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A TECHNIQUE FOR CULTURING CELLS FROM AMNIOTIC FLUID
84 established 5 and (c) the difficulties in distinguishing heterozygous from homozygous individuals.5 It seems, therefore, that if prenatal diagnosis is to become a practical clinical method for the prevention of mental retardation, simpler procedures employing chemical or enzymic methods are highly desirable. Accordingly we have sought to develop microprocedures for the determination of constituents of amniotic fluid. The acid-mucopolysaccharide content was determined in 40 samples of amniotic fluid from volunteers in the 12th to 22nd weeks of pregnancies leading to the delivery of normal infants. The average content of acid mucopolysaccharide was found to be approximately 0-02 mg. per ml. of fluid, with a range of 0.006 to 0-035. In contrast, amniotic fluid obtained at the 14th week of a pregnancy which resulted in the delivery of an infant with Hurler’s syndrome showed 0-087 mg. of mucopolysaccharide per ml. Fractionation of the polysaccharide obtained from a pool of normal amniotic fluid showed approximately 80% hyaluronic acid, 13% chondroitin-6-sulphate, and 7% dermatan sulphate. In contrast, the polysaccharide in the abnormal amniotic fluid was composed of approximately 63% heparitin sulphate-a compound not detectable in the normal amniotic fluid. The remaining material was a mixture of hyaluronic acid, dermatan sulphate, and chondroitin-6-sulphate. Studies of the urine of the patient showed the presence of heparitin sulphate as the major component. In both amniotic fluid and urine the presence of increased amounts of heparitin sulphate could be detected directly by the nitrous acid reaction for N-sulphated hexosamine.66 These results suggest that prenatal diagnosis of Hurler’s syndrome can be carried out by a rapid and relatively simple method. Possibly such a method may also permit a more specific diagnosis than the use of cultured cells. Obviously much more experience must be obtained as more amniotic-fluid samples become available. Department of Pediatrics, Joseph P. Kennedy, Jr., Mental Retardation Research Center, REUBEN MATALON The Pritzker School of Medicine, ALBERT DORFMAN. Chicago, Illinois. HENRY L. NADLER. Children’s Memorial Hospital, Chicago. Department of Obstetrics and Gynecology, George Washington University, Medical Center, Washington, D.C.
CECIL B.
amniotic fluid is centrifuged at 1000 r.p.m. for 5 minutes and the cells are resuspended in medium containing 75% Glaxo TC 199 and 25% fetal-calf serum, to allow the setting up of 2 leighton tubes. Small leighton tubes are seeded with 0-6 ml. of this cell-suspension, and 1 ml. is used in standard leighton tubes. The tubes are gassed with 5% CO2 in air, closed with silicone-rubber bungs and incubated at 37°C. If there are large numbers of cells in the amniotic fluid, they are resuspended in sufficient medium for 3-4 leighton tubes to be set up. After 3 days, 0-6 ml. of fresh medium is added, and the cultures are again gassed. 2-3 days later the cultures are examined for growth. The medium, with any cells in suspension, is removed, spun at 1000 r.p.m. for five minutes, the supernatant poured off, and the cells resuspended in 0-6 ml. of fresh medium. If no growth has been observed, this cell suspension is replaced in the primary tube. If growth has been seen in the original culture, 1 ml. of fresh medium is added to the primary tube, and the above cell suspension is put into a separate tube, where it often begins to grow 3-4 days later. Growing cultures have their medium changed three times a week, whereas cultures which do not show growth have medium added at one inspection and changed at the next.
Growth normally begins in small colonies and is epithelial in nature. Harvesting is carried out on a primary culture unless growth has occurred in only one tube, when subculturing is performed a few days before harvesting, The small leighton tubes are particularly advantageous when subculturing, since the suspension of cells obtained from the primary culture can be split into more tubes giving a higher cell concentration than when a standard tube has been used. The cultures are given 6 hours’ colcemid treatment before the cells are digested off the glass with 1-2 ml. of 0-025% trypsin/versene (Worthington x 2 crystallised trypsin) solution. Then, immediately after the addition of 2 ml. of distilled water per ml. of trypsin for hypotonic treatment, the cells are centrifuged at 500 r.p.m. for 5 minutes and fixed in 3/1 methanol/glacial acetic acid. The fixative is changed once, and the preparations made by the standard air-drying method. In most cultures the mitotic-rate is high and the chromosome quality excellent. Cytogenetics Unit, School of Medicine, University of Liverpool.
NINA M. GREGSON.
HYPERTENSION DURING IMIPRAMINE TREATMENT
JACOBSON.
SIR,-Orthostatic hypotension during imipramine treatis not unusual; it was described by Kuhn in the first
ment
A
TECHNIQUE FOR CULTURING CELLS FROM AMNIOTIC FLUID
SIR,-Methods for the rapid culture of amniotic-fluid cells for chromosome studies are being developed in a number of laboratories. Most of these are modifications of the techniques described by Jacobson and Barterand by Nadler.8 In this laboratory, cultures of 22 amniotic-fluid specimens have so far been attempted. The specimens were obtained by amniocentesis or at hysterotomy, the duration of the pregnancies ranging from 12 to 34 weeks. Good chromosome preparations were obtained from all samples which were not heavily contaminated with red blood-cells. Of the 5 specimens which were contaminated, culture was unsuccessful in 3. As we become more experienced with the technique, the time taken to make chromosome preparations is decreasing-it is now usually less than 14 days. Our method is outlined below. The culture vessels used are standard leighton tubes with a 40 x 10 mm. chamber, and leighton tubes which have been modified to a chamber size of 20 x 10 mm. by a glass-blower. The 5. 6. 7. 8.
Matalon, R., Dorfman, A. Lancet, 1969, ii, 838. Lagunoff, D., Warren, G. Archs Biochem. Biophys. 1962, 33, 396. Jacobson, C. B., Barter, R. H. Am. J. Obstet. Gynec. 1967, 99, 795. Nadler, H. L. Pediatrics, Springfield, 1968, 42, 912.
publication on imipramine in 1957.1 A few cases of rises in but the developblood-pressure have been published,2>3 ment of hypertension in patients who were initially normotensive and were taking no drug other than imipramine has not previously been described. I should like, therefore, to report the following case. A woman of 57 was admitted to hospital with endogenous depression. On a previous occasion she had been given amitryptiline for her depression, and had developed precordial pain and dyspnoea, with inverted x2-waves on the electrocardiogram, but no changes in blood-pressure. She was started on imipramine. The two blood-pressure meastaken before treatment were 140/90 mm. Hg and Hg. After three days of treatment the bloodpressure had risen to 150/110 mm. Hg; after nine days on a daily dose of 150 mg. imipramine it was 175/120 mm. Hg; and on the three subsequent days it was 160/125, 165/115, and 170/115 mm. Hg. A reduction of the dose of imipramine to 100 mg. daily did not lead to a fall in the bloodpressure, so the drug was withdrawn. The patient was discharged, and after three weeks the blood-pressure was 140/105 mm. Hg. Electrocardiograms done before and urements
140/85
mm.
1. Kuhn, R. Schweiz. med. Wschr. 1957, 87, 1135. 2. Frick, B. Atti Soc. med.-chir. Bolzano, 1959, 3, 1. 3. Tellez, A., Brzovic, J. Revta. méd. Chile, 1960, 88, 659.
CECIL B.
amniotic fluid is centrifuged at 1000 r.p.m. for 5 minutes and the cells are resuspended in medium containing 75% Glaxo TC 199 and 25% fetal-calf serum, to allow the setting up of 2 leighton tubes. Small leighton tubes are seeded with 0-6 ml. of this cell-suspension, and 1 ml. is used in standard leighton tubes. The tubes are gassed with 5% CO2 in air, closed with silicone-rubber bungs and incubated at 37°C. If there are large numbers of cells in the amniotic fluid, they are resuspended in sufficient medium for 3-4 leighton tubes to be set up. After 3 days, 0-6 ml. of fresh medium is added, and the cultures are again gassed. 2-3 days later the cultures are examined for growth. The medium, with any cells in suspension, is removed, spun at 1000 r.p.m. for five minutes, the supernatant poured off, and the cells resuspended in 0-6 ml. of fresh medium. If no growth has been observed, this cell suspension is replaced in the primary tube. If growth has been seen in the original culture, 1 ml. of fresh medium is added to the primary tube, and the above cell suspension is put into a separate tube, where it often begins to grow 3-4 days later. Growing cultures have their medium changed three times a week, whereas cultures which do not show growth have medium added at one inspection and changed at the next.
Growth normally begins in small colonies and is epithelial in nature. Harvesting is carried out on a primary culture unless growth has occurred in only one tube, when subculturing is performed a few days before harvesting, The small leighton tubes are particularly advantageous when subculturing, since the suspension of cells obtained from the primary culture can be split into more tubes giving a higher cell concentration than when a standard tube has been used. The cultures are given 6 hours’ colcemid treatment before the cells are digested off the glass with 1-2 ml. of 0-025% trypsin/versene (Worthington x 2 crystallised trypsin) solution. Then, immediately after the addition of 2 ml. of distilled water per ml. of trypsin for hypotonic treatment, the cells are centrifuged at 500 r.p.m. for 5 minutes and fixed in 3/1 methanol/glacial acetic acid. The fixative is changed once, and the preparations made by the standard air-drying method. In most cultures the mitotic-rate is high and the chromosome quality excellent. Cytogenetics Unit, School of Medicine, University of Liverpool.
NINA M. GREGSON.
HYPERTENSION DURING IMIPRAMINE TREATMENT
JACOBSON.
SIR,-Orthostatic hypotension during imipramine treatis not unusual; it was described by Kuhn in the first
ment
A
TECHNIQUE FOR CULTURING CELLS FROM AMNIOTIC FLUID
SIR,-Methods for the rapid culture of amniotic-fluid cells for chromosome studies are being developed in a number of laboratories. Most of these are modifications of the techniques described by Jacobson and Barterand by Nadler.8 In this laboratory, cultures of 22 amniotic-fluid specimens have so far been attempted. The specimens were obtained by amniocentesis or at hysterotomy, the duration of the pregnancies ranging from 12 to 34 weeks. Good chromosome preparations were obtained from all samples which were not heavily contaminated with red blood-cells. Of the 5 specimens which were contaminated, culture was unsuccessful in 3. As we become more experienced with the technique, the time taken to make chromosome preparations is decreasing-it is now usually less than 14 days. Our method is outlined below. The culture vessels used are standard leighton tubes with a 40 x 10 mm. chamber, and leighton tubes which have been modified to a chamber size of 20 x 10 mm. by a glass-blower. The 5. 6. 7. 8.
Matalon, R., Dorfman, A. Lancet, 1969, ii, 838. Lagunoff, D., Warren, G. Archs Biochem. Biophys. 1962, 33, 396. Jacobson, C. B., Barter, R. H. Am. J. Obstet. Gynec. 1967, 99, 795. Nadler, H. L. Pediatrics, Springfield, 1968, 42, 912.
publication on imipramine in 1957.1 A few cases of rises in but the developblood-pressure have been published,2>3 ment of hypertension in patients who were initially normotensive and were taking no drug other than imipramine has not previously been described. I should like, therefore, to report the following case. A woman of 57 was admitted to hospital with endogenous depression. On a previous occasion she had been given amitryptiline for her depression, and had developed precordial pain and dyspnoea, with inverted x2-waves on the electrocardiogram, but no changes in blood-pressure. She was started on imipramine. The two blood-pressure meastaken before treatment were 140/90 mm. Hg and Hg. After three days of treatment the bloodpressure had risen to 150/110 mm. Hg; after nine days on a daily dose of 150 mg. imipramine it was 175/120 mm. Hg; and on the three subsequent days it was 160/125, 165/115, and 170/115 mm. Hg. A reduction of the dose of imipramine to 100 mg. daily did not lead to a fall in the bloodpressure, so the drug was withdrawn. The patient was discharged, and after three weeks the blood-pressure was 140/105 mm. Hg. Electrocardiograms done before and urements
140/85
mm.
1. Kuhn, R. Schweiz. med. Wschr. 1957, 87, 1135. 2. Frick, B. Atti Soc. med.-chir. Bolzano, 1959, 3, 1. 3. Tellez, A., Brzovic, J. Revta. méd. Chile, 1960, 88, 659.