1217 G-BANDING WITH HEAT TREATMENT
SIR,—The variety of methods’-’ described
for
G-banding ot
human chromosomes indicates that a generally satisfactory method has not yet been found. In the past 2 years we have tried different techniques to obtain a method for G-banding which is simple, quick, cheap, and reliable. In order to shorten the time between the preparation of slides and G-band staining, hydrogen peroxide has been usedbut in our experience the quality of banding is not satisfactory. Giemsa solution in a dilution of 1/100 has been used for G-banding, and the number of satisfactory preparations was greatly increased when slides were stored for 7 or more days in a dry atmosphere before staining. Collagenase7has also been used to shorten the prestaining period, but at$30 for 100 mg of collagenase this method is expensive for routine use. The number of methods which require prolonged air-drying suggested that a quicker result could be obtained by heating the slides before treatment with trypsin.
leucocytes were cultured to obtain metaphases and transglass slides. The slides were then placed either on a hot-plate
Human
ferred
to
75-80°C for 4-6 hours or in an oven at 120°C for 15 minutes. The slides were allowed to cool and were treated for 20 seconds at 350C with 0.05% solution of trypsin in McIlvaine’s buffer (pH 7-2). They were then passed through 50%, 70%, and 100% ethanol, air-dried, and stained for 5 minutes in 10% Giemsa stain in phosphate buffer (pH at
6.8). Controlled trials have shown that the lower the pretreattemperature, the longer the time required for satisfactory G-banding. When the pretreatment temperature exceeds 120°C, staining of chromosomes diminishes, and it is lost above 140°C. This method has now been used for over 6 ment
months with
consistently satisfactory G-banding of all chromo-
somes.
Chromosome Laboratory, St. Nicholas Hospital,
Carlton, Victoria, Australia.
RAQUELLA BIRNER SAUL WIENER
some cases of which may have been caused by bacterial infection and others by mechanical and chemical factors. Mechanical, chemical, or other factors might predispose the urethra to become infected with bacteria; in this instance, the syndrome would probably respond to antibiotic therapy. Patients who have urethral syndrome without infection would not respond to antibiotic therapy, and symptoms would disappear only after the source of urethral irritation had been removed and the inflammatory response had subsided.
syndrome,
Carney Hospital, Boston, Massachusetts 02124, U.S.A.
ROBERT P. YOUNES
SIMULTANEOUS DIAGNOSIS AND TREATMENT OF ACUTE ADRENOCORTICAL INSUFFICIENCY
SIR,—We should like to make some observations on the paper by Dr Sheridan and Dr Mattingly (Oct. 11, p. 676). All their patients with confirmed Addison’s disease had plasma-levels of hydroxycorticosteroids below 10 µg/dl before stimulation with tetracosactrin. By contrast, all the patients who to the stimulus had baseline levels above 10 µg/dl. In our series of 29 patients with proved Addison’s disease the highest level of plasma-glucocorticoids found was 7.6 µg/dl in a 56-year-old woman with Schmidt’s syndrome. Determinations by fluorimetry and a competitive protein-binding technique2 agreed very well. We agreed that a low concentration of circulating glucocorticoids does not establish adrenocortical insufficiency: this seems obvious if one takes into account the striking cyclical (circadian and longer) variations of corticotrophin (A.C.T.H.)/cortisol secretion .3In this regard we were surprised to find no information about the time when the subjects were examined. On the other hand, it is conceivable that in cases of primary and secondary adrenocortical insufficiency plasmacortisol might be in the "normal" range: this might occur in patients with enhanced transcortin binding (in pregnancy and
subsequently responded
oestrogen administration)
RELEVANCE OF "SIGNIFICANT BACTERIURIA" TO ÆTIOLOGY AND DIAGNOSIS OF URINARY-TRACT INFECTION
SIR,—Ishould like to comment on the article by Dr Tapsall and others (Oct. 4, p. 637). Kass’s criterion for the diagnosis of urinary-tract infection (> 100 000 viable organisms per ml)*’requires at least two or three positive clean voided specimens (c.v.s.). Dr Tapsall and his colleagues, however, obtained only one c.v.s., and they therefore ran a significant risk of misdiagnosis. A false-positive rate in Tapsall’s patients could run as high as 20%. Therefore, the conclusions drawn from the data are questionable. There is no doubt that the urethral syndrome does exist, but diagnosis must be based on symptoms and not on Kass’s criterion. Urethral cultures can be obtained but are impractical in the clinical situation. I agree with Dr Tapsall that there are many causes of the urethral syndrome, bacterial infection being one of them. Sexual intercourse, cold weather, emotional are stress, allergy, bubble bath, detergent, pinworms, &c., other possible causes. If Tapsall’s group had obtained two or three c.v.s. from each patient, I believe that they would have found fewer individuals with significant bacteriuria. Those without significant bacteriuria would have had the urethral
or
presenting
severe
hypothyroidism
leading big reduction of the hormonal metabolic clearance. Nevertheless, it seems to us that the finding of a concentration of circulating glucocorticoids above 10 µg/dl is sufficient to exclude the diagnosis of acute hypoadrenalism and to avoid incorrect treatment. Our second point concerns the stimulus with corticotrophin. Acute intravenous injection of tetracosactrin causes a significant increase of plasma-cortisol within minutes. 6 In our experience, estimation of cortisol on plasma drawn 15 minutes after injection of 0-25 mg of ’Synacthen’ was adequate for diagnosing acute hypoadrenalism in 4 patients, in whom prompt rehydration and treatment with large doses of hydrocortisone were required. No false-negative responses in seriously ill patients have been noted. The only limitation of such a "rapid" test is that differentiation between primary and secondary insufficiency is uncertain; it is agreed that in patients with prolonged failure of endogenous stimulation the response to exogenous A.C.T.H. is often particularly sluggish and below normal.67In such cases the pituitary defect can be easily documented once the threatening phase of acute glucocorticoid insufficiency has been resolved with adequate support. Finally, we wish to point out that, with the widely used comto
1. 2.
a
Mattingly, D.J. clin. Path. 1962, 15, 374. Angeli, A., Bisbocci, D., Melo’, F., Frajria, R., Gaidano,
G. P. Chim. clin.
Acta, 1975, 61, 279. 3. Kneger, D. T., Allen, W., Rizzo, F., Krieger, H. P. J. clin. Endocr. 1971, 1. Sumner, A. T, Evans, H. J., Buckland, R. A. Nature, 1971, 2. Drets, M. E., Shaw, M. W. Proc. natn. Acad. Sci., U.S.A. 3. Seabright, M. Lancet, 1971, ii, 971. 4. Utakoji, T. Nature, 1972, 239, 168. 5. Seabright, M. Lancet, 1973, i, 1249. 6 Yunis, J. J., Sanchez, O. Chromosoma, 1973, 44, 15. 7. Trusler, S. Lancet, 1975, i, 44. 8. Kass, E. H. Trans. Ass. Am. Physns, 1956, 69, 56. 9. Kass, E. H. Archs intern. Med. 1957, 100, 709.
232, 31 1971, 68, 2073.
32, 266. 4. Weitzman, E. D., Fukushima, D., Nogeire, F., Hellman, L. ibid. 1971, 33, 14. 5. London, J., James, V. H. T., Wharton, M.
L., Roffwarg, H., Gallagher, T.
J., Friedman, M. Lancet, 1967, ii, 697. 6. Angeli, A., Frajria, R., Boccuzzi, G., Bisbocci, D., Ceresa, F. Acta endocr.,
7.
Copenh. 1973, 74, 250. Frawley, T. F. in Adrenal Cortex (edited by don, 1967.
A. B.
Eisenstein); p. 439. Lon-
SIR,—The variety of methods’-’ described
for
G-banding ot
human chromosomes indicates that a generally satisfactory method has not yet been found. In the past 2 years we have tried different techniques to obtain a method for G-banding which is simple, quick, cheap, and reliable. In order to shorten the time between the preparation of slides and G-band staining, hydrogen peroxide has been usedbut in our experience the quality of banding is not satisfactory. Giemsa solution in a dilution of 1/100 has been used for G-banding, and the number of satisfactory preparations was greatly increased when slides were stored for 7 or more days in a dry atmosphere before staining. Collagenase7has also been used to shorten the prestaining period, but at$30 for 100 mg of collagenase this method is expensive for routine use. The number of methods which require prolonged air-drying suggested that a quicker result could be obtained by heating the slides before treatment with trypsin.
leucocytes were cultured to obtain metaphases and transglass slides. The slides were then placed either on a hot-plate
Human
ferred
to
75-80°C for 4-6 hours or in an oven at 120°C for 15 minutes. The slides were allowed to cool and were treated for 20 seconds at 350C with 0.05% solution of trypsin in McIlvaine’s buffer (pH 7-2). They were then passed through 50%, 70%, and 100% ethanol, air-dried, and stained for 5 minutes in 10% Giemsa stain in phosphate buffer (pH at
6.8). Controlled trials have shown that the lower the pretreattemperature, the longer the time required for satisfactory G-banding. When the pretreatment temperature exceeds 120°C, staining of chromosomes diminishes, and it is lost above 140°C. This method has now been used for over 6 ment
months with
consistently satisfactory G-banding of all chromo-
somes.
Chromosome Laboratory, St. Nicholas Hospital,
Carlton, Victoria, Australia.
RAQUELLA BIRNER SAUL WIENER
some cases of which may have been caused by bacterial infection and others by mechanical and chemical factors. Mechanical, chemical, or other factors might predispose the urethra to become infected with bacteria; in this instance, the syndrome would probably respond to antibiotic therapy. Patients who have urethral syndrome without infection would not respond to antibiotic therapy, and symptoms would disappear only after the source of urethral irritation had been removed and the inflammatory response had subsided.
syndrome,
Carney Hospital, Boston, Massachusetts 02124, U.S.A.
ROBERT P. YOUNES
SIMULTANEOUS DIAGNOSIS AND TREATMENT OF ACUTE ADRENOCORTICAL INSUFFICIENCY
SIR,—We should like to make some observations on the paper by Dr Sheridan and Dr Mattingly (Oct. 11, p. 676). All their patients with confirmed Addison’s disease had plasma-levels of hydroxycorticosteroids below 10 µg/dl before stimulation with tetracosactrin. By contrast, all the patients who to the stimulus had baseline levels above 10 µg/dl. In our series of 29 patients with proved Addison’s disease the highest level of plasma-glucocorticoids found was 7.6 µg/dl in a 56-year-old woman with Schmidt’s syndrome. Determinations by fluorimetry and a competitive protein-binding technique2 agreed very well. We agreed that a low concentration of circulating glucocorticoids does not establish adrenocortical insufficiency: this seems obvious if one takes into account the striking cyclical (circadian and longer) variations of corticotrophin (A.C.T.H.)/cortisol secretion .3In this regard we were surprised to find no information about the time when the subjects were examined. On the other hand, it is conceivable that in cases of primary and secondary adrenocortical insufficiency plasmacortisol might be in the "normal" range: this might occur in patients with enhanced transcortin binding (in pregnancy and
subsequently responded
oestrogen administration)
RELEVANCE OF "SIGNIFICANT BACTERIURIA" TO ÆTIOLOGY AND DIAGNOSIS OF URINARY-TRACT INFECTION
SIR,—Ishould like to comment on the article by Dr Tapsall and others (Oct. 4, p. 637). Kass’s criterion for the diagnosis of urinary-tract infection (> 100 000 viable organisms per ml)*’requires at least two or three positive clean voided specimens (c.v.s.). Dr Tapsall and his colleagues, however, obtained only one c.v.s., and they therefore ran a significant risk of misdiagnosis. A false-positive rate in Tapsall’s patients could run as high as 20%. Therefore, the conclusions drawn from the data are questionable. There is no doubt that the urethral syndrome does exist, but diagnosis must be based on symptoms and not on Kass’s criterion. Urethral cultures can be obtained but are impractical in the clinical situation. I agree with Dr Tapsall that there are many causes of the urethral syndrome, bacterial infection being one of them. Sexual intercourse, cold weather, emotional are stress, allergy, bubble bath, detergent, pinworms, &c., other possible causes. If Tapsall’s group had obtained two or three c.v.s. from each patient, I believe that they would have found fewer individuals with significant bacteriuria. Those without significant bacteriuria would have had the urethral
or
presenting
severe
hypothyroidism
leading big reduction of the hormonal metabolic clearance. Nevertheless, it seems to us that the finding of a concentration of circulating glucocorticoids above 10 µg/dl is sufficient to exclude the diagnosis of acute hypoadrenalism and to avoid incorrect treatment. Our second point concerns the stimulus with corticotrophin. Acute intravenous injection of tetracosactrin causes a significant increase of plasma-cortisol within minutes. 6 In our experience, estimation of cortisol on plasma drawn 15 minutes after injection of 0-25 mg of ’Synacthen’ was adequate for diagnosing acute hypoadrenalism in 4 patients, in whom prompt rehydration and treatment with large doses of hydrocortisone were required. No false-negative responses in seriously ill patients have been noted. The only limitation of such a "rapid" test is that differentiation between primary and secondary insufficiency is uncertain; it is agreed that in patients with prolonged failure of endogenous stimulation the response to exogenous A.C.T.H. is often particularly sluggish and below normal.67In such cases the pituitary defect can be easily documented once the threatening phase of acute glucocorticoid insufficiency has been resolved with adequate support. Finally, we wish to point out that, with the widely used comto
1. 2.
a
Mattingly, D.J. clin. Path. 1962, 15, 374. Angeli, A., Bisbocci, D., Melo’, F., Frajria, R., Gaidano,
G. P. Chim. clin.
Acta, 1975, 61, 279. 3. Kneger, D. T., Allen, W., Rizzo, F., Krieger, H. P. J. clin. Endocr. 1971, 1. Sumner, A. T, Evans, H. J., Buckland, R. A. Nature, 1971, 2. Drets, M. E., Shaw, M. W. Proc. natn. Acad. Sci., U.S.A. 3. Seabright, M. Lancet, 1971, ii, 971. 4. Utakoji, T. Nature, 1972, 239, 168. 5. Seabright, M. Lancet, 1973, i, 1249. 6 Yunis, J. J., Sanchez, O. Chromosoma, 1973, 44, 15. 7. Trusler, S. Lancet, 1975, i, 44. 8. Kass, E. H. Trans. Ass. Am. Physns, 1956, 69, 56. 9. Kass, E. H. Archs intern. Med. 1957, 100, 709.
232, 31 1971, 68, 2073.
32, 266. 4. Weitzman, E. D., Fukushima, D., Nogeire, F., Hellman, L. ibid. 1971, 33, 14. 5. London, J., James, V. H. T., Wharton, M.
L., Roffwarg, H., Gallagher, T.
J., Friedman, M. Lancet, 1967, ii, 697. 6. Angeli, A., Frajria, R., Boccuzzi, G., Bisbocci, D., Ceresa, F. Acta endocr.,
7.
Copenh. 1973, 74, 250. Frawley, T. F. in Adrenal Cortex (edited by don, 1967.
A. B.
Eisenstein); p. 439. Lon-
