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DIFFERENTIATION OF IRON DEFICIENCY FROM THALASSÆMIA TRAIT
455
laboratory comparing karyotypes from amniotic-fluid-cell cultures where ultrasound was used with those where it was not used showed no differences in the infrequent finding of a chromosome aberration nor in the number of aneuploid cells per culture. We typically examine 20 cells and have about one hypodiploid cell per culture and one hyperdiploid cell per 10 cultures. We believe that prior sonography is advantageous with middle-trimester amniocentesis and, at the energy levels cited herein, does not interfere with successful culture of fetal cells for diagnostic genetic studies. We also believe that exposure should be minimised in light of known effects of ultrasound on animal tissues while we remain ignorant of possible effects on the growing fetus. This work 12579.
was
supported in part by U.S.P.H.S. grant AM-
Departments of Human Genetics, Pediatrics, and Obstetrics and Gynecology, Yale University, New Haven, Connecticut 06510, U.S.A.
J. MAHONEY JOHN C. HOBBINS.
MAURICE
GIEMSA BANDING OF MEIOTIC CHROMOSOMES
SIR,-Although specific banding of mitotic chromosomes produced by various techniques,l-4 little has been
has been
identification of meiotic chromosomes. on To date, the majority of papers deal with chromomere patterns along pachytene bivalents.5-8 This method has
published
Caspersson, T., Zech, L., Johansson, C., Modest, E. J. Chromosoma, 1970, 30, 215. 2. Sumner, A. T., Evans, H. J., Buckland, R. A. Nature New Biol. 1971, 232, 31. 3. Seabright, M. Lancet, 1971, ii, 971. 4. Shafer, D. A. ibid. 1973, i, 828. 5. Hungerford, D. A. Cytogenetics, 1971, 10, 23. 6. Hungerford, D. A., La Bodie, G. U., Balaban, G. B. ibid. p. 33. 7. Hungerford, D. A., La Bodie, G. U., Balaban, G. B., Messatzzia, L. R., Haller, G., Miller, A. E. Ann. Genet. 1971, 14, 257. 8. Bordjadze, V. K., Prokofieva-Belgovskaya, A. A. Cytogenetics, 1971, 10, 38.
been used extensively, mainly because of technical problems in obtaining adequate spreads and the difficulty of microscopic analysis. One report has been published not
on identification of meiotic bivalents from spermatocytes in diplotene/diakinesis.9 We have, therefore, attempted to obtain banding on meiotic material using Giemsa staining.
Cytological preparations were made from tissue of orchidectomised patients with prostatic cancer. Spermatogenic cells were dispersed in 0 075Af potassium chloride adjusted to pH 8-5 with borax buffer and allowed to remain in the hypotonic solution for 60 minutes before fixation in methanol/acetic-acid (3/1). After several washes in fixative, cells were flame-dried on clean wet slides which had been stored in distilled water. After drying for an hour, the preparations were immersed in 0-2M hydrochloric acid for 30-60 minutes. Slides were then rinsed in 2 x SSC (0’3M sodium chloride-0’03M sodium citrate) and incubated in buffered 2 x SSC (pH 7-0) at 60°C for 4-6 hours. At the end of the incubation period, slides were rinsed in distilled water and stained for 30 minutes in 2% Giemsa in MacIlvain’s buffer at pH 7-0. Good banding has been obtained on pachytene and diplotene/diakinesis stages of meiosis. Banding is especially good along the XY bivalent in the latter stages. It is also possible to identify the centromere in the majority of bivalents. Analysis of the banded bivalents is now in progress to determine
a
consistent and reliable pattern for
identification. Cytogenetics Research Laboratory, Research and Medical Services, Veterans Administration Hospital, 1030 Jefferson Avenue, Memphis, Tennessee 38104, U.S.A.
DANIEL W. BATH B. R. GENDEL.
1.
ACQUIRED TRISOMY 9 SIR,—Professor Davidson and Dr Knight 10 report on two patients with myelosclerosis having an extra chromosome 9 in all cells of the bone-marrow. Rowley 11 has found a possible translocation between the long arm of chromosome 22 and the long arm of chromosome 9. The two observations prompt us to mention that we recently discovered a case of acute myelomonoblastic leukæmia in a man having a trisomy of chromosome 9 in about 35% of the mitotic bone-marrow cells examined directly after 9 aspiration. The methods used to identify chromosome 12 and were a modified trypsin-Giemsa banding technique the Giemsa-11 staining method.13 A
more
detailed report will appear later.
Departments of Human Genetics and Haematology, Katholieke Universiteit
Nijmegen, Nijmegen, Netherlands.
F. J. RUTTEN T. W. J. HUSTINX J. M. J. C. SCHERES D. J. T. WAGENER.
DIFFERENTIATION OF IRON DEFICIENCY FROM THALASSÆMIA TRAIT
SiR—Dr England and his colleagues 14 have missed my point. The polyeythaemic patient that I described 15 with a discriminant function (D.F.1) in the -thalassaemia range had not been venesected, but had presented with iron deficiency. This is of course a common presentation in polycythsemia rubra vera, presumably because of the Caspersson, T., Hulten, M., Lindsten, J., Zech, L. Hereditas, 1971, 67, 147. 10. Davidson, W. M., Knight, L. A. Lancet, 1973, i, 1510. 11. Rowley, J. D. Nature, 1973, 243, 290. 12. Scheres, J. M. J. C. Lancet, 1972, i, 849. 13. Bobrow, M., et al. Nature, 1972, 238, 663. 14. England, J. M., Bain, B. J., Fraser, P. M. Lancet, 1973, i, 1514. 15. Hamblin, T. J. ibid. p. 636. 9.
Banded bivalents from primary spermatocytes.
A. Pachytene. B. Diplotene’diakinesis showing banding and Arrows indicate association of the X and Y chromosomes. centromeres.
456 abnormal cases
bleeding state. Nor is the diagnosis in these always self-evident, since the hxmoglobin may be in
the normal range or even reduced. I estimated the D.F.1 in venesected patients merely to demonstrate that in patients with definite polycythxmia rubra vera and undisputed iron deficiency this value is
consistently negative. I am grateful to Dr Srivastava (July 21, p. 154) for pointing out further exceptions to their rule. No doubt in uncomplicated cases the formula does differentiate between iron deficiency and p-thalassaemia trait. Unfortunately it is in just these cases, as Dr Schriever (July 21, p. 154) suggests, that the distinction usually is self-evident. Department of Pathology, General Hospital, Poole, Dorset.
T.
J. HAMBLIN.
DISPOSABLE SYRINGES
SiR,—Itrust you will allow a further comment on the correspondence which followed my earlier letter.1 It has been more than once said that bacterial sterility is absolute state, one of the few. It shares this distinction with death and virginity-there are no half-measures. One is, therefore, surprised to see that no attention is paid to the long-known criteria that apply to the sterilisation of syringes, or other materials. In 1897 Epstein showed that alcohol was not a germicide. In 1898 it was also shown that it had no effect on spores. It has also been shown that if one must use alcohol, 80% isopropylalcohol is a less bad agent than alcohol itself. I am astounded to see Dr Sheldon (July 21, p. 148) stating categorically that it is in order to store syringes intended for human use in industrial spirit, and that it is only necessary to change the spirit about once a week. Surely this is no way of maintaining sterility, let alone in syringes intended to be used in vulnerable beings. It is also stated that the syringes should be boiled once a week. It has been shown over and over again that boiling syringes is an unsatisfactory method of sterilisation. These are matters that are of the most common knowledge. If one wishes to trace authoritative statements these can be found in any standard textbook on bacteriology. Again, it is suggested by Dr Sheldon that in exceptional cases, where the general practitioner considers that disposable needles and syringes are needed on medical grounds, they can be obtained through hospitals, with the agreement of the consultant ". How many more barriers could be placed against free access to sterile syringes and needles ? The British Diabetic Association, according to Dr Sheldon, has made several pleas to the Department of Health and Social Security for such provision ", " but the decision of the Department remains that the expenditure This is a supine act on the part of cannot be justified ". the Association, which must be regarded as a denigration of the seriousness of the diabetic state. It is a matter of record, of course, that in certain of the Swiss cantons a special allowance is made against taxation because of the expense of acquiring suitable foods for diabetics. This is another matter which requires consideration, apart from disposable syringes and needles. On the whole, diabetics receive a raw deal at the hands of the Department, and one wishes to direct attention once again to the importance of proper and careful consideration of this serious condition. an
N.B.T. TEST IN LEPROMATOUS LEPROSY
SIR,-Using the Gifford and Malawista technique1 in different diseases, we found that lepromatous patients, who harbour a great number of bacilli, had a normal percentage of formazan-positive cells in polymorphoThere was. no nuclear leucocytes of peripheral blood. increase above control levels, as might be expected from such a massive bacterial infection. As this technique has an inherent in-vitro activation of polymorphonuclear leucocytes and a rather wide range of normal values, we repeated our experiments using Matula and Paterson’s technique.2 The results obtained
are
shown in the
accompanying
figure. Patients with lepromatous leprosy had consistently lower values than normal controls. However, when patients with reactional lepromatous leprosy were tested, the percentage of formazan-positive cells was as high or higher than that in severe septic conditions due to pyogenic bacteria. We were able, in 2 cases, to test lepromatous patients before and during reaction, and the low values rose to extremely high ones. In an isolated case a patient with lepromatous leprosy had a high proportion of formazanpositive cells when he developed osteomyelitis of a metatarsal bone. We are in process of
investigating in-vitro activation of polymorphonuclear leucocytes from lepromatous patients. It seems that while Mycobacterium leprae is unable to activate neutrophils of lepromatous patients in vivo, something takes place during lepromatous reaction that is able to do so. Disintegration products of bacilli, antigenantibody complexes, or substances liberated from macro1. 2.
Gifford, R. H., Malawista, S. E. J. Lab. clin. Med. 1970, 75, 511. Matula, G., Paterson, P. Y. New Engl. J. Med. 1971, 285, 311.
"
"
3
Upper Wimpole Street, London W1.
1.
Haler, D. Lancet, 1973, i, 1130.
DAVID HALER.
Percentage of formazan-positive neutrophils in peripheral blood.
Mean and standard errors indicated correspond to a series of Dots correspond to individual values for 19 normal adults. patients with lepromatous leprosy (a), reactional lepromatous leprosy (b), septic processes due to pyogenic bacteria (c), and non-septic skin diseases (d).
laboratory comparing karyotypes from amniotic-fluid-cell cultures where ultrasound was used with those where it was not used showed no differences in the infrequent finding of a chromosome aberration nor in the number of aneuploid cells per culture. We typically examine 20 cells and have about one hypodiploid cell per culture and one hyperdiploid cell per 10 cultures. We believe that prior sonography is advantageous with middle-trimester amniocentesis and, at the energy levels cited herein, does not interfere with successful culture of fetal cells for diagnostic genetic studies. We also believe that exposure should be minimised in light of known effects of ultrasound on animal tissues while we remain ignorant of possible effects on the growing fetus. This work 12579.
was
supported in part by U.S.P.H.S. grant AM-
Departments of Human Genetics, Pediatrics, and Obstetrics and Gynecology, Yale University, New Haven, Connecticut 06510, U.S.A.
J. MAHONEY JOHN C. HOBBINS.
MAURICE
GIEMSA BANDING OF MEIOTIC CHROMOSOMES
SIR,-Although specific banding of mitotic chromosomes produced by various techniques,l-4 little has been
has been
identification of meiotic chromosomes. on To date, the majority of papers deal with chromomere patterns along pachytene bivalents.5-8 This method has
published
Caspersson, T., Zech, L., Johansson, C., Modest, E. J. Chromosoma, 1970, 30, 215. 2. Sumner, A. T., Evans, H. J., Buckland, R. A. Nature New Biol. 1971, 232, 31. 3. Seabright, M. Lancet, 1971, ii, 971. 4. Shafer, D. A. ibid. 1973, i, 828. 5. Hungerford, D. A. Cytogenetics, 1971, 10, 23. 6. Hungerford, D. A., La Bodie, G. U., Balaban, G. B. ibid. p. 33. 7. Hungerford, D. A., La Bodie, G. U., Balaban, G. B., Messatzzia, L. R., Haller, G., Miller, A. E. Ann. Genet. 1971, 14, 257. 8. Bordjadze, V. K., Prokofieva-Belgovskaya, A. A. Cytogenetics, 1971, 10, 38.
been used extensively, mainly because of technical problems in obtaining adequate spreads and the difficulty of microscopic analysis. One report has been published not
on identification of meiotic bivalents from spermatocytes in diplotene/diakinesis.9 We have, therefore, attempted to obtain banding on meiotic material using Giemsa staining.
Cytological preparations were made from tissue of orchidectomised patients with prostatic cancer. Spermatogenic cells were dispersed in 0 075Af potassium chloride adjusted to pH 8-5 with borax buffer and allowed to remain in the hypotonic solution for 60 minutes before fixation in methanol/acetic-acid (3/1). After several washes in fixative, cells were flame-dried on clean wet slides which had been stored in distilled water. After drying for an hour, the preparations were immersed in 0-2M hydrochloric acid for 30-60 minutes. Slides were then rinsed in 2 x SSC (0’3M sodium chloride-0’03M sodium citrate) and incubated in buffered 2 x SSC (pH 7-0) at 60°C for 4-6 hours. At the end of the incubation period, slides were rinsed in distilled water and stained for 30 minutes in 2% Giemsa in MacIlvain’s buffer at pH 7-0. Good banding has been obtained on pachytene and diplotene/diakinesis stages of meiosis. Banding is especially good along the XY bivalent in the latter stages. It is also possible to identify the centromere in the majority of bivalents. Analysis of the banded bivalents is now in progress to determine
a
consistent and reliable pattern for
identification. Cytogenetics Research Laboratory, Research and Medical Services, Veterans Administration Hospital, 1030 Jefferson Avenue, Memphis, Tennessee 38104, U.S.A.
DANIEL W. BATH B. R. GENDEL.
1.
ACQUIRED TRISOMY 9 SIR,—Professor Davidson and Dr Knight 10 report on two patients with myelosclerosis having an extra chromosome 9 in all cells of the bone-marrow. Rowley 11 has found a possible translocation between the long arm of chromosome 22 and the long arm of chromosome 9. The two observations prompt us to mention that we recently discovered a case of acute myelomonoblastic leukæmia in a man having a trisomy of chromosome 9 in about 35% of the mitotic bone-marrow cells examined directly after 9 aspiration. The methods used to identify chromosome 12 and were a modified trypsin-Giemsa banding technique the Giemsa-11 staining method.13 A
more
detailed report will appear later.
Departments of Human Genetics and Haematology, Katholieke Universiteit
Nijmegen, Nijmegen, Netherlands.
F. J. RUTTEN T. W. J. HUSTINX J. M. J. C. SCHERES D. J. T. WAGENER.
DIFFERENTIATION OF IRON DEFICIENCY FROM THALASSÆMIA TRAIT
SiR—Dr England and his colleagues 14 have missed my point. The polyeythaemic patient that I described 15 with a discriminant function (D.F.1) in the -thalassaemia range had not been venesected, but had presented with iron deficiency. This is of course a common presentation in polycythsemia rubra vera, presumably because of the Caspersson, T., Hulten, M., Lindsten, J., Zech, L. Hereditas, 1971, 67, 147. 10. Davidson, W. M., Knight, L. A. Lancet, 1973, i, 1510. 11. Rowley, J. D. Nature, 1973, 243, 290. 12. Scheres, J. M. J. C. Lancet, 1972, i, 849. 13. Bobrow, M., et al. Nature, 1972, 238, 663. 14. England, J. M., Bain, B. J., Fraser, P. M. Lancet, 1973, i, 1514. 15. Hamblin, T. J. ibid. p. 636. 9.
Banded bivalents from primary spermatocytes.
A. Pachytene. B. Diplotene’diakinesis showing banding and Arrows indicate association of the X and Y chromosomes. centromeres.
456 abnormal cases
bleeding state. Nor is the diagnosis in these always self-evident, since the hxmoglobin may be in
the normal range or even reduced. I estimated the D.F.1 in venesected patients merely to demonstrate that in patients with definite polycythxmia rubra vera and undisputed iron deficiency this value is
consistently negative. I am grateful to Dr Srivastava (July 21, p. 154) for pointing out further exceptions to their rule. No doubt in uncomplicated cases the formula does differentiate between iron deficiency and p-thalassaemia trait. Unfortunately it is in just these cases, as Dr Schriever (July 21, p. 154) suggests, that the distinction usually is self-evident. Department of Pathology, General Hospital, Poole, Dorset.
T.
J. HAMBLIN.
DISPOSABLE SYRINGES
SiR,—Itrust you will allow a further comment on the correspondence which followed my earlier letter.1 It has been more than once said that bacterial sterility is absolute state, one of the few. It shares this distinction with death and virginity-there are no half-measures. One is, therefore, surprised to see that no attention is paid to the long-known criteria that apply to the sterilisation of syringes, or other materials. In 1897 Epstein showed that alcohol was not a germicide. In 1898 it was also shown that it had no effect on spores. It has also been shown that if one must use alcohol, 80% isopropylalcohol is a less bad agent than alcohol itself. I am astounded to see Dr Sheldon (July 21, p. 148) stating categorically that it is in order to store syringes intended for human use in industrial spirit, and that it is only necessary to change the spirit about once a week. Surely this is no way of maintaining sterility, let alone in syringes intended to be used in vulnerable beings. It is also stated that the syringes should be boiled once a week. It has been shown over and over again that boiling syringes is an unsatisfactory method of sterilisation. These are matters that are of the most common knowledge. If one wishes to trace authoritative statements these can be found in any standard textbook on bacteriology. Again, it is suggested by Dr Sheldon that in exceptional cases, where the general practitioner considers that disposable needles and syringes are needed on medical grounds, they can be obtained through hospitals, with the agreement of the consultant ". How many more barriers could be placed against free access to sterile syringes and needles ? The British Diabetic Association, according to Dr Sheldon, has made several pleas to the Department of Health and Social Security for such provision ", " but the decision of the Department remains that the expenditure This is a supine act on the part of cannot be justified ". the Association, which must be regarded as a denigration of the seriousness of the diabetic state. It is a matter of record, of course, that in certain of the Swiss cantons a special allowance is made against taxation because of the expense of acquiring suitable foods for diabetics. This is another matter which requires consideration, apart from disposable syringes and needles. On the whole, diabetics receive a raw deal at the hands of the Department, and one wishes to direct attention once again to the importance of proper and careful consideration of this serious condition. an
N.B.T. TEST IN LEPROMATOUS LEPROSY
SIR,-Using the Gifford and Malawista technique1 in different diseases, we found that lepromatous patients, who harbour a great number of bacilli, had a normal percentage of formazan-positive cells in polymorphoThere was. no nuclear leucocytes of peripheral blood. increase above control levels, as might be expected from such a massive bacterial infection. As this technique has an inherent in-vitro activation of polymorphonuclear leucocytes and a rather wide range of normal values, we repeated our experiments using Matula and Paterson’s technique.2 The results obtained
are
shown in the
accompanying
figure. Patients with lepromatous leprosy had consistently lower values than normal controls. However, when patients with reactional lepromatous leprosy were tested, the percentage of formazan-positive cells was as high or higher than that in severe septic conditions due to pyogenic bacteria. We were able, in 2 cases, to test lepromatous patients before and during reaction, and the low values rose to extremely high ones. In an isolated case a patient with lepromatous leprosy had a high proportion of formazanpositive cells when he developed osteomyelitis of a metatarsal bone. We are in process of
investigating in-vitro activation of polymorphonuclear leucocytes from lepromatous patients. It seems that while Mycobacterium leprae is unable to activate neutrophils of lepromatous patients in vivo, something takes place during lepromatous reaction that is able to do so. Disintegration products of bacilli, antigenantibody complexes, or substances liberated from macro1. 2.
Gifford, R. H., Malawista, S. E. J. Lab. clin. Med. 1970, 75, 511. Matula, G., Paterson, P. Y. New Engl. J. Med. 1971, 285, 311.
"
"
3
Upper Wimpole Street, London W1.
1.
Haler, D. Lancet, 1973, i, 1130.
DAVID HALER.
Percentage of formazan-positive neutrophils in peripheral blood.
Mean and standard errors indicated correspond to a series of Dots correspond to individual values for 19 normal adults. patients with lepromatous leprosy (a), reactional lepromatous leprosy (b), septic processes due to pyogenic bacteria (c), and non-septic skin diseases (d).