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Needle muscle biopsy in infants and children
Needle muscle biopsy in infants and children Percutaneous muscle biopsies were performed in 77 infants and children using the Bergstrom muscle biopsy needle and local anesthesia. Satisfactory specimens for histochemistry and electron microscopy were obtained in all but one case, and the procedure was well tolerated by all patients. No complications were Observed. The percutaneous biopsy is a useful technique for the diagnosis of neuromuscular disorders in children. (J PEDIATR 103:566, 1983)
John H. DiLiberti, M.D., A. N. D'Agostino, M.D., and George Cole, B.A. P o r t l a n d , Ore.
THE ACCURATE DIAGNOSIS of neuromuscular disease in
children generally requires histochemical and sometimes ultrastructural analysis of skeletal muscle in addition to other methods of evaluation. Although satisfactory techniques for securing skeletal muscle biopsies have been available for several years, the open biopsy method is still most commonly used. We review the technique of needle biopsy of skeletal muscle in children, the indications for its use, and our experience with the procedure in 77 pediatric patients. MATERIALS
AND METHODS
Muscle biopsies were obtained from 77 children who were referred for the evaluation of neuromuscular disease or Reye syndrome. They ranged in age from 1 week to 15 years. Two or more biopsies were performed in five patients, resulting in a total of 86 procedures in this series. With the exception of those in hospitalized children, all procedures were done in the outpatient clinic. Most children younger than 8 years were sedated with chloral hydrate 50 mg/kg and meperidine 1 mg/kg, both given orally about 40 to 60 minutes prior to the procedure. Parents were asked not to feed the younger children for four to six hours before coming to the hospital, Most of the biopsies were obtained from the quadriceps femoris muscle, but the gastrocnemius was occasionally sampled in patients with predominantly distal weakness. After preparation of the skin of the mid-lateral thigh From the Departments of Pediatrics, Emanuel Hospital and Oregon Health Sciences University, and the Department o f Pathology, Good Samaritan Hospital. Reprint requests: John DiLiberli, M.D., Chief o f Pediatrics, Oregon Children's" Medical Center~ Emanuel Hospital, 2801 N. Gantenbein Ave.. Portland, OR 97227.
566
The Journal o f P E D I A T R I C S
with povidone iodine, and shaving the site if necessary, a 25- to 27-gauge needle was used to infiltrate the skin with 1% xylocaine. After a satisfactory wheal was produced, this needle was withdrawn, and a 21-gauge needle was used to infiltrate the subcutaneous tissue and muscle sheath. The large needle facilitated identification of the muscle sheath and decreased the likelihood of infiltrating the muscle itself. A total of 3 to 10 ml xylocaine was used, depending on the size of the patient. After a few minutes, a 5 to 8 mm incision was made through the skin of the mid-lateral thigh with a No. 11 scalpel blade, aiming at the center of the shaft of the femur. The knife blade was then pushed through the subcutaneous tissue down to the muscle sheath, which was identified by its tough, resistant character. The sheath was incised by moving the tip of the blade along the surface of the muscle, taking care not to penetrate deeply. All of the biopsies were obtained using Bergstrom muscle biopsy needles ranging in diameter from 3.0 to 5.0 mm. The 3.0 irma needle was modified by reducing the distance from the tip to the cutting window; this modified needle was used in young infants, The biopsy needle was inserted through the skin incision and subcutaneous tissue. It was often necessary to move the tip to identify the incision in the muscle sheath. The needle was then pushed into the muscle to a depth su~cient to cover the cutting window. The experienced operator can usuaily tell if the needle has penetrated muscle by the quality and degree of resistance to the advancing needle. Muscle is more difficult to penetrate that fat and feels more viscous than fascia. After the needle was thought to be securely placed in muscle tissue, the cutting blade was pulled back sufficiently to expose the window completely, the operator's free hand was used to force muscle into the window by applying pressure from
Volume 103 Number 4
Needle muscle biopsy
567
Fig. 2. Low magnification view of part of biopsy sample from 8-year-old boy with congenital myopathy. (ATPase; original magnification X54.) Table. Clinical diagnosis in 77 infants and children Fig. 1. Low magnification view of part of biopsy sample from 3-year-old boy with muscular dystrophy. (Trichrome; original magnification X54.) above, and finally the cutting blade was quickly pushed in and the entire needle removed. Muscle tissue can usually be distinguished from fat and fascia by its color, texture, and consistency, but for small samples obtained from infants, a dissecting microscope at the bedside helps to confirm that an adequate sample has been obtained before terminating the procedure. Additional samples were usually obtained though the same incision, but the needle was inserted at different angles in order to sample other sites within the same muscle. After the biopsy, pressure was applied at the site for a short time, and then the wound was closed using sterile, adhesive tape strips. A large bandage was applied, and an elastic bandage wrapped around the thigh in older children. The elastic bandage allegedly reduces the likelihood of hematoma formation, but we have not seen this complization in patients in whom the elastic bandage was not used. Muscle samples for histochemieal analysis were placed
Patients Myopathy Dystrophy Spinal muscular atrophy Myositis Reye syndrome Normal
25 9 5 2 10 26 77
1 Biopsies 27 9 5 2 17 26 86
in gauze saturated with isotonic saline solution, and transported to the laboratory immediately. After preliminary examination through the dissecting microscope, the sample was placed in gum tragacanth, oriented for appropriate fiber direction, and then frozen in isopentane chilled in liquid nitrogen. Frozen sections 5 and 10 # in thickness were then made and stained using hematoxylin-eosin, trichrome, ATPase, N A D H , acid phosphatase, periodic acid Schiff, oil red O, and phosphorylase. A small sample for electron microscopy was pinned at resting length onto a small piece of tongue depressor blade or cork, using fine needles. This procedure was usually done at the bedside, and the sample was placed immediate-
568
DiLiberti, D'Agostino, and Cole
The Journal of Pediatrics October 1983
Fig. 3. Duchenne muscular dystrophy; increased fat and connective tissue with degenerating fibers. (H & E; original magnification X400.) ly in glutaraldehyde fixative. Unless ultrastructural evaluation was known to be required, this sample was saved and a decision to perform electron microscopy made after histochemical analysis was completed. RESULTS The biopsy samples were adequate for complete histochemical and ultrastructural evaluation in all but one patient, an infant in whom biopsy was done early in the course of the series. In that case enough tissue was obtained to identify clearly an abnormality on hernatoxylin-eosin sections, however, and we plan to obtain a follow-up biopsy in the future. The,'diagnoses are grouped by general category in the Table. The procedure was well tolerated by all patients, although ther e obviously was some discomfort. The older patients said that they did not feel much pain, but a vague, uncomfortable sensation. The infants who were sedated
often slept through the biopsy after crying briefly during the xylocaine injection, which is the most painful part of the procedure,,;]No complications from the procedure were observed. A few of the older patients described some mild pain for one or two days at the biopsy site when asked specifically about it, but none volunteered this information. Despite markedly prolonged prothrombin and partial thromboplastin times in the patients with Reye syndrome, no hemorrhages or hematomas were detected. Representative sections from several of the patients are illustrated in Figs. 1 through 5 and demonstrate the adequacy of the technique for diagnostic purposes. DISCUSSION Since Duchenne first devised and used a muscle biopsy needle over a century ago, several other needles designed for this purpose have evolved?-3 The two most important features in such instruments are the ability to obtain an
Volume 103 Number 4
Needle muscle biopsy
569
Fig. 4. Polymyositis;note large number of inflammatory cells. (H & E; original magnification • adequate sample size and a minimal tendency to crush the tissue. We have found the Bergstrom needle to be quite satisfactory in meeting both of these criteria, The principal difficulty we have had with the Bergstrom needle is the excessive distance from the tip of the needle to the cutting window when used in small infants. As noted above, the 3.0 mm needle has been modified to decrease the tip-towindow distance. This modification has greatly facilitated biopsies in infants younger than 6 months. In addition to the needles designed specifically for muscle biopsy, other instruments, such as the Silverman-type needle, have been used. 4 The tissue samples obtained with these needles have generally been too small for proper orientation for sectioning, and also tend to be crushed. An adequate sample may be obtained, however, for ultrastructural or microbiologic studies in many cases. The conchotome is probably unsatisfactory for use in children and infants because of their small muscle mass? Although Edwards et al. 6 have described a large series of needle muscle biopsies in adults, and Dubowitz7 and
Curliss and Nelson 8 have reported use of the technique in children, open biopsies continue to be standard in North American pediatric centers. An informal survey among colleagues at eight major pediatric programs throughout the United States and Canada revealed that none was using the needle technique; all were performing open biopsies under general anesthesia in young children. The reasons given were a lack of familiarity with the needle procedure and concern about the adequacy of the tissue sample. We believe that the technique is relatively easy to learn, although in infants and small children it requires considerable skill and attention to technique. Our experience suggests that an adequate sample can be obtained in almost all patients, even neonates. The smallest patient studied to date weighed 1800 gm; the procedure was difficult, but a satisfactory sample was obtained. The sample of tissue obtained using the Bergstrom needle is undoubtedly smaller than if an open procedure were performed. In addition, it arrives in the laboratory without obvious orientation of the muscle fibers. The
570
DiLiberti, D'Agostino, and Cole
The Journal of Pediatrics October 1983
Fig. 5. Central core disease. (NADH-tetrazolium reductase; original magnification X400.) average sample obtained with the 3.0 m m needle fortunately provides an adequate number of fibers in cross section for accurate diagnosis. A typical biopsy from the 3.0 mm needle in an infant has about 5000 to 10,000 fibers in cross section; a 5.0 mm needle in an adolescent provides about 4000 to 5000. The ability to sample several sites during the same procedure lowers the probability of sampling error to the point that there is little reason to believe that the larger, open biopsy is more accurate diagnostically?'7 Difficulty in establishing proper orientation of the tissue for sectioning is unusual. This does not imply, however, that it is an exceptionally easy skill to learn. Considerable practice and experience are necessary to master the technique. Percutaneous needle biopsy of skeletal muscle is a fast, safe method for obtaining samples of tissue adequate for routine histochemical and ultrast'fu~tural diagnosis. The biopsy size is also large enough for some metabolic evaluations to be done. Needle biopsy is relatively painless and has few complications. W e believe it is a useful procedure for the diagnosis of neuromuscular disorders in infants and children.
REFERENCES 1. Charriere M, Duchenne GB: Emporte piece histologique. Bull Acad Med 30:1050, 1865. 2. Young A, Wiles CM, Edwards RHT: University College Hospital "muscle-biopsy" needle. Lancet 2:1285, 1978. 3. Nichols BL, Hazelwood CF, Barnes D J: Percutaneous needle biopsy of quadriceps muscle: Potassium analysis in normal children. J P~']~IATR72"840, 1968. 4. Siperstein MD, Unger RH, Madison LL: Studies of muscle capillary basement membranes in normal subjects, diabetic and prediabetic patients. J Clin Invest 47:1973, 1968. 5. Henrichsson KG: "Semi-open" muscle biopsy technique: A simple outpatient procedure. Acta Neurol Scand 59:317, 1979. 6. Edwards R, Young A, Wiles M: Needle biopsy of skcletat muscle in the diagnosis of myopathy and the clinical study of muscle function and repair. N Engl J Med 302:261, 1980. 7.' Dubowitz V: Muscle disorders in childhood. Philadelphia, 1978, WB Saunders, pp 13-18. 8. Curless RG, Nelson M: Needle biopsies of muscle in infants for diagnosis and research. Dev Med Child Neurol 17:592, 1975.
John H. DiLiberti, M.D., A. N. D'Agostino, M.D., and George Cole, B.A. P o r t l a n d , Ore.
THE ACCURATE DIAGNOSIS of neuromuscular disease in
children generally requires histochemical and sometimes ultrastructural analysis of skeletal muscle in addition to other methods of evaluation. Although satisfactory techniques for securing skeletal muscle biopsies have been available for several years, the open biopsy method is still most commonly used. We review the technique of needle biopsy of skeletal muscle in children, the indications for its use, and our experience with the procedure in 77 pediatric patients. MATERIALS
AND METHODS
Muscle biopsies were obtained from 77 children who were referred for the evaluation of neuromuscular disease or Reye syndrome. They ranged in age from 1 week to 15 years. Two or more biopsies were performed in five patients, resulting in a total of 86 procedures in this series. With the exception of those in hospitalized children, all procedures were done in the outpatient clinic. Most children younger than 8 years were sedated with chloral hydrate 50 mg/kg and meperidine 1 mg/kg, both given orally about 40 to 60 minutes prior to the procedure. Parents were asked not to feed the younger children for four to six hours before coming to the hospital, Most of the biopsies were obtained from the quadriceps femoris muscle, but the gastrocnemius was occasionally sampled in patients with predominantly distal weakness. After preparation of the skin of the mid-lateral thigh From the Departments of Pediatrics, Emanuel Hospital and Oregon Health Sciences University, and the Department o f Pathology, Good Samaritan Hospital. Reprint requests: John DiLiberli, M.D., Chief o f Pediatrics, Oregon Children's" Medical Center~ Emanuel Hospital, 2801 N. Gantenbein Ave.. Portland, OR 97227.
566
The Journal o f P E D I A T R I C S
with povidone iodine, and shaving the site if necessary, a 25- to 27-gauge needle was used to infiltrate the skin with 1% xylocaine. After a satisfactory wheal was produced, this needle was withdrawn, and a 21-gauge needle was used to infiltrate the subcutaneous tissue and muscle sheath. The large needle facilitated identification of the muscle sheath and decreased the likelihood of infiltrating the muscle itself. A total of 3 to 10 ml xylocaine was used, depending on the size of the patient. After a few minutes, a 5 to 8 mm incision was made through the skin of the mid-lateral thigh with a No. 11 scalpel blade, aiming at the center of the shaft of the femur. The knife blade was then pushed through the subcutaneous tissue down to the muscle sheath, which was identified by its tough, resistant character. The sheath was incised by moving the tip of the blade along the surface of the muscle, taking care not to penetrate deeply. All of the biopsies were obtained using Bergstrom muscle biopsy needles ranging in diameter from 3.0 to 5.0 mm. The 3.0 irma needle was modified by reducing the distance from the tip to the cutting window; this modified needle was used in young infants, The biopsy needle was inserted through the skin incision and subcutaneous tissue. It was often necessary to move the tip to identify the incision in the muscle sheath. The needle was then pushed into the muscle to a depth su~cient to cover the cutting window. The experienced operator can usuaily tell if the needle has penetrated muscle by the quality and degree of resistance to the advancing needle. Muscle is more difficult to penetrate that fat and feels more viscous than fascia. After the needle was thought to be securely placed in muscle tissue, the cutting blade was pulled back sufficiently to expose the window completely, the operator's free hand was used to force muscle into the window by applying pressure from
Volume 103 Number 4
Needle muscle biopsy
567
Fig. 2. Low magnification view of part of biopsy sample from 8-year-old boy with congenital myopathy. (ATPase; original magnification X54.) Table. Clinical diagnosis in 77 infants and children Fig. 1. Low magnification view of part of biopsy sample from 3-year-old boy with muscular dystrophy. (Trichrome; original magnification X54.) above, and finally the cutting blade was quickly pushed in and the entire needle removed. Muscle tissue can usually be distinguished from fat and fascia by its color, texture, and consistency, but for small samples obtained from infants, a dissecting microscope at the bedside helps to confirm that an adequate sample has been obtained before terminating the procedure. Additional samples were usually obtained though the same incision, but the needle was inserted at different angles in order to sample other sites within the same muscle. After the biopsy, pressure was applied at the site for a short time, and then the wound was closed using sterile, adhesive tape strips. A large bandage was applied, and an elastic bandage wrapped around the thigh in older children. The elastic bandage allegedly reduces the likelihood of hematoma formation, but we have not seen this complization in patients in whom the elastic bandage was not used. Muscle samples for histochemieal analysis were placed
Patients Myopathy Dystrophy Spinal muscular atrophy Myositis Reye syndrome Normal
25 9 5 2 10 26 77
1 Biopsies 27 9 5 2 17 26 86
in gauze saturated with isotonic saline solution, and transported to the laboratory immediately. After preliminary examination through the dissecting microscope, the sample was placed in gum tragacanth, oriented for appropriate fiber direction, and then frozen in isopentane chilled in liquid nitrogen. Frozen sections 5 and 10 # in thickness were then made and stained using hematoxylin-eosin, trichrome, ATPase, N A D H , acid phosphatase, periodic acid Schiff, oil red O, and phosphorylase. A small sample for electron microscopy was pinned at resting length onto a small piece of tongue depressor blade or cork, using fine needles. This procedure was usually done at the bedside, and the sample was placed immediate-
568
DiLiberti, D'Agostino, and Cole
The Journal of Pediatrics October 1983
Fig. 3. Duchenne muscular dystrophy; increased fat and connective tissue with degenerating fibers. (H & E; original magnification X400.) ly in glutaraldehyde fixative. Unless ultrastructural evaluation was known to be required, this sample was saved and a decision to perform electron microscopy made after histochemical analysis was completed. RESULTS The biopsy samples were adequate for complete histochemical and ultrastructural evaluation in all but one patient, an infant in whom biopsy was done early in the course of the series. In that case enough tissue was obtained to identify clearly an abnormality on hernatoxylin-eosin sections, however, and we plan to obtain a follow-up biopsy in the future. The,'diagnoses are grouped by general category in the Table. The procedure was well tolerated by all patients, although ther e obviously was some discomfort. The older patients said that they did not feel much pain, but a vague, uncomfortable sensation. The infants who were sedated
often slept through the biopsy after crying briefly during the xylocaine injection, which is the most painful part of the procedure,,;]No complications from the procedure were observed. A few of the older patients described some mild pain for one or two days at the biopsy site when asked specifically about it, but none volunteered this information. Despite markedly prolonged prothrombin and partial thromboplastin times in the patients with Reye syndrome, no hemorrhages or hematomas were detected. Representative sections from several of the patients are illustrated in Figs. 1 through 5 and demonstrate the adequacy of the technique for diagnostic purposes. DISCUSSION Since Duchenne first devised and used a muscle biopsy needle over a century ago, several other needles designed for this purpose have evolved?-3 The two most important features in such instruments are the ability to obtain an
Volume 103 Number 4
Needle muscle biopsy
569
Fig. 4. Polymyositis;note large number of inflammatory cells. (H & E; original magnification • adequate sample size and a minimal tendency to crush the tissue. We have found the Bergstrom needle to be quite satisfactory in meeting both of these criteria, The principal difficulty we have had with the Bergstrom needle is the excessive distance from the tip of the needle to the cutting window when used in small infants. As noted above, the 3.0 mm needle has been modified to decrease the tip-towindow distance. This modification has greatly facilitated biopsies in infants younger than 6 months. In addition to the needles designed specifically for muscle biopsy, other instruments, such as the Silverman-type needle, have been used. 4 The tissue samples obtained with these needles have generally been too small for proper orientation for sectioning, and also tend to be crushed. An adequate sample may be obtained, however, for ultrastructural or microbiologic studies in many cases. The conchotome is probably unsatisfactory for use in children and infants because of their small muscle mass? Although Edwards et al. 6 have described a large series of needle muscle biopsies in adults, and Dubowitz7 and
Curliss and Nelson 8 have reported use of the technique in children, open biopsies continue to be standard in North American pediatric centers. An informal survey among colleagues at eight major pediatric programs throughout the United States and Canada revealed that none was using the needle technique; all were performing open biopsies under general anesthesia in young children. The reasons given were a lack of familiarity with the needle procedure and concern about the adequacy of the tissue sample. We believe that the technique is relatively easy to learn, although in infants and small children it requires considerable skill and attention to technique. Our experience suggests that an adequate sample can be obtained in almost all patients, even neonates. The smallest patient studied to date weighed 1800 gm; the procedure was difficult, but a satisfactory sample was obtained. The sample of tissue obtained using the Bergstrom needle is undoubtedly smaller than if an open procedure were performed. In addition, it arrives in the laboratory without obvious orientation of the muscle fibers. The
570
DiLiberti, D'Agostino, and Cole
The Journal of Pediatrics October 1983
Fig. 5. Central core disease. (NADH-tetrazolium reductase; original magnification X400.) average sample obtained with the 3.0 m m needle fortunately provides an adequate number of fibers in cross section for accurate diagnosis. A typical biopsy from the 3.0 mm needle in an infant has about 5000 to 10,000 fibers in cross section; a 5.0 mm needle in an adolescent provides about 4000 to 5000. The ability to sample several sites during the same procedure lowers the probability of sampling error to the point that there is little reason to believe that the larger, open biopsy is more accurate diagnostically?'7 Difficulty in establishing proper orientation of the tissue for sectioning is unusual. This does not imply, however, that it is an exceptionally easy skill to learn. Considerable practice and experience are necessary to master the technique. Percutaneous needle biopsy of skeletal muscle is a fast, safe method for obtaining samples of tissue adequate for routine histochemical and ultrast'fu~tural diagnosis. The biopsy size is also large enough for some metabolic evaluations to be done. Needle biopsy is relatively painless and has few complications. W e believe it is a useful procedure for the diagnosis of neuromuscular disorders in infants and children.
REFERENCES 1. Charriere M, Duchenne GB: Emporte piece histologique. Bull Acad Med 30:1050, 1865. 2. Young A, Wiles CM, Edwards RHT: University College Hospital "muscle-biopsy" needle. Lancet 2:1285, 1978. 3. Nichols BL, Hazelwood CF, Barnes D J: Percutaneous needle biopsy of quadriceps muscle: Potassium analysis in normal children. J P~']~IATR72"840, 1968. 4. Siperstein MD, Unger RH, Madison LL: Studies of muscle capillary basement membranes in normal subjects, diabetic and prediabetic patients. J Clin Invest 47:1973, 1968. 5. Henrichsson KG: "Semi-open" muscle biopsy technique: A simple outpatient procedure. Acta Neurol Scand 59:317, 1979. 6. Edwards R, Young A, Wiles M: Needle biopsy of skcletat muscle in the diagnosis of myopathy and the clinical study of muscle function and repair. N Engl J Med 302:261, 1980. 7.' Dubowitz V: Muscle disorders in childhood. Philadelphia, 1978, WB Saunders, pp 13-18. 8. Curless RG, Nelson M: Needle biopsies of muscle in infants for diagnosis and research. Dev Med Child Neurol 17:592, 1975.