596
Brief clinical and laboratory observations
physical examination had suggested that a previously normal child had had a simple febrile convulsion, skull roentgenograms were not of help. For the patients surveyed in this study this entailed an approximate overall expenditure of $24,400.00. The yield, compared with the high cost of skull radiography as well as the unnecessary exposure to ionizing radiation may not justify the use of skull radiography in the management of patients with simple febrile seizures. The authors acknowledge the assistance of Dr. Sally Roth of Boston, Mass., during this study. We also express gratitude to the Pediatric Radiology Departments of Boston City Hospital, Babies Hospital in New York, and Variety Children's Hospital in Miami for their help and cooperation.
Isolation of Gaucher cells from bone marrow Robert Cohn, M.D.,* John R. Yandrasitz, B.S., Beverly Lange, M.D., Jeffrey Rosenstock, M.D., James Gerson, M.D., and Stanton Segal, M.D., Philadelphia, Pa. REPLACEMENT of missing or defective enzymes by systemic injection offers the potential for relief of the symptoms of lysosomal storage diseases. The development of such therapy requires evaluation of its efficacy on tissue storage pools as well as on circulating levels of the accumulating metabolite. In Gaucher disease the bone marrow provides a pool of characteristic storage cells that may be collected by aspiration. To use bone marrow to assess changes in storage levels, the isolation of these cells is essential. Bone marrow obtained from the total hip arthroplasty of an adolescent patient with Gaucher disease provided us with sufficient material to investigate the sedimentation From the Division of Biochemical Development and Molecular Diseases and the Division of Oncology, Children's Hospital of Philadelphia, and the Departments of Pediatrics and Medicine, University of Pennsylvania School of Medicine. Supported by Grant 5 P01 HD 08536 of the National Institutes of Health. Robert M. Cohn is a recipient of a Research Career Development A ward from the National Institutes of Health, No. 5 K04 HDO0008. *Reprint address: The Children "s Hospital of Philadelphia, 34th & Civic Center Blvd, Philadelphia, PA 19104.
The Journal of Pediatrics April 1977
REFERENCES 1. Ouellette EM: The child who convulses with fever, Pediatr Clin North Am 21:467, 1974. 2. Livingston S: Infantile febrile convulsions, Dev Med Child Neurol 10:374, 1968. 3. Hammill J, and Carter S: Febrile convulsions, N Engl J Med 274:563, 1966. 4. Asnes RA, Novick LF, Nealis JGT, and Nguyen M: The first febrile seizure: A study of current pediatric practice, J ~PEDIATR87:485, 1975. 5. Hayes WG, and Shopfner CE: Plain skull roentgcnographic findings in infants and children with convulsions, Am J Dis Child 126:785, 1973.
characteristics of Gaucher cells. Employing velocity sedimentation on a discontinuous gradient of bovine serum albumin, we obtained an essentially pure fraction of these cells. MATERIALS
AND METHODS
Bovine serum albumin was prepared from Fraction V powder (Miles-Pentex) according to Worton and associates~ with the following modifications: the 40% (w:v) solution (40 gm BSA plus 78 ml water) was deonized with 4 gm Amberlite MB-3 resin; the long centrifugation and filtration steps were omitted owing to lack of time before receipt of the sample; the phosphate-buffered saline contained 1 mM EDTA (final) and had a pH of 6.8; addition of tenfold concentrated PBS to the BSA solution brought its final concentration to 37% and its pH to 6.8 rather than 5.4. This solution was diluted (v:v) with isotonic PBS to form the solutions for the gradient. Abbreviation used BSA: bovine serum albumin PBS: phosphate-buffered saline Discontinuous gradients were prepared in 30 ml cellulose nitrate tubes by layering 4 ml each of 37, 30, 25, 20, 15, 10% BSA solutions. Gradients were incubated at 37 ~ for 30 minutes and then cooled in an ice bath; this incubation accomplished no visible softening of boundaries. Approximately 25 ml of bone marrow was obtained at surgery and was suspended in sterile Hank's medium containing heparin, penicillin, and streptomycin. About half of this volume was comprised of bone and fibrous material, most of which was removed by washing with PBS through nylon (stocking) mesh. The suspension
Volume 90 Number 4
Brief clinical and laboratory observations
597
Table I. Differential cell counts in freshly obtained marrow and in gradient fractions Fraction No.
Gaucher Myeloid precursors Erythroid precursors Lymphoid Smudge
Marrow
1
2
3
4
13 33
97 1
40 30
5 40
5 59
0 80
45
0
5
43
35
12
10 0
2 +
25 +
12 ++
1 +++
8 ++++
Table II. Quantity of Gaucher cells isolated Gradient 1
Gradient H
(cells/ml of marrow suspension)
Fraction 1 Fraction 2 Total
2.96 x 105 0.52 x 10,~' 3.48 x 10~
2.52 • 105 1.20 x t02 3.71 x 10~
Bone marrow was diluted with PBS and filtered several times through nylon. Aliquotsof this suspensionwere separated on BSA gradients at an average centrifugalforce of 1,280 gm. Gradient I was centrifugedfor 45 minutes and gradient II for 60 minutes. obtained was filtered several times through double layers of nylon; however, much fibrous material remained. Contact of the cells with glass was avoided throughout the procedure. Five milliliters of the final suspension was layered onto each gradient, and these were centrifuged at 3,500 rpm (750 to 1,800 g top to bottom) for 45 minutes in a cold (04 ~ Spinco SW 25. t rotor. After a preliminary evaluation of this first experiment, a second set of gradients was prepared in an identical fashion. These were centrifuged at the same speed for 60 minutes rather than 45, but the layering pattern was visually identical. The fractions were collected by aspiration. Corresponding layers from three tubes were pooled, diluted three to four fold with PBS, and centrifuged at 1,000 g for 10 minutes in a refrigerated centrifuge. The pellets were gently suspended with sterile isotonic saline. Samples were taken for cell counts and for differential cell counts. The remaining material was frozen for subsequent chemical assays. RESULTS Centrifugation of the gradients produced bands at every interface and a large red cell pellet. The fibrous material which was not removed by filtration was scattered throughout the gradient. Bands at the 30/37 (fraction 5) and 25/30 (fraction 4) interfaces were red and
Fig. 1. Photomicrograph of gradient fraction 1 demonstrating clumps of typical Gaucher cells (Wright-Giemsa stain; original magnification 500 x ). appeared clumped. Bands at 20/25 (fraction 3) and 15/20 (fraction 2) interfaces were tan and somewhat smoother, but contained some red fibrous material. The band at the 10/15 (fraction 1) interface was similar in appearance to the two below but was less compact-diffusing throughout the 10% layer. The supernatant was clear, light red, and topped by a fairly compact layer of fat. Results of a microscopic examination of the gradient fractions are presented in Table I. For comparison the composition of the freshly obtained marrow is also given. The same ratio of myeloid to erythroid precursors has been seen previously in an aspirate from this patient. Fraction 1 represented a nearly pure preparation of Gaucher cells (Fig. 1). The n u m b e r of Gaucher cells isolated in the two centrifugations is presented in Table II. Although the two sets of gradients were visually identical, more spillover of Gaucher cells into fraction 2 occurred during the 60-minute centrifugation. For the most part, the Gaucher cells did not cross into the 15% BSA layer at 1,100 g (effective force at the 10/15% interface). The n u m b e r of Gaucher cells in fraction 1 was fairly consistent even with this variation in centrifugation time. Apparently the density of material stored in Gaucher ceils, and perhaps in histiocytes in other lipid storage diseases, makes their sedimentation characteristics suffi-
598
Brief cfinical and laboratory observations
ciently different from other cells occurring in marrow that their isolation may be readily accomplished by velocity sedimentation. COMMENTS Gaucher cells are tissue histocytes that are found in organs possessing a significant reticuloendothelial component, viz., liver, spleen and bone marrow. Although these cells have been considered pathognomonic for glucocerebrosidase deficiency, they have been reported in a number of cases of chronic myelocytic leukemia, presumably as a result of overproduction of glucocerebroside. 2 Enzyme placement therapy either by direct injection of purified human placental enzyme 3 or enzyme enclosed in antigenically compatible liposomes ' has been proposed as a rational means of ameliorating the inexorable progression of lysosomal storage diseases. Such therapy has been attempted in Gaucher disease, z Success of therapy from a biochemical viewpoint must be judged by its effects on both tissue storage pools and circulating levels of the accumulating glycolipid. Up to now liver biopsy ~has been employed for the estimation of tissue storage levels, but such procedures are liable to patient morbidity. Bone marrow represents a readily available source of cells, and its aspiration is a safe method for obtaining tissue from the patient. In order to use a bone marrow aspirate for quantitative evaluation of storage pools, one
The (?) value of routine skull radiograph); in clinical evaluation of children with recurrent convulsions Wulfred Berman, M.D.,* and Burton A. Johnson, M.D., Baltimore, Md. S K U L L R A D I O G R A P H Y is often an integral part of a routine work-up in patients with convulsions. Since there is scanty documentation of the expected diagnostic yield from simple radiography, we studied the skull roentgeno-
From the Department of Pediatrics and Radiology, Johns Hopkins University, and The Epilepsy Clinic Johns Hopkins Hospital. *Reprint address: Rosewood Center, Owings Mills. MD 21117.
The Journal of Pediatrics April 1977
must know the concentration of storage cells in the marrow as well as the level of stored material per cell. The former data may reasonably be obtained from differential cell counts; the latter, however, depends upon a method for the isolation of these cells from the heterogenous population of cells from bone marrow. Adaptation of the methods described in this paper should permit an equivalent separation of cells from a usual bone marrow aspirate. These cells can serve as a convenient gauge to assess the success of enzyme replacement therapy in this disease, as well as other lysosomal storage diseases in which storage of material occurs in the reticuloendothelial system.
REFERENCES 1. Worton RG, McCulloch EA, and Till JE: Physical separation of hemopoietic stem ceils from cells forming colonies in culture, J Cell Physiol 74:171, 1969. 2. Kattlove HE, Williams JC, Gaynor E, Spivak M, Bradley RM, and Brady RO: Gaucher cells in chronic myelocytic leukemia: An acquired abnormality, Blood 33:379, 1969. 3. Pentchev PG, Brady RO, Gal AE, and Hibbert SR: Replacement therapy for inherited enzyme deficiency. Sustained clearance of accumulated glucocerebroside in Gaucher's disease following infusion of purified glucocerebrosidase, J Molec Med 1:73, 1975. 4. Gregoriadis G: Carrier potential of liposomes in biology and Medicine, N Engl J Med 295:704, 765, 1976.
grams performed as part of the routine work-up of children attending the Johns Hopkins Epilepsy Clinic. MATERIAL
AND METHOD
This report presents the positive radiographic findings of 130 consecutive patients who had routine skull roent-
See related article, p. 595. genograms performed as part of their seizure work-up. Skull radiography included Towne, Caldwell, and lateral views and was performed on all patients with seizures whether of epileptic origin or secondary to other causes. The period of study extend ed over 12 months (1972-1973). Children who had simple febrile convulsions, breathholding spells, hysterical spells, fainting spells, and whose roentgenograms of the skull were unsatisfactory or unobtainable for technical reasons were excluded from the study. RESULTS Abnormal roentgenograms were present in nine (6.9%) of the 130 patients. Microcephaly (3) and calvarial thick-
Brief clinical and laboratory observations
physical examination had suggested that a previously normal child had had a simple febrile convulsion, skull roentgenograms were not of help. For the patients surveyed in this study this entailed an approximate overall expenditure of $24,400.00. The yield, compared with the high cost of skull radiography as well as the unnecessary exposure to ionizing radiation may not justify the use of skull radiography in the management of patients with simple febrile seizures. The authors acknowledge the assistance of Dr. Sally Roth of Boston, Mass., during this study. We also express gratitude to the Pediatric Radiology Departments of Boston City Hospital, Babies Hospital in New York, and Variety Children's Hospital in Miami for their help and cooperation.
Isolation of Gaucher cells from bone marrow Robert Cohn, M.D.,* John R. Yandrasitz, B.S., Beverly Lange, M.D., Jeffrey Rosenstock, M.D., James Gerson, M.D., and Stanton Segal, M.D., Philadelphia, Pa. REPLACEMENT of missing or defective enzymes by systemic injection offers the potential for relief of the symptoms of lysosomal storage diseases. The development of such therapy requires evaluation of its efficacy on tissue storage pools as well as on circulating levels of the accumulating metabolite. In Gaucher disease the bone marrow provides a pool of characteristic storage cells that may be collected by aspiration. To use bone marrow to assess changes in storage levels, the isolation of these cells is essential. Bone marrow obtained from the total hip arthroplasty of an adolescent patient with Gaucher disease provided us with sufficient material to investigate the sedimentation From the Division of Biochemical Development and Molecular Diseases and the Division of Oncology, Children's Hospital of Philadelphia, and the Departments of Pediatrics and Medicine, University of Pennsylvania School of Medicine. Supported by Grant 5 P01 HD 08536 of the National Institutes of Health. Robert M. Cohn is a recipient of a Research Career Development A ward from the National Institutes of Health, No. 5 K04 HDO0008. *Reprint address: The Children "s Hospital of Philadelphia, 34th & Civic Center Blvd, Philadelphia, PA 19104.
The Journal of Pediatrics April 1977
REFERENCES 1. Ouellette EM: The child who convulses with fever, Pediatr Clin North Am 21:467, 1974. 2. Livingston S: Infantile febrile convulsions, Dev Med Child Neurol 10:374, 1968. 3. Hammill J, and Carter S: Febrile convulsions, N Engl J Med 274:563, 1966. 4. Asnes RA, Novick LF, Nealis JGT, and Nguyen M: The first febrile seizure: A study of current pediatric practice, J ~PEDIATR87:485, 1975. 5. Hayes WG, and Shopfner CE: Plain skull roentgcnographic findings in infants and children with convulsions, Am J Dis Child 126:785, 1973.
characteristics of Gaucher cells. Employing velocity sedimentation on a discontinuous gradient of bovine serum albumin, we obtained an essentially pure fraction of these cells. MATERIALS
AND METHODS
Bovine serum albumin was prepared from Fraction V powder (Miles-Pentex) according to Worton and associates~ with the following modifications: the 40% (w:v) solution (40 gm BSA plus 78 ml water) was deonized with 4 gm Amberlite MB-3 resin; the long centrifugation and filtration steps were omitted owing to lack of time before receipt of the sample; the phosphate-buffered saline contained 1 mM EDTA (final) and had a pH of 6.8; addition of tenfold concentrated PBS to the BSA solution brought its final concentration to 37% and its pH to 6.8 rather than 5.4. This solution was diluted (v:v) with isotonic PBS to form the solutions for the gradient. Abbreviation used BSA: bovine serum albumin PBS: phosphate-buffered saline Discontinuous gradients were prepared in 30 ml cellulose nitrate tubes by layering 4 ml each of 37, 30, 25, 20, 15, 10% BSA solutions. Gradients were incubated at 37 ~ for 30 minutes and then cooled in an ice bath; this incubation accomplished no visible softening of boundaries. Approximately 25 ml of bone marrow was obtained at surgery and was suspended in sterile Hank's medium containing heparin, penicillin, and streptomycin. About half of this volume was comprised of bone and fibrous material, most of which was removed by washing with PBS through nylon (stocking) mesh. The suspension
Volume 90 Number 4
Brief clinical and laboratory observations
597
Table I. Differential cell counts in freshly obtained marrow and in gradient fractions Fraction No.
Gaucher Myeloid precursors Erythroid precursors Lymphoid Smudge
Marrow
1
2
3
4
13 33
97 1
40 30
5 40
5 59
0 80
45
0
5
43
35
12
10 0
2 +
25 +
12 ++
1 +++
8 ++++
Table II. Quantity of Gaucher cells isolated Gradient 1
Gradient H
(cells/ml of marrow suspension)
Fraction 1 Fraction 2 Total
2.96 x 105 0.52 x 10,~' 3.48 x 10~
2.52 • 105 1.20 x t02 3.71 x 10~
Bone marrow was diluted with PBS and filtered several times through nylon. Aliquotsof this suspensionwere separated on BSA gradients at an average centrifugalforce of 1,280 gm. Gradient I was centrifugedfor 45 minutes and gradient II for 60 minutes. obtained was filtered several times through double layers of nylon; however, much fibrous material remained. Contact of the cells with glass was avoided throughout the procedure. Five milliliters of the final suspension was layered onto each gradient, and these were centrifuged at 3,500 rpm (750 to 1,800 g top to bottom) for 45 minutes in a cold (04 ~ Spinco SW 25. t rotor. After a preliminary evaluation of this first experiment, a second set of gradients was prepared in an identical fashion. These were centrifuged at the same speed for 60 minutes rather than 45, but the layering pattern was visually identical. The fractions were collected by aspiration. Corresponding layers from three tubes were pooled, diluted three to four fold with PBS, and centrifuged at 1,000 g for 10 minutes in a refrigerated centrifuge. The pellets were gently suspended with sterile isotonic saline. Samples were taken for cell counts and for differential cell counts. The remaining material was frozen for subsequent chemical assays. RESULTS Centrifugation of the gradients produced bands at every interface and a large red cell pellet. The fibrous material which was not removed by filtration was scattered throughout the gradient. Bands at the 30/37 (fraction 5) and 25/30 (fraction 4) interfaces were red and
Fig. 1. Photomicrograph of gradient fraction 1 demonstrating clumps of typical Gaucher cells (Wright-Giemsa stain; original magnification 500 x ). appeared clumped. Bands at 20/25 (fraction 3) and 15/20 (fraction 2) interfaces were tan and somewhat smoother, but contained some red fibrous material. The band at the 10/15 (fraction 1) interface was similar in appearance to the two below but was less compact-diffusing throughout the 10% layer. The supernatant was clear, light red, and topped by a fairly compact layer of fat. Results of a microscopic examination of the gradient fractions are presented in Table I. For comparison the composition of the freshly obtained marrow is also given. The same ratio of myeloid to erythroid precursors has been seen previously in an aspirate from this patient. Fraction 1 represented a nearly pure preparation of Gaucher cells (Fig. 1). The n u m b e r of Gaucher cells isolated in the two centrifugations is presented in Table II. Although the two sets of gradients were visually identical, more spillover of Gaucher cells into fraction 2 occurred during the 60-minute centrifugation. For the most part, the Gaucher cells did not cross into the 15% BSA layer at 1,100 g (effective force at the 10/15% interface). The n u m b e r of Gaucher cells in fraction 1 was fairly consistent even with this variation in centrifugation time. Apparently the density of material stored in Gaucher ceils, and perhaps in histiocytes in other lipid storage diseases, makes their sedimentation characteristics suffi-
598
Brief cfinical and laboratory observations
ciently different from other cells occurring in marrow that their isolation may be readily accomplished by velocity sedimentation. COMMENTS Gaucher cells are tissue histocytes that are found in organs possessing a significant reticuloendothelial component, viz., liver, spleen and bone marrow. Although these cells have been considered pathognomonic for glucocerebrosidase deficiency, they have been reported in a number of cases of chronic myelocytic leukemia, presumably as a result of overproduction of glucocerebroside. 2 Enzyme placement therapy either by direct injection of purified human placental enzyme 3 or enzyme enclosed in antigenically compatible liposomes ' has been proposed as a rational means of ameliorating the inexorable progression of lysosomal storage diseases. Such therapy has been attempted in Gaucher disease, z Success of therapy from a biochemical viewpoint must be judged by its effects on both tissue storage pools and circulating levels of the accumulating glycolipid. Up to now liver biopsy ~has been employed for the estimation of tissue storage levels, but such procedures are liable to patient morbidity. Bone marrow represents a readily available source of cells, and its aspiration is a safe method for obtaining tissue from the patient. In order to use a bone marrow aspirate for quantitative evaluation of storage pools, one
The (?) value of routine skull radiograph); in clinical evaluation of children with recurrent convulsions Wulfred Berman, M.D.,* and Burton A. Johnson, M.D., Baltimore, Md. S K U L L R A D I O G R A P H Y is often an integral part of a routine work-up in patients with convulsions. Since there is scanty documentation of the expected diagnostic yield from simple radiography, we studied the skull roentgeno-
From the Department of Pediatrics and Radiology, Johns Hopkins University, and The Epilepsy Clinic Johns Hopkins Hospital. *Reprint address: Rosewood Center, Owings Mills. MD 21117.
The Journal of Pediatrics April 1977
must know the concentration of storage cells in the marrow as well as the level of stored material per cell. The former data may reasonably be obtained from differential cell counts; the latter, however, depends upon a method for the isolation of these cells from the heterogenous population of cells from bone marrow. Adaptation of the methods described in this paper should permit an equivalent separation of cells from a usual bone marrow aspirate. These cells can serve as a convenient gauge to assess the success of enzyme replacement therapy in this disease, as well as other lysosomal storage diseases in which storage of material occurs in the reticuloendothelial system.
REFERENCES 1. Worton RG, McCulloch EA, and Till JE: Physical separation of hemopoietic stem ceils from cells forming colonies in culture, J Cell Physiol 74:171, 1969. 2. Kattlove HE, Williams JC, Gaynor E, Spivak M, Bradley RM, and Brady RO: Gaucher cells in chronic myelocytic leukemia: An acquired abnormality, Blood 33:379, 1969. 3. Pentchev PG, Brady RO, Gal AE, and Hibbert SR: Replacement therapy for inherited enzyme deficiency. Sustained clearance of accumulated glucocerebroside in Gaucher's disease following infusion of purified glucocerebrosidase, J Molec Med 1:73, 1975. 4. Gregoriadis G: Carrier potential of liposomes in biology and Medicine, N Engl J Med 295:704, 765, 1976.
grams performed as part of the routine work-up of children attending the Johns Hopkins Epilepsy Clinic. MATERIAL
AND METHOD
This report presents the positive radiographic findings of 130 consecutive patients who had routine skull roent-
See related article, p. 595. genograms performed as part of their seizure work-up. Skull radiography included Towne, Caldwell, and lateral views and was performed on all patients with seizures whether of epileptic origin or secondary to other causes. The period of study extend ed over 12 months (1972-1973). Children who had simple febrile convulsions, breathholding spells, hysterical spells, fainting spells, and whose roentgenograms of the skull were unsatisfactory or unobtainable for technical reasons were excluded from the study. RESULTS Abnormal roentgenograms were present in nine (6.9%) of the 130 patients. Microcephaly (3) and calvarial thick-
