 3 instances did leg paralysis (normal in some monkeys after vaccine inoculation) accompany the scoliosis. There seems to be no correlation between scoliosis and vaccine manufacturer, poliovirus type, cell substrate, or dilution of vaccine inoculated. Radiographs of five spines examined by Prof. L. C. Vaughan (Royal Veterinary College), Dr P. A. Zorab (Brompton and St. Mary’s Hospitals), and Dr Min Mehta (Royal National Orthopxdic Hospital) showed no bone abnormality, thus the condition might be associated with muscle and/or neuronal damage. As might be expected after the inoculation of live virus, histological examination of the grey-matter of the lumbar cord showed varying degrees of cellular infiltration and destruction of nerve cells through needle trauma and viral activity. Perhaps, however, the most interesting findings were that 2 monkeys, 1 (fig. 1) with marked scoliosis, had few or no nerve cells destroyed through viral activity at the site of inoculation in the grey-matter of the lumbar cord (fig. 2); and that the needle track (trauma) occurred in every case on the same side of the cord as the convex side of the spinal curve. It is therefore possible that the scoliosis seen infrequently in monkeys following intraspinal inoculation may be due to mechanical damage of the dorsal nerve root and/or posterior horn caused by the needle as it passes into the spinal cord, or to the failure of the spinal cord to rotate in company with the vertebrx during the violent muscular contractions of one or both legs which on occasion accompany the injection. This possibility is noBB being
was
Fig. I-Radiograph of cynomolgus monkey. Severe right lateral curvature and rotation of spine, first observed 2 davs after intraspinal inoculation with 0.1 ml of a type m live, attenuated poliomyelitis vaccine. Sedated and X-rayed 22 days after inoculation.
explored. Whatever the
wause
and mechanism of monkey section.
