- Email: [email protected]
Y-FLUORESCENCE IN FORMALIN-FIXED TISSUES
769
quantitative differences were noted between normal and leukxmic myeloblasts or monoblasts. Recent ultrastructural studies in our laboratories between lymphoblasts from normal lymph follicles (bursa-dependent lymphoblasts) and lymphoblasts from acute lymphoblastic leukaemia demonstrated size differences (see accompanying table). It can be seen that the small size of the leukxmic lymphoblast is primarily due to small cytoplasmic area. It is of interest that only 1 of our 6 patients with acute lymphoblastic leukxmia had a mediastinal tumour at the onset of his disease. None of the measurements for this patient were different from the measurements obtained from the other leukxmic patients. The real difference in the cell types with or without prior mediastinal involvement may not be a difference between B versus T cells, but one of T-cell origin. Possibly this variability of their origin may predestine T cells to variable physiological responses towards mitogens and sheep erythrocytes. If size does have importance
in
determining T
or
B cell
origin
in
acute
lympho-
blastic leukaemia, then, on the basis of such data as shown in the table, it seems that the majority will probably be of T-cell origin. Harrisburg Hospital, Harrisburg, Pennsylvania 17101, U.S.A.
I. E. SZEKELY D. R. FISHER H. R. SCHUMACHER.
Potato
blight
and
anencephaly deaths in Quebec.
quantities.8 tetratogenic
Assuming a 24-38-week interval between effect and births, the births from blight in year N would occur from January N+l to February N+2, the calendar year N-t-1 gives a reasonable approximation to this, as a detailed Quebec study has shown no monthly variation in anencephaly incidence.9 The accompanying figure shows the Quebec results; there is no association demonstrated. Correlation coefficients calculated for each province were small and not significant, all values of P being >0-10. Prince Edward Island, having only 2500 births per year, was combined with its neighbour New Brunswick in the following data:
ANENCEPHALY AND POTATO BLIGHT IN EASTERN CANADA
SIR,-The Eastern provinces of Canada (Nova Scotia, Prince Edward Island, New Brunswick, and Quebec) have a higher mortality from spina bifida than the rest of the countryand are also major potato-producing areas with a high prevalence of late potato blight.If anencephaly and spina bifida are both related to the consumption of blighted potatoes, as Renwick3 has suggested, these provinces should show a time correlation between mortality from anencephaly and blight severity, such has been shown in Scotland 4 and England and Wales.5 The per-caput consumption of potatoes in Canada in 1966 was 1-55 kg. per week, which is about 80% of the British consumption.s To test this idea, the number of infant deaths and stillbirths from anencephaly per 1000 total live and stillbirths was calculated for the named provinces, and for Ontario, which has considerable potato production but little blight,2 for the years 1944 to 1968 from data supplied by Statistics Canada. These rates were compared to an estimate of potato-blight severity taken from the Canadian Annual Plant Disease Reports.’ These record the amount of blight present both on plants and in harvested tubers from selected fields in each province. The severity of blight is recorded by a descriptive grade from " absent " to very severe ". These grades were converted to a 0-5 scale, for plant (haulm) and tuber blight separately, and compared to the anencephaly mortality in the following year. Blight epidemics occur from mid-July to early September2 and blighted tubers may be consumed from then until about May the following year, when the new crop becomes available. Imports, from the U.S.A., are only in very small "
1. 2.
3. 4. 5. 6. 7.
Hewitt, D. Br. J. prev. soc. Med. 1963, 17, 13. Cox, A. E., Large, E. C. Potato Blight Epidemics throughout the World. Agricultural Handbook no. 174, Agricultural Research Service, U.S. Department of Agriculture. Washington, 1960. Renwick, J. H. Br. J. prev. soc. Med. 1972, 26, 67. Renwick, J. H. Lancet, 1972, ii, 336, 967. Renwick, J. H. Br. med. J. 1973, i, 172. Apparent Per Capita Domestic Disappearance of Food in Canada. Dominion Bureau of Statistics 32-226, Ottawa, 1967. 23rd-39th Annual Report of the Canadian Plant Disease Survey; Canadian Plant Disease Survey vols. 41-47 and 50. Department of Agriculture, Canada. Ottawa, 1944-1970.
Thus, the annual variations in anencephaly mortality in these provinces do not appear to be related to changes in blight severity. Prince Edward Island and New Brunswick export potatoes to the other provinces which might mask a local effect, but then one would still expect a correlation within these provinces. Whether the east-west decline in anencephaly and spina-bifida mortality is related to potatoes or to other concomitantly varying factors 10 -e.g., genetic influences 1 or softness of water-supplies -has yet to be determined. Royal Ottawa Hospital, Ottawa, Canada K1Z 7K4.
J. MARK ELWOOD.
Y-FLUORESCENCE IN FORMALIN-FIXED TISSUES
SiR,—Dr Laszlo and Dr Gaal (March 3, p. 488) reported technique for the detection of Barr bodies in paraffinembedded embryonic tissues. We have developed a similar technique 11 that displayed Y-fluorescent bodies well in paraffin-embedded tissues of fetuses, placentas, placental site, and decidua, prostate, a
ovaries, testis, and ovotestis. We have been unable to get good results with cutaneous tissues. After the removal of the paraffin, the sections were washed well through alcohol series and kept in absolute alcohol for at least 24 hours. Fixation in glacial-aceticacid/methanol (1/3) was carried out for 10 minutes. The slides buffer
then incubated in a modified McIlvaine’s 650C for 1 hour, and then they were stained with
were
at
8. Trade of Canada, Imports by Commodities. Dominion Bureau of Statistics 65-007. Ottawa, 1966. 9. Horowitz, I., McDonald, A. D. Can. med. Ass. J. 1969, 100, 748. 10. Neri, L. C., Mandel, J. S., Hewitt, D. Lancet, 1972, i, 931. 11. Khudr, G., Benirschke, K. Stain Tech. (in the press).
770
0-05 °u quinacrine hydrochloride for 5 minutes. After 10 minutes’ differentiation in running tap-water, the slides were mounted in buffer. This method has been helpful in determining fetal sex in accidentally formalin-fixed tissues, assessing the percentage of XY to XX cells in tissues of proven mosaics or hermaphrodites, and evaluating the origin of X-cells and placental-site giant cells.
Support by grant UFPHS GM 18839-01 is gratefully acknowledged. Department of Obstetrics and Gynecology, University of California School of Medicine, La Jolla, California 92037, U.S.A.
SiR,—II should like wholeheartedly to support Dr Evans (March 31, p. 721). With Dr Cornes’s letter I would also agree except on the question of centrifuging. Centrifugation will pick up sperms not found in uncentrifuged specimens. But those of us seeking
to promote vasectomy as an efficient method of birth control will be disturbed by the letter from Mr Craft and Mr Diggory (March 24, p. 663).
insisting on two consecutive, completely negative being required before a vasectomised man may be regarded as sterile, they are on dangerous ground. It may
By
not
counts
GABRIEL KHUDR KURT BENIRSCHKE.
CLICKING RIB
SIR,-I was surprised to read (March 24, p. 674) that clicking rib or cartilage " is a condition not well known. May I draw attention to Hamilton Bailey’s account1 of a very similar " affection of the thoracic cage " ? Quoting R. Davies-Colley, Bailey states that the usual sufferer is a lady, as in the three cases so well described by Mr Mynors. "
Liverpool Royal Infirmary, Pembroke Place, Liverpool L3 5PU.
SPERM-COUNTS AFTER VASECTOMY
be true that the presence of an occasional non-motile sperm is " unlikely to be of great significance ", but nobody can yet say that it is of no significance. It is not good enough to be content with probabilities. The object of vasectomy is to make it impossible, not merely improbable, for a vasectomised man to beget a child. It is better to be safe than sorry. That is why many of us urge the importance of a double negative count. To say " provided the operation has been performed correctly " is merely to beg the ques-
tion. The Crediton Project, West Longsight,
Crediton, Devon.
L. N. JACKSON.
R. J. BRERETON. SEMINAL UREA LEVEL AND SPERM-COUNT
OUTCOME AFTER SEVERE BRAIN DAMAGE
SIR,-your leading article of March 10 (p. 523) is certainly timely. Although it concentrates on the question of the late effects of head injury, many of the comments apply equally to brain damage from other causes. Similar problems of prediction face us and other electroencephalographers in patients after cardiac arrest. We have been concerned not only with the significance of the isoelectric E.E.G. as an
indicator of total neocortical necrosis but also
early recognition of patients in whom survival in a vegetative state is likely. The patient suspected of brain damage in whom some, albeit abnormal, E.E.G. activity persists presents a great prognostic problem in terms of the quality of survival possible. However, exploration of some refinements of E.E.G. evaluation, such as the application of computer-aided discriminant function analysis, 2,3 suggests that it is possible to delineate patterns of abnormality indicating the degrees of brain damage compatible with survival. These fall between those characteristic of patients dying with severe irreversible brain damage and those recovering fully. Neuropathological examinations have shown that a scale of degrees of brain damage from total with the
cortical necrosis to mild selective neuronal necrosis with degrees of cortical sparing, correlates closely with the amount of E.E.G. abnormality found after hypoxic brain damage.4 These studies have concerned patients with cardiac arrest, but in those with coexisting cerebral lesions, such as head injury, accurate predictions were still possible. This suggests that such E.E.G. criteria may be applicable to patients with head injury per se, although our experience is that these present some particular problem and that their E.E.G. assessment is more fraught with difficulty. The London
Hospital, Whitechapel,
London E1 1BB.
PAMELA F. PRIOR D. F. SCOTT.
1. Demonstrations of Physical Signs in Clinical Surgery; p. 303. Bristol, 1960. 2. Binnie, C. D., Prior, P. F., Lloyd, D. S. L., Scott, D. F., Margerison, J. H. Br. med. J. 1970, iv, 265. 3. Maynard, D., Prior, P. F. Electroenceph. clin. Neurophysiol. 1973,
74, 108. 4. Prior, P. F.
SiR,—The presence of an intrauterine contraceptive device is said to cause a rise of 300-400 % in the urea level of uterine fluid.l It was later demonstrated that a urea solution of comparable higher concentration instantaneously immobilised live sperms. Based on these observations, an intravaginal contraceptive device has been developed. It also seemed worth examining the urea level of seminal plasma with variable sperm-counts, to detect any correlation between the two. Semen samples were collected by masturbation from husbands of primary sterile couples reporting to the pathology laboratory of the Central Command Military Hospital, Lucknow. Routine semen analyses including sperm-counts were conducted immediately after collection at the laboratory, and urea estimations were done at the Central Drug Research Institute, Lucknow, by the method described previously.l The results were:
The total with sperm-counts of 100 million per ml. or below was 53. Of these, 29 (55%) had a urea level at 50 mg. per 100 ml. and above, and 14 cases (26%) at 60 mg. per 100 ml. and above. This sample of 101 cases of primary sterility has been divided arbitrarily into: (1) husbands with sperm-counts of 100 million per ml. and below and (2) those with counts above 100 million per ml. According to our findings, a significantly greater number with seminal-urea levels of 50 mg. per 100 ml. and above are in group 1 than in group 2. This trend is maintained even if 60 mg. per 100 ml. and above is taken as the demarcation line. Yet more significant is the fact that 14 cases out of 15 recording seminal urea concentration above 60 mg. per 100 ml. were in group 1. Thus, a negative correlation seems to exist between 1. 2.
Excerpta med. Amst. (in the press).
Kar, A. B., Engineer, A. D., Dasgupta, P. R., Srivastava, A. K. Am. J. Obstet. Gynec. 1969, 104, 607. Dasgupta, P. R., Kar, A. B., Dhar, M. L. Ind. J. exp. Biol. 1971, 9, 413.
quantitative differences were noted between normal and leukxmic myeloblasts or monoblasts. Recent ultrastructural studies in our laboratories between lymphoblasts from normal lymph follicles (bursa-dependent lymphoblasts) and lymphoblasts from acute lymphoblastic leukaemia demonstrated size differences (see accompanying table). It can be seen that the small size of the leukxmic lymphoblast is primarily due to small cytoplasmic area. It is of interest that only 1 of our 6 patients with acute lymphoblastic leukxmia had a mediastinal tumour at the onset of his disease. None of the measurements for this patient were different from the measurements obtained from the other leukxmic patients. The real difference in the cell types with or without prior mediastinal involvement may not be a difference between B versus T cells, but one of T-cell origin. Possibly this variability of their origin may predestine T cells to variable physiological responses towards mitogens and sheep erythrocytes. If size does have importance
in
determining T
or
B cell
origin
in
acute
lympho-
blastic leukaemia, then, on the basis of such data as shown in the table, it seems that the majority will probably be of T-cell origin. Harrisburg Hospital, Harrisburg, Pennsylvania 17101, U.S.A.
I. E. SZEKELY D. R. FISHER H. R. SCHUMACHER.
Potato
blight
and
anencephaly deaths in Quebec.
quantities.8 tetratogenic
Assuming a 24-38-week interval between effect and births, the births from blight in year N would occur from January N+l to February N+2, the calendar year N-t-1 gives a reasonable approximation to this, as a detailed Quebec study has shown no monthly variation in anencephaly incidence.9 The accompanying figure shows the Quebec results; there is no association demonstrated. Correlation coefficients calculated for each province were small and not significant, all values of P being >0-10. Prince Edward Island, having only 2500 births per year, was combined with its neighbour New Brunswick in the following data:
ANENCEPHALY AND POTATO BLIGHT IN EASTERN CANADA
SIR,-The Eastern provinces of Canada (Nova Scotia, Prince Edward Island, New Brunswick, and Quebec) have a higher mortality from spina bifida than the rest of the countryand are also major potato-producing areas with a high prevalence of late potato blight.If anencephaly and spina bifida are both related to the consumption of blighted potatoes, as Renwick3 has suggested, these provinces should show a time correlation between mortality from anencephaly and blight severity, such has been shown in Scotland 4 and England and Wales.5 The per-caput consumption of potatoes in Canada in 1966 was 1-55 kg. per week, which is about 80% of the British consumption.s To test this idea, the number of infant deaths and stillbirths from anencephaly per 1000 total live and stillbirths was calculated for the named provinces, and for Ontario, which has considerable potato production but little blight,2 for the years 1944 to 1968 from data supplied by Statistics Canada. These rates were compared to an estimate of potato-blight severity taken from the Canadian Annual Plant Disease Reports.’ These record the amount of blight present both on plants and in harvested tubers from selected fields in each province. The severity of blight is recorded by a descriptive grade from " absent " to very severe ". These grades were converted to a 0-5 scale, for plant (haulm) and tuber blight separately, and compared to the anencephaly mortality in the following year. Blight epidemics occur from mid-July to early September2 and blighted tubers may be consumed from then until about May the following year, when the new crop becomes available. Imports, from the U.S.A., are only in very small "
1. 2.
3. 4. 5. 6. 7.
Hewitt, D. Br. J. prev. soc. Med. 1963, 17, 13. Cox, A. E., Large, E. C. Potato Blight Epidemics throughout the World. Agricultural Handbook no. 174, Agricultural Research Service, U.S. Department of Agriculture. Washington, 1960. Renwick, J. H. Br. J. prev. soc. Med. 1972, 26, 67. Renwick, J. H. Lancet, 1972, ii, 336, 967. Renwick, J. H. Br. med. J. 1973, i, 172. Apparent Per Capita Domestic Disappearance of Food in Canada. Dominion Bureau of Statistics 32-226, Ottawa, 1967. 23rd-39th Annual Report of the Canadian Plant Disease Survey; Canadian Plant Disease Survey vols. 41-47 and 50. Department of Agriculture, Canada. Ottawa, 1944-1970.
Thus, the annual variations in anencephaly mortality in these provinces do not appear to be related to changes in blight severity. Prince Edward Island and New Brunswick export potatoes to the other provinces which might mask a local effect, but then one would still expect a correlation within these provinces. Whether the east-west decline in anencephaly and spina-bifida mortality is related to potatoes or to other concomitantly varying factors 10 -e.g., genetic influences 1 or softness of water-supplies -has yet to be determined. Royal Ottawa Hospital, Ottawa, Canada K1Z 7K4.
J. MARK ELWOOD.
Y-FLUORESCENCE IN FORMALIN-FIXED TISSUES
SiR,—Dr Laszlo and Dr Gaal (March 3, p. 488) reported technique for the detection of Barr bodies in paraffinembedded embryonic tissues. We have developed a similar technique 11 that displayed Y-fluorescent bodies well in paraffin-embedded tissues of fetuses, placentas, placental site, and decidua, prostate, a
ovaries, testis, and ovotestis. We have been unable to get good results with cutaneous tissues. After the removal of the paraffin, the sections were washed well through alcohol series and kept in absolute alcohol for at least 24 hours. Fixation in glacial-aceticacid/methanol (1/3) was carried out for 10 minutes. The slides buffer
then incubated in a modified McIlvaine’s 650C for 1 hour, and then they were stained with
were
at
8. Trade of Canada, Imports by Commodities. Dominion Bureau of Statistics 65-007. Ottawa, 1966. 9. Horowitz, I., McDonald, A. D. Can. med. Ass. J. 1969, 100, 748. 10. Neri, L. C., Mandel, J. S., Hewitt, D. Lancet, 1972, i, 931. 11. Khudr, G., Benirschke, K. Stain Tech. (in the press).
770
0-05 °u quinacrine hydrochloride for 5 minutes. After 10 minutes’ differentiation in running tap-water, the slides were mounted in buffer. This method has been helpful in determining fetal sex in accidentally formalin-fixed tissues, assessing the percentage of XY to XX cells in tissues of proven mosaics or hermaphrodites, and evaluating the origin of X-cells and placental-site giant cells.
Support by grant UFPHS GM 18839-01 is gratefully acknowledged. Department of Obstetrics and Gynecology, University of California School of Medicine, La Jolla, California 92037, U.S.A.
SiR,—II should like wholeheartedly to support Dr Evans (March 31, p. 721). With Dr Cornes’s letter I would also agree except on the question of centrifuging. Centrifugation will pick up sperms not found in uncentrifuged specimens. But those of us seeking
to promote vasectomy as an efficient method of birth control will be disturbed by the letter from Mr Craft and Mr Diggory (March 24, p. 663).
insisting on two consecutive, completely negative being required before a vasectomised man may be regarded as sterile, they are on dangerous ground. It may
By
not
counts
GABRIEL KHUDR KURT BENIRSCHKE.
CLICKING RIB
SIR,-I was surprised to read (March 24, p. 674) that clicking rib or cartilage " is a condition not well known. May I draw attention to Hamilton Bailey’s account1 of a very similar " affection of the thoracic cage " ? Quoting R. Davies-Colley, Bailey states that the usual sufferer is a lady, as in the three cases so well described by Mr Mynors. "
Liverpool Royal Infirmary, Pembroke Place, Liverpool L3 5PU.
SPERM-COUNTS AFTER VASECTOMY
be true that the presence of an occasional non-motile sperm is " unlikely to be of great significance ", but nobody can yet say that it is of no significance. It is not good enough to be content with probabilities. The object of vasectomy is to make it impossible, not merely improbable, for a vasectomised man to beget a child. It is better to be safe than sorry. That is why many of us urge the importance of a double negative count. To say " provided the operation has been performed correctly " is merely to beg the ques-
tion. The Crediton Project, West Longsight,
Crediton, Devon.
L. N. JACKSON.
R. J. BRERETON. SEMINAL UREA LEVEL AND SPERM-COUNT
OUTCOME AFTER SEVERE BRAIN DAMAGE
SIR,-your leading article of March 10 (p. 523) is certainly timely. Although it concentrates on the question of the late effects of head injury, many of the comments apply equally to brain damage from other causes. Similar problems of prediction face us and other electroencephalographers in patients after cardiac arrest. We have been concerned not only with the significance of the isoelectric E.E.G. as an
indicator of total neocortical necrosis but also
early recognition of patients in whom survival in a vegetative state is likely. The patient suspected of brain damage in whom some, albeit abnormal, E.E.G. activity persists presents a great prognostic problem in terms of the quality of survival possible. However, exploration of some refinements of E.E.G. evaluation, such as the application of computer-aided discriminant function analysis, 2,3 suggests that it is possible to delineate patterns of abnormality indicating the degrees of brain damage compatible with survival. These fall between those characteristic of patients dying with severe irreversible brain damage and those recovering fully. Neuropathological examinations have shown that a scale of degrees of brain damage from total with the
cortical necrosis to mild selective neuronal necrosis with degrees of cortical sparing, correlates closely with the amount of E.E.G. abnormality found after hypoxic brain damage.4 These studies have concerned patients with cardiac arrest, but in those with coexisting cerebral lesions, such as head injury, accurate predictions were still possible. This suggests that such E.E.G. criteria may be applicable to patients with head injury per se, although our experience is that these present some particular problem and that their E.E.G. assessment is more fraught with difficulty. The London
Hospital, Whitechapel,
London E1 1BB.
PAMELA F. PRIOR D. F. SCOTT.
1. Demonstrations of Physical Signs in Clinical Surgery; p. 303. Bristol, 1960. 2. Binnie, C. D., Prior, P. F., Lloyd, D. S. L., Scott, D. F., Margerison, J. H. Br. med. J. 1970, iv, 265. 3. Maynard, D., Prior, P. F. Electroenceph. clin. Neurophysiol. 1973,
74, 108. 4. Prior, P. F.
SiR,—The presence of an intrauterine contraceptive device is said to cause a rise of 300-400 % in the urea level of uterine fluid.l It was later demonstrated that a urea solution of comparable higher concentration instantaneously immobilised live sperms. Based on these observations, an intravaginal contraceptive device has been developed. It also seemed worth examining the urea level of seminal plasma with variable sperm-counts, to detect any correlation between the two. Semen samples were collected by masturbation from husbands of primary sterile couples reporting to the pathology laboratory of the Central Command Military Hospital, Lucknow. Routine semen analyses including sperm-counts were conducted immediately after collection at the laboratory, and urea estimations were done at the Central Drug Research Institute, Lucknow, by the method described previously.l The results were:
The total with sperm-counts of 100 million per ml. or below was 53. Of these, 29 (55%) had a urea level at 50 mg. per 100 ml. and above, and 14 cases (26%) at 60 mg. per 100 ml. and above. This sample of 101 cases of primary sterility has been divided arbitrarily into: (1) husbands with sperm-counts of 100 million per ml. and below and (2) those with counts above 100 million per ml. According to our findings, a significantly greater number with seminal-urea levels of 50 mg. per 100 ml. and above are in group 1 than in group 2. This trend is maintained even if 60 mg. per 100 ml. and above is taken as the demarcation line. Yet more significant is the fact that 14 cases out of 15 recording seminal urea concentration above 60 mg. per 100 ml. were in group 1. Thus, a negative correlation seems to exist between 1. 2.
Excerpta med. Amst. (in the press).
Kar, A. B., Engineer, A. D., Dasgupta, P. R., Srivastava, A. K. Am. J. Obstet. Gynec. 1969, 104, 607. Dasgupta, P. R., Kar, A. B., Dhar, M. L. Ind. J. exp. Biol. 1971, 9, 413.