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Lymphocytic inclusions in I-cell disease
88
B r i e f clinical and laboratory observations
agnosis a n d describes m e t h o d s for diagnosis a n d treat-
The Journal Of Pediatrics July 1974
4.
m e n t in t h e n e o n a t a l period. We are indebted to Vivian Shih, M.D., for the argininosuccinase assay and ASA determination on cord blood plasma. Paul Blankenship provided technical assistance.
5.
REFERENCES
6.
1. Shih, V. E., and Efron, M. L.: Urea cycle disorders, in Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., editors: The metabolic basis of inherited disease, ed. 3, New York, 1972, McGraw Hill Book Company, Inc. p. 282. 2. Shih, V. E.: Early dietary management in an infant with argininosuccinase deficiency: Preliminary report, J. PEDlATR. 80: 645,1972. 3. Shih, V. E., and Littlefield, J. W.: Argininosuccinase activity in amniotic-fluid cells, Lancet 2: 45, 1970.
Lymphocytic inclusions in 1-cell disease Juhani Rapola, M.D.,* Seppo Autio, M.D., Pertti Aula, M.D., and Veikko Nanto, M.D., Helsinki, Finland
L E R 0 Y a n d D e M a r s t c o i n e d t h e t e r m "I-cell d i s e a s e " to describe a p a t i e n t w i t h a Hurler-like s y n d r o m e , w h o s e skin fibroblasts in culture c o n t a i n e d a b u n d a n t inclusions in p h a s e - c o n t r a s t microscopy. S u b s e q u e n t reports h a v e c o n f i r m e d t h a t I-cell disease is a clinical entity. 2, 3 A l t h o u g h the clinical a n d r o e n t g e n o g r a p h i c findings are relatively u n i f o r m , the final diagnosis is usually b a s e d on e l a b o r a t e e n z y m e studies s u p p l e m e n t e d by tissue culture. T h e c o n s i s t e n t p r e s e n c e o f c y t o p l a s m i c vacuoles in t h e blood l y m p h o c y t e s of p a t i e n t s w i t h I-cell disease led us to study t h e m by e l e c t r o n m i c r o s c o p y ; the results m a y be useful for diagnostic purposes. CASE REPORTS Case 1. T. O., a girl, the only child of healthy and non-consanguineous parents, was born after an uneventful pregnancy by cesarean section because of suspected intrauterine asphyxia. Coarse facies, broad and low nasal bridge, and prominent upper From the Children ~"Hospital, University of HelsinkL Supported by grants .h'om the National Council for Medical Sciences and the Sigrid Jusglius Foundation, Finland. *Reprint address: Children's Hospital, Stenbiickinkatu 11, SF00290, Hels#~ki 29, Finland.
7.
8.
Goodman, S. L, Mace, J. W., Turner, B., and Garrett, W. J.: Antenatal diagnosis of argininosuccinic aciduria, Clin. Genet. 4: 236, 1973. Coryell, M. E., Hall, W. K., Thevaos, T. G., Welter, D. A., Gatz, A. J., Horton, B. F., Sisson, B. D., Looper, J. W., Jr., and Farrow, R. T.: A familial study of a human enzyme defect, argininosuccinic aciduria, Biochem. Biophys. Res. Commun. 14: 307, 1964. Rather, S., and Kunkemueller, M.: Separation and properties of argininosuccinate and its two anhydrides and their detection in biological material, Biochemistry 5: 1821, 1966. Cusworth, D. C., and Westall, R. G.: Determination of argininosuccinic acid by ion-exchange chromatography, Nature 192: 555, 1961. O'Brien, D., Ibbott, F. A., and Rogerson, D. O.: Laboratory Manual of Pediatrics: Micro Biochemical Techniques, ed. 4, New York, 1968, Hoeber Medical Division, Harper and Row, Publishers, p. 35.
lip, together with unusually curved legs, were noted at birth. Smears of peripheral blood showed numerous lymp hocytes with cytoplasmic vacuoles. During the next two years increasingly coarse facies, progressive skeletal changes, slow psychomotor development, and diffuse opacities in the corneal stroma and epithelium raised suspicion of a disorder such as Hurler's syndrome, though determinations of urinary glycosaminoglycans were repeatedly within normal limits. At the age of seven years she is unable to speak, the movements of her shoulder, elbow, knee, and hip joints are severely restricted, and she moves by crawling. Her weight and height have been constantly below the standard 2.5 percentile curve. She suffers from recurrent respiratory infections. The diagnosis of l-cell disease was made from cultures of skin fibroblasts with typical cytoplasmic inclusions by both light and electron microscopy. 4 Determination of the activities of several lysosomal enzymes (N-acetyl-/3-glucosaminidase,/3-glucosidase, /3-galactosidase, r arylsulphatase-A, N-aspartyl-/3-glucosaminidase) revealed abnormally low activities in cell homogenates of the fibroblasts, but markedly increased activities, up to 20 to 50 fold as compared with normal values, in the serum and urine. Case 2. J. A., a boy, the only child of healthy and nonconsanguineous parents, was born by vacuum extraction at the thirty-sixth gestational week. Skeletal abnormalities were suspected from a radiograph taken at the thirty-fifth week of pregnancy because of vaginal bleeding of the mother. The child was hypotonic at birth. He had facial features similar to those of T. O. (case 1), thoracic deformity, and hepatomegaly. Vacuolized lymphocytes were found in his peripheral blood. Radiographs showed skeletal changes, particularly in the long bones; these included irregularities of structure, periosteal reaction, and osteolytic changes. Similar but milder abnormalities were found in the calvarium, ribs, and vertebral bodies. Urinary excretion of glycosaminoglycans was normal. At the age of 11 months his psychomotor development is clearly retarded, facial coarseness
Volume 85 Number 1
Fig. 1. A lymphocyte from the peripheral blood of T. O,, case 1. Several cytosomes with polymorphic contents are present. The nucleus and other cytoplasmic organelles appear normal. (x 20, 000.) is more evident and the movements of his shoulder joints are restricted. A systolic murmur of Grade 3/6 was heard maximally at the lower left sternal bodies, but there is no clear evidence of a structural heart anomaly. He suffers from recurrent respiratory infections. The diagnosis of l-cell disease was made from cultured skin fibroblasts showing the same abnormalities as in case 1. Similarly, low intracellular and high extracellular activities of lysosomal enzymes were recorded. For lymphocyte studies, 10 ml. of freshly drawn heparinized venous blood was centrifuged at 1,000 r.p.m. After removal of the serum, the burly layer was fixed with two per cent glutaraldehyde solution; the resulting leukocyte-rich disk was cut into blocks about 1 ram. 3, postfixed with one per cent OsO4, and processed for electron microscopy. 5 RESULTS Many peripheral blood lymphocytes contained numerous cytoplasmic inclusions that appeared similar to those which were observed in the fibroblasts (Fig. 1). T h e inclusions were bounded by a unit m e m b r a n e and had polymorphic, m e m b r a n o u s , and a m o r p h o u s contents. Clear-centered spherules, 60 to 200 rim. in diameter, were particularly frequent (Fig. 2). Occasionally structures suggesting degenerating mitochondria and other cell constituents were seen within the cytosomes. Abnormal c y t o s o m e s were seen in about 10 per cent of the lymphocytes in two samples o f peripheral blood in case
Brief clinical and laboratory observations
89
Fig. 2. Higher magnification of two cytosomes. Numerous spherules and other membranous structures are evident. (• 60 00o.) 1 and in more than 50 per c e n t of the lymphocytes in case 2. They were most f r e q u e n t in the large lymphocytes, but were also present in the small dark lymphocytes and the monocytes. T h e n u m b e r o f c y t o s o m e s in each section of a cell varied f r o m one to about eight. P o l y m o r p h o n u c l e a r leukocytes and rare plasmocytes did not s h o w similar changes. COMMENT I-cell disease may be suspected during the first week of life f r o m the facial deformity and skeletal changes which r e s e m b l e those seen in Hurler's syndrome. N o r m a l excretion o f urinary glycosaminoglycans and the presence of n u m e r o u s vacuolated l y m p h o c y t e s are additional features o f the disease. Hitherto, the final diagnosis has b e e n based on fibroblast cultures with typical light and electron microscopic changes as well as disturbances in the activities of several lysosomal hydrolytic enzymes. High s e r u m levels of lysosomal e n z y m e s in I-cell disease led s o m e investigators to propose the determination of s e r u m arylsulphatase-A activity for screening of the disease. 6 Since the report o f Abt and Bloom 7 in 1928, describing vacuolated lymphocytes and m o n o c y t e s in N i e m a n n Pick disease, similar cells have been found in a few other
90
Brief clin&al and laboratory observations
The Journal of Pediatricw July 1974
storage disorders. The vacuoles apparently reflect the same storage p h e n o m e n o n which is present in many solid tissues. In the light of extensive investigations on the ultrastructure of affected organs in lysosomal storage disorders, it is surprising that only rare reports on the ultrastructure of the readily available teukocytes have appeared. Lazarus and associates 8 reported cytosomes with lamellar membranous inclusions in the lymphocytes in Niemann-Pick disease. Similar findings have been reported in juvenile amaurotic idiocy,9 several types of mucopolysaccharidoses, ~~ and in GM~gangliosidosis.1~In these reports the ultrastructure of the lymphocytic cytosomes bore evident similarity to the storage inclusions in solid tissues of the respective disorder. Likewise, the lymphocytic cytosomes of the present cases are very much like those of cultured fibroblasts and of cells from other tissues. 3,4 Electron microscopy of the peripheral leukocytes, a relatively simple procedure, as compared with cell culture, is suggested as an aid in the diagnosis of I-cell disease and in similar disorders.
10.
REFERENCES
11.
1. Leroy,J. G., and DeMars, R. I.: Mutant enzymatic and cytological phenotypes in cultured human fibroblasts, Science 157: 804, 1967. 2. Leroy, J. G., Spranger, J. W., Feingold, M., Opitz, J. M.,
Inner canthal and intermamillary indices in the newborn infant K~iroly M(~hes, M.D., and Erzs~bet Kitzv~ger, M.D., Gy6r, Hungary HYPERTELORISM means an excessive distance between any paired organs. Ocular hypertelorism is regarded as a characteristic sign of at least twenty congenital syndromes, whereas widely set nipples are common in T u r n e r a n d Noonan phenotypes and in about ten other conditions. Since inspection alone might be misleading, exact determination of these two forms of hypertelorism would be i m p o r t a n t , especially in newborn infants. However, no adequate data on normal variation of the distance between the eyes and nipples in Reprint address: Departmento/' Pediatrics, County Hospital, H-9002Gyol, Hungary.
3.
4.
5.
6. 7. 8.
9.
and Crocker, A. C.: I-cell disease. A clinical picture, J. PEDIATR.79: 360, 1971. Tondeur, M., Vamos-Hurwitz, E, Mockel-Pohl, S., Dereume, J. P., Cremer, N., arid Loeb, H.: Clinical, biochemical and ultrastructural studies in a case of chondrodystrophy presenting the l-cell phenotype in tissue culture, J. PEt)~ATR.79: 366, 1971. Hanai, J., Leroy, J. G., and O'Brien, J.: Ultrastructure of cultured fibroblasts in I-celldisease, Am. J. Dis. Child. 122: 34, 1971: Anderson, D. R.: A method for preparing peripheral leucocytes for electron microscopy, J. Ultrastruct. Res. 13: 263, 1965. Kelly, T. E., Thomas, G. H., and Taylor, H. A., Jr.: Screening for mucolipidosis, Lancet 2: 1089, 1973. Abt, A. F., and Bloom, V.: Essential lipoid histiocytosis (type Niemann-Pick), J. A. M. A. 90: 2076, 1928. Lazarus, S. S., Vethamany, V. G., Schneck, L., and Volk, B. V.: Fine structure and histochemistry of peripheral blood cells in Niemann-Pick disease, Lab. Invest. 17: 155, 1967. Witzleben, C. L., Smith, K., Nelson, J. S., Monteleone, P. L., and Livingston, D.: Ultrastructural studies in late-onset arnaurotic idiocy. Lymphocyte inclusions as a diagnostic marker, J. PEDIATR.79: 285, 1971, Belcher, R. W.: Ultrastructure and cytochemistry of lymphocytes in the genetic mucopolysaccharidoses, Arch. Pathol. 93: 1, 1972. Heyne, K., Kemmer, Ch., Simon, Ch., and Trtibsbach, A.: Generalisierte GMi-Gangliosidose. Feinstruktur und differentialdiagnostische Bedeutung speichernder Lymphozyten und Knocbenmarkszellen, P~diatr. P~dol. 8: 272, 1973.
neonates of different gestational ages and birth weights are available. For the determination of ocular hypertelorism the measurement of the interpupillary distance 1 is often recommended, yet this cannot be reliably carried out in newborn infants. The distance between the inner canthi
Seerelated articles, pp. 141 and 148. may easily be measured with a caliper ruler graduated in millimeters,2 even in the presence of epicanthic folds. However, when considering the inner canthal distance alone, the constitution of the head is disregarded. It seems to be more practicable to employ the inner canthal index calculated by the following formula: Inner canthal index =
Inner canthal distance (cm.) • 100 Head circumference at the level of the canthi (cm.)
The position of the mamillae can be objectively expressed by the intermamillary index, introduced by Pelz.3The principle of computing this index is the same as that of the inner canthal index:
B r i e f clinical and laboratory observations
agnosis a n d describes m e t h o d s for diagnosis a n d treat-
The Journal Of Pediatrics July 1974
4.
m e n t in t h e n e o n a t a l period. We are indebted to Vivian Shih, M.D., for the argininosuccinase assay and ASA determination on cord blood plasma. Paul Blankenship provided technical assistance.
5.
REFERENCES
6.
1. Shih, V. E., and Efron, M. L.: Urea cycle disorders, in Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., editors: The metabolic basis of inherited disease, ed. 3, New York, 1972, McGraw Hill Book Company, Inc. p. 282. 2. Shih, V. E.: Early dietary management in an infant with argininosuccinase deficiency: Preliminary report, J. PEDlATR. 80: 645,1972. 3. Shih, V. E., and Littlefield, J. W.: Argininosuccinase activity in amniotic-fluid cells, Lancet 2: 45, 1970.
Lymphocytic inclusions in 1-cell disease Juhani Rapola, M.D.,* Seppo Autio, M.D., Pertti Aula, M.D., and Veikko Nanto, M.D., Helsinki, Finland
L E R 0 Y a n d D e M a r s t c o i n e d t h e t e r m "I-cell d i s e a s e " to describe a p a t i e n t w i t h a Hurler-like s y n d r o m e , w h o s e skin fibroblasts in culture c o n t a i n e d a b u n d a n t inclusions in p h a s e - c o n t r a s t microscopy. S u b s e q u e n t reports h a v e c o n f i r m e d t h a t I-cell disease is a clinical entity. 2, 3 A l t h o u g h the clinical a n d r o e n t g e n o g r a p h i c findings are relatively u n i f o r m , the final diagnosis is usually b a s e d on e l a b o r a t e e n z y m e studies s u p p l e m e n t e d by tissue culture. T h e c o n s i s t e n t p r e s e n c e o f c y t o p l a s m i c vacuoles in t h e blood l y m p h o c y t e s of p a t i e n t s w i t h I-cell disease led us to study t h e m by e l e c t r o n m i c r o s c o p y ; the results m a y be useful for diagnostic purposes. CASE REPORTS Case 1. T. O., a girl, the only child of healthy and non-consanguineous parents, was born after an uneventful pregnancy by cesarean section because of suspected intrauterine asphyxia. Coarse facies, broad and low nasal bridge, and prominent upper From the Children ~"Hospital, University of HelsinkL Supported by grants .h'om the National Council for Medical Sciences and the Sigrid Jusglius Foundation, Finland. *Reprint address: Children's Hospital, Stenbiickinkatu 11, SF00290, Hels#~ki 29, Finland.
7.
8.
Goodman, S. L, Mace, J. W., Turner, B., and Garrett, W. J.: Antenatal diagnosis of argininosuccinic aciduria, Clin. Genet. 4: 236, 1973. Coryell, M. E., Hall, W. K., Thevaos, T. G., Welter, D. A., Gatz, A. J., Horton, B. F., Sisson, B. D., Looper, J. W., Jr., and Farrow, R. T.: A familial study of a human enzyme defect, argininosuccinic aciduria, Biochem. Biophys. Res. Commun. 14: 307, 1964. Rather, S., and Kunkemueller, M.: Separation and properties of argininosuccinate and its two anhydrides and their detection in biological material, Biochemistry 5: 1821, 1966. Cusworth, D. C., and Westall, R. G.: Determination of argininosuccinic acid by ion-exchange chromatography, Nature 192: 555, 1961. O'Brien, D., Ibbott, F. A., and Rogerson, D. O.: Laboratory Manual of Pediatrics: Micro Biochemical Techniques, ed. 4, New York, 1968, Hoeber Medical Division, Harper and Row, Publishers, p. 35.
lip, together with unusually curved legs, were noted at birth. Smears of peripheral blood showed numerous lymp hocytes with cytoplasmic vacuoles. During the next two years increasingly coarse facies, progressive skeletal changes, slow psychomotor development, and diffuse opacities in the corneal stroma and epithelium raised suspicion of a disorder such as Hurler's syndrome, though determinations of urinary glycosaminoglycans were repeatedly within normal limits. At the age of seven years she is unable to speak, the movements of her shoulder, elbow, knee, and hip joints are severely restricted, and she moves by crawling. Her weight and height have been constantly below the standard 2.5 percentile curve. She suffers from recurrent respiratory infections. The diagnosis of l-cell disease was made from cultures of skin fibroblasts with typical cytoplasmic inclusions by both light and electron microscopy. 4 Determination of the activities of several lysosomal enzymes (N-acetyl-/3-glucosaminidase,/3-glucosidase, /3-galactosidase, r arylsulphatase-A, N-aspartyl-/3-glucosaminidase) revealed abnormally low activities in cell homogenates of the fibroblasts, but markedly increased activities, up to 20 to 50 fold as compared with normal values, in the serum and urine. Case 2. J. A., a boy, the only child of healthy and nonconsanguineous parents, was born by vacuum extraction at the thirty-sixth gestational week. Skeletal abnormalities were suspected from a radiograph taken at the thirty-fifth week of pregnancy because of vaginal bleeding of the mother. The child was hypotonic at birth. He had facial features similar to those of T. O. (case 1), thoracic deformity, and hepatomegaly. Vacuolized lymphocytes were found in his peripheral blood. Radiographs showed skeletal changes, particularly in the long bones; these included irregularities of structure, periosteal reaction, and osteolytic changes. Similar but milder abnormalities were found in the calvarium, ribs, and vertebral bodies. Urinary excretion of glycosaminoglycans was normal. At the age of 11 months his psychomotor development is clearly retarded, facial coarseness
Volume 85 Number 1
Fig. 1. A lymphocyte from the peripheral blood of T. O,, case 1. Several cytosomes with polymorphic contents are present. The nucleus and other cytoplasmic organelles appear normal. (x 20, 000.) is more evident and the movements of his shoulder joints are restricted. A systolic murmur of Grade 3/6 was heard maximally at the lower left sternal bodies, but there is no clear evidence of a structural heart anomaly. He suffers from recurrent respiratory infections. The diagnosis of l-cell disease was made from cultured skin fibroblasts showing the same abnormalities as in case 1. Similarly, low intracellular and high extracellular activities of lysosomal enzymes were recorded. For lymphocyte studies, 10 ml. of freshly drawn heparinized venous blood was centrifuged at 1,000 r.p.m. After removal of the serum, the burly layer was fixed with two per cent glutaraldehyde solution; the resulting leukocyte-rich disk was cut into blocks about 1 ram. 3, postfixed with one per cent OsO4, and processed for electron microscopy. 5 RESULTS Many peripheral blood lymphocytes contained numerous cytoplasmic inclusions that appeared similar to those which were observed in the fibroblasts (Fig. 1). T h e inclusions were bounded by a unit m e m b r a n e and had polymorphic, m e m b r a n o u s , and a m o r p h o u s contents. Clear-centered spherules, 60 to 200 rim. in diameter, were particularly frequent (Fig. 2). Occasionally structures suggesting degenerating mitochondria and other cell constituents were seen within the cytosomes. Abnormal c y t o s o m e s were seen in about 10 per cent of the lymphocytes in two samples o f peripheral blood in case
Brief clinical and laboratory observations
89
Fig. 2. Higher magnification of two cytosomes. Numerous spherules and other membranous structures are evident. (• 60 00o.) 1 and in more than 50 per c e n t of the lymphocytes in case 2. They were most f r e q u e n t in the large lymphocytes, but were also present in the small dark lymphocytes and the monocytes. T h e n u m b e r o f c y t o s o m e s in each section of a cell varied f r o m one to about eight. P o l y m o r p h o n u c l e a r leukocytes and rare plasmocytes did not s h o w similar changes. COMMENT I-cell disease may be suspected during the first week of life f r o m the facial deformity and skeletal changes which r e s e m b l e those seen in Hurler's syndrome. N o r m a l excretion o f urinary glycosaminoglycans and the presence of n u m e r o u s vacuolated l y m p h o c y t e s are additional features o f the disease. Hitherto, the final diagnosis has b e e n based on fibroblast cultures with typical light and electron microscopic changes as well as disturbances in the activities of several lysosomal hydrolytic enzymes. High s e r u m levels of lysosomal e n z y m e s in I-cell disease led s o m e investigators to propose the determination of s e r u m arylsulphatase-A activity for screening of the disease. 6 Since the report o f Abt and Bloom 7 in 1928, describing vacuolated lymphocytes and m o n o c y t e s in N i e m a n n Pick disease, similar cells have been found in a few other
90
Brief clin&al and laboratory observations
The Journal of Pediatricw July 1974
storage disorders. The vacuoles apparently reflect the same storage p h e n o m e n o n which is present in many solid tissues. In the light of extensive investigations on the ultrastructure of affected organs in lysosomal storage disorders, it is surprising that only rare reports on the ultrastructure of the readily available teukocytes have appeared. Lazarus and associates 8 reported cytosomes with lamellar membranous inclusions in the lymphocytes in Niemann-Pick disease. Similar findings have been reported in juvenile amaurotic idiocy,9 several types of mucopolysaccharidoses, ~~ and in GM~gangliosidosis.1~In these reports the ultrastructure of the lymphocytic cytosomes bore evident similarity to the storage inclusions in solid tissues of the respective disorder. Likewise, the lymphocytic cytosomes of the present cases are very much like those of cultured fibroblasts and of cells from other tissues. 3,4 Electron microscopy of the peripheral leukocytes, a relatively simple procedure, as compared with cell culture, is suggested as an aid in the diagnosis of I-cell disease and in similar disorders.
10.
REFERENCES
11.
1. Leroy,J. G., and DeMars, R. I.: Mutant enzymatic and cytological phenotypes in cultured human fibroblasts, Science 157: 804, 1967. 2. Leroy, J. G., Spranger, J. W., Feingold, M., Opitz, J. M.,
Inner canthal and intermamillary indices in the newborn infant K~iroly M(~hes, M.D., and Erzs~bet Kitzv~ger, M.D., Gy6r, Hungary HYPERTELORISM means an excessive distance between any paired organs. Ocular hypertelorism is regarded as a characteristic sign of at least twenty congenital syndromes, whereas widely set nipples are common in T u r n e r a n d Noonan phenotypes and in about ten other conditions. Since inspection alone might be misleading, exact determination of these two forms of hypertelorism would be i m p o r t a n t , especially in newborn infants. However, no adequate data on normal variation of the distance between the eyes and nipples in Reprint address: Departmento/' Pediatrics, County Hospital, H-9002Gyol, Hungary.
3.
4.
5.
6. 7. 8.
9.
and Crocker, A. C.: I-cell disease. A clinical picture, J. PEDIATR.79: 360, 1971. Tondeur, M., Vamos-Hurwitz, E, Mockel-Pohl, S., Dereume, J. P., Cremer, N., arid Loeb, H.: Clinical, biochemical and ultrastructural studies in a case of chondrodystrophy presenting the l-cell phenotype in tissue culture, J. PEt)~ATR.79: 366, 1971. Hanai, J., Leroy, J. G., and O'Brien, J.: Ultrastructure of cultured fibroblasts in I-celldisease, Am. J. Dis. Child. 122: 34, 1971: Anderson, D. R.: A method for preparing peripheral leucocytes for electron microscopy, J. Ultrastruct. Res. 13: 263, 1965. Kelly, T. E., Thomas, G. H., and Taylor, H. A., Jr.: Screening for mucolipidosis, Lancet 2: 1089, 1973. Abt, A. F., and Bloom, V.: Essential lipoid histiocytosis (type Niemann-Pick), J. A. M. A. 90: 2076, 1928. Lazarus, S. S., Vethamany, V. G., Schneck, L., and Volk, B. V.: Fine structure and histochemistry of peripheral blood cells in Niemann-Pick disease, Lab. Invest. 17: 155, 1967. Witzleben, C. L., Smith, K., Nelson, J. S., Monteleone, P. L., and Livingston, D.: Ultrastructural studies in late-onset arnaurotic idiocy. Lymphocyte inclusions as a diagnostic marker, J. PEDIATR.79: 285, 1971, Belcher, R. W.: Ultrastructure and cytochemistry of lymphocytes in the genetic mucopolysaccharidoses, Arch. Pathol. 93: 1, 1972. Heyne, K., Kemmer, Ch., Simon, Ch., and Trtibsbach, A.: Generalisierte GMi-Gangliosidose. Feinstruktur und differentialdiagnostische Bedeutung speichernder Lymphozyten und Knocbenmarkszellen, P~diatr. P~dol. 8: 272, 1973.
neonates of different gestational ages and birth weights are available. For the determination of ocular hypertelorism the measurement of the interpupillary distance 1 is often recommended, yet this cannot be reliably carried out in newborn infants. The distance between the inner canthi
Seerelated articles, pp. 141 and 148. may easily be measured with a caliper ruler graduated in millimeters,2 even in the presence of epicanthic folds. However, when considering the inner canthal distance alone, the constitution of the head is disregarded. It seems to be more practicable to employ the inner canthal index calculated by the following formula: Inner canthal index =
Inner canthal distance (cm.) • 100 Head circumference at the level of the canthi (cm.)
The position of the mamillae can be objectively expressed by the intermamillary index, introduced by Pelz.3The principle of computing this index is the same as that of the inner canthal index: