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FIBROBLAST CHROMOSOME PREPARATIONS
897
FIBROBLAST CHROMOSOME PREPARATIONS SIR,-One of us (T. K.) has reported the use of ’Varidase ’ in bone-marrow (Lederle) for prevention of fibrin formation preparations of human chromosomes.I Since then the technique has been modified for making chromosome preparations from fibroblast cells. This procedure has been successful, using both human and animal tissue. The technique is as follows: 1. Colchicine is added to the cultures (final concentration 4 x 10-7 g. per ml.) 16-18 hours before harvesting. 2. The cultures are gently agitated for 1 minute, and the medium is decanted into a centrifuge tube. 3. 0-25% trypsin solution is added,and the culture flask is left for 20 minutes at 37°C, with occasional shaking. A rubber-tipped spatula (" rubber policeman ") may be used if necessary to aid in detaching the cells from the glass. Decant this into the centrifuge-tube con-
the medium. 4. The trypsinised tissue is centrifuged at 800 r.p.m. for 8 minutes and the supernatant is decanted. 5. 2 ml. of a 0-075 M potassium-chloride solution containing 16 units per ml. of heparin and an amount of varidase solution representing 500 units streptokinase and 125 units of streptodornase are added. The varidase solution should be divided into 0-1 ml. portions, frozen at -20°C, and thawed when needed by adding the 2 ml. of potassium-chloride solution. The cells are gently resuspended
taining
C6 is involved in the formation of the nucleolus, and that this association can be observed when such things as therapy and chromosome stickiness are not problems. National Center for
Radiological Health, Department of Health, Education, and Welfare, 1901 Chapman Avenue, Rockville, Maryland 20852.
HOWARD NANKIN.
SERVING THE MENTALLY SUBNORMAL SIR,-The number of cases referred to a hospital for the mentally subnormal gives a measure of the use being made of the hospital, and a consideration of this should enable the hospital to improve the effectiveness of the service it offers. The bed-bureau for the mentally subnormal at this hospital serves a population of 840,986. In the service area are 2 county boroughs, 5 municipal boroughs, 16 urban districts, and 6 rural districts. The new cases which were referred to the bureau from April 1, 1966, to March 31, 1967, are related to these areas as follows:
by pipetting. 6. The mixture is immediately centrifuged at 800 r.p.m. for 8 minutes and the supernatant is decanted. 7. 2 ml. of fixative (1 part glacial acetic acid plus 3 parts absolute methyl alcohol, freshly prepared) is added, and the mixture gently pipetted, resuspending the cells. 8. The mixture is again centrifuged at 800 r.p.m. for 8 minutes and the supernatant decanted. 9. Enough fresh fixative is added to give the desired concentration of cells for making slides. 10. Slides are made and gently flamed dry. 11. Slides are treated with Giemsa’s stain (2 ml. stain, 2 ml. phosphate buffer, and 47 ml. distilled water) for 10 minutes, passed through acetone and acetone/xylene (1/2) for 10 seconds each, and left in xylene for 5 minutes before mounting in ’Permount’.
The
hypotonic potassium-chloride solution2 improves the of chromosomes in that they appear to be better fixed, better stained, and more sharply defined. The varidase prevents the formation of fibrin clots, which cause the clumping of cells often seen in chromosome preparations from fibroblast
quality
cultures. This study was supported by research grant AM-02504, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, and United States Public Health Service. Genetic and Endocrine Unit, Department of Pediatrics, TADASHI KAJII State University of New RICHARD L. NEU Medical Center, York, Upstate LYTT I. GARDNER. York 13210. Syracuse, New
CHROMOSOMAL SATELLITE ASSOCIATION SiR,-Dr. Khan and Professor Martin (March 11, p. 567) propose " that the chromatin knobs just above the secondary constrictions of C6 chromosome possess the property of adhesion and participate in satellite association ", after studies in two patients in the acute phase of leukaemia with a total of eight cells demonstrating the "association. Acrocentrics probably do associate " with other chromosomes but as a chance occurrence. Our ability to recognise this association is limited. Nucleoli can adhere to any chromosome in mitotic cells.3 If nucleolar material is responsible for satellite association it is therefore not unreasonable that acrocentrics can be found associated with subcentrics and metacentrics. Shaw has reported an association between satellited chromosomes and no. Al.4 It would be of interest to show that 1. 2. 3.
Kajii, T., Imai, T., Homma, T., Oikawa, K. Lancet, 1966, i, 1323. Hungerford, D. A. Stain Technol. 1965, 40, 333. Heneen, W. K., Nichols, W. W. J. Cell Biol. 1966, 31, 543. Hsu, T. C., Arrighi, F. E., Klevecz, R. R., Brinkley, B. R. ibid. 1965, 26,
4.
Shaw,
539.
M. W.
Lancet, 1961, i,
1351.
From these figures the number of cases with which the hospital should be prepared to cope in a given period can be roughly estimated. Each new referral represents a request from a family doctor or a local health authority for help from the hospital service in the management of a mentally subnormal person who needs investigation, assessment, treatment for a behaviour problem, nursing-care, or hospital accommodation. Per head of population more mentally subnormal people are referred to hospital in the towns than in the rural areas. It would be of interest to know how the patterns of referrals to hospitals for the mentally subnormal vary in different parts of the country. Such studies should be of value in planning the types and location of hospital facilities for the mentally subnormal. Westwood Hospital, D. A. SPENCER. Bradford 6.
SYSTEMATIC DESENSITISATION FOR PHOBIAS SIR,-The results reported by Dr. Friedman and Dr. Silverstone (March 4, p. 470) are impressive. They compare their results with those published by other workers. But it would have been more informative if they had compared their technique of systematic desensitisation using intravenous methohexitone sodium with established methods of treatment -e.g., desensitisation without methohexitone, or with the effects of drug therapy alone. Sargant and Dallyhave described the use of monoamine-oxidase inhibitor (M.A.o.l.)
drugs in treating certain anxiety states. It is still not generally recognised how extremely effective these drugs can be in some patients with very severe anxiety, including severe agarophobia. The following two cases are good examples of the effectiveness of M.A.O.I.S in treating severe anxiety. Case 1.-I was called to see a woman of fifty-four on a domiciliary visit. She was in bed, petrified by anxiety, unable to move out of the bedroom. She was feeling her pulse and expecting to die at any moment, because she felt her heart beat any faster and would suddenly stop. She was quinidine 200 mg. b.d. The pulse-rate was 120 per minute. There had been no previous history of neurotic illness. I took her off quinidine and substituted isocarboxazid (’Marplan’) 10 mg. t.d.s. A week later she was feeling very
could
not
already
on
1.
Sargant, W., Dally, P. Br. med. J. 1962, i, 6.
FIBROBLAST CHROMOSOME PREPARATIONS SIR,-One of us (T. K.) has reported the use of ’Varidase ’ in bone-marrow (Lederle) for prevention of fibrin formation preparations of human chromosomes.I Since then the technique has been modified for making chromosome preparations from fibroblast cells. This procedure has been successful, using both human and animal tissue. The technique is as follows: 1. Colchicine is added to the cultures (final concentration 4 x 10-7 g. per ml.) 16-18 hours before harvesting. 2. The cultures are gently agitated for 1 minute, and the medium is decanted into a centrifuge tube. 3. 0-25% trypsin solution is added,and the culture flask is left for 20 minutes at 37°C, with occasional shaking. A rubber-tipped spatula (" rubber policeman ") may be used if necessary to aid in detaching the cells from the glass. Decant this into the centrifuge-tube con-
the medium. 4. The trypsinised tissue is centrifuged at 800 r.p.m. for 8 minutes and the supernatant is decanted. 5. 2 ml. of a 0-075 M potassium-chloride solution containing 16 units per ml. of heparin and an amount of varidase solution representing 500 units streptokinase and 125 units of streptodornase are added. The varidase solution should be divided into 0-1 ml. portions, frozen at -20°C, and thawed when needed by adding the 2 ml. of potassium-chloride solution. The cells are gently resuspended
taining
C6 is involved in the formation of the nucleolus, and that this association can be observed when such things as therapy and chromosome stickiness are not problems. National Center for
Radiological Health, Department of Health, Education, and Welfare, 1901 Chapman Avenue, Rockville, Maryland 20852.
HOWARD NANKIN.
SERVING THE MENTALLY SUBNORMAL SIR,-The number of cases referred to a hospital for the mentally subnormal gives a measure of the use being made of the hospital, and a consideration of this should enable the hospital to improve the effectiveness of the service it offers. The bed-bureau for the mentally subnormal at this hospital serves a population of 840,986. In the service area are 2 county boroughs, 5 municipal boroughs, 16 urban districts, and 6 rural districts. The new cases which were referred to the bureau from April 1, 1966, to March 31, 1967, are related to these areas as follows:
by pipetting. 6. The mixture is immediately centrifuged at 800 r.p.m. for 8 minutes and the supernatant is decanted. 7. 2 ml. of fixative (1 part glacial acetic acid plus 3 parts absolute methyl alcohol, freshly prepared) is added, and the mixture gently pipetted, resuspending the cells. 8. The mixture is again centrifuged at 800 r.p.m. for 8 minutes and the supernatant decanted. 9. Enough fresh fixative is added to give the desired concentration of cells for making slides. 10. Slides are made and gently flamed dry. 11. Slides are treated with Giemsa’s stain (2 ml. stain, 2 ml. phosphate buffer, and 47 ml. distilled water) for 10 minutes, passed through acetone and acetone/xylene (1/2) for 10 seconds each, and left in xylene for 5 minutes before mounting in ’Permount’.
The
hypotonic potassium-chloride solution2 improves the of chromosomes in that they appear to be better fixed, better stained, and more sharply defined. The varidase prevents the formation of fibrin clots, which cause the clumping of cells often seen in chromosome preparations from fibroblast
quality
cultures. This study was supported by research grant AM-02504, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, and United States Public Health Service. Genetic and Endocrine Unit, Department of Pediatrics, TADASHI KAJII State University of New RICHARD L. NEU Medical Center, York, Upstate LYTT I. GARDNER. York 13210. Syracuse, New
CHROMOSOMAL SATELLITE ASSOCIATION SiR,-Dr. Khan and Professor Martin (March 11, p. 567) propose " that the chromatin knobs just above the secondary constrictions of C6 chromosome possess the property of adhesion and participate in satellite association ", after studies in two patients in the acute phase of leukaemia with a total of eight cells demonstrating the "association. Acrocentrics probably do associate " with other chromosomes but as a chance occurrence. Our ability to recognise this association is limited. Nucleoli can adhere to any chromosome in mitotic cells.3 If nucleolar material is responsible for satellite association it is therefore not unreasonable that acrocentrics can be found associated with subcentrics and metacentrics. Shaw has reported an association between satellited chromosomes and no. Al.4 It would be of interest to show that 1. 2. 3.
Kajii, T., Imai, T., Homma, T., Oikawa, K. Lancet, 1966, i, 1323. Hungerford, D. A. Stain Technol. 1965, 40, 333. Heneen, W. K., Nichols, W. W. J. Cell Biol. 1966, 31, 543. Hsu, T. C., Arrighi, F. E., Klevecz, R. R., Brinkley, B. R. ibid. 1965, 26,
4.
Shaw,
539.
M. W.
Lancet, 1961, i,
1351.
From these figures the number of cases with which the hospital should be prepared to cope in a given period can be roughly estimated. Each new referral represents a request from a family doctor or a local health authority for help from the hospital service in the management of a mentally subnormal person who needs investigation, assessment, treatment for a behaviour problem, nursing-care, or hospital accommodation. Per head of population more mentally subnormal people are referred to hospital in the towns than in the rural areas. It would be of interest to know how the patterns of referrals to hospitals for the mentally subnormal vary in different parts of the country. Such studies should be of value in planning the types and location of hospital facilities for the mentally subnormal. Westwood Hospital, D. A. SPENCER. Bradford 6.
SYSTEMATIC DESENSITISATION FOR PHOBIAS SIR,-The results reported by Dr. Friedman and Dr. Silverstone (March 4, p. 470) are impressive. They compare their results with those published by other workers. But it would have been more informative if they had compared their technique of systematic desensitisation using intravenous methohexitone sodium with established methods of treatment -e.g., desensitisation without methohexitone, or with the effects of drug therapy alone. Sargant and Dallyhave described the use of monoamine-oxidase inhibitor (M.A.o.l.)
drugs in treating certain anxiety states. It is still not generally recognised how extremely effective these drugs can be in some patients with very severe anxiety, including severe agarophobia. The following two cases are good examples of the effectiveness of M.A.O.I.S in treating severe anxiety. Case 1.-I was called to see a woman of fifty-four on a domiciliary visit. She was in bed, petrified by anxiety, unable to move out of the bedroom. She was feeling her pulse and expecting to die at any moment, because she felt her heart beat any faster and would suddenly stop. She was quinidine 200 mg. b.d. The pulse-rate was 120 per minute. There had been no previous history of neurotic illness. I took her off quinidine and substituted isocarboxazid (’Marplan’) 10 mg. t.d.s. A week later she was feeling very
could
not
already
on
1.
Sargant, W., Dally, P. Br. med. J. 1962, i, 6.