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LEUCOCYTE CULTURES WITH PHYTOHÆMAGGLUTININ IN CHRONIC LYMPHATIC LEUKÆMIA
563 TABLE III-C0113PARISON OF FLOW BY XENON-133 METHOD AND BROMSULPHTHALEIN METHOD IN TWO DOGS. RESULTS OVER ONE HOUR
artery and how much by portal vein, well as the flow-rates in each. The calculation assumes that the flow-paths remain separate. The hepatic artery supplies about 30% of the liver mass and about 15% of the liver blood-flow: the portal vein supplies the remainder
supplied by hepatic
as
(table iv). SUMMARY
measured in dogs by deterthe disappearance-rate of xenon 133 from the mining liver. Flow-rates were greater when the isotope was given by the portal vein than when it was given by the hepatic artery. We conclude that portal-venous and arterial streams remain functionally separate as they perfuse the capillaries and sinusoids of the liver.
Hepatic blood-flow
TABLE IV-PROPORTION OF LIVER MASS SUPPLIED BY HEPATIC ARTERY AND PORTAL VEIN, AND PROPORTION OF LIVER BLOOD-FLOW SUPPLIED BY BACH. RESULTS IN TWO DOGS
was
We are grateful for grants from the research fund of the board of governors of the Westminster Hospital, from the British Heart Foundation, and from the Peel Medical Research Trust. Mr. Richard Robbins gave valuable technical help throughout. We are grateful to Mr. J. P. Nicholson, of the physics department, for arranging supplies of xenon 133, and to Mr. H. S. Grainger for supplies of drugs. We thank Dr. C. J. Gavey for his encouragement.
J. RUSSELL REES Cantab., M.R.C.P. V. J. REDDING
M.A., M.D.
M.B.
Departments of Cardiology and Clinical Measurement, Westminster Hospital, London, S.W.1
Lond.
RICHARD ASHFIELD M.B. Lond., M.R.C.P.
LEUCOCYTE CULTURES WITH PHYTOHÆMAGGLUTININ IN CHRONIC LYMPHATIC LEUKÆMIA
comparison of flows by the isotope method and bromsulphthalein clearance over the same period of one hour is shown in table III. In both, as might be predicted, flow-rates by dye clearance lie between flow-rates by the isotope method and closer to the values via the portal venous route. In dog no. 11, P02 measurements were made. Mean results showed hepatic-arterial P02 levels of 120 mm. Hg; portal venous P02 levels of 41 mm. Hg, and hepatic-venous Po2 levels of 24 mm. Hg. A
DISCUSSION
Current views about the intrahepatic vascular network have been reviewed by Bradley.3 The precise arrangement is still disputed, but the portal-venous and arterial systems probably meet in the sinusoids and in the capillary network of the portal tracts. The arterioles may join the terminal portal veins as they enter the sinusoids, or may join the latter directly. If there is functional mixing of the two streams, then the rates of xenon clearance should be equal, regardless of the route of administration. Our findings suggest that the main hepatic-arterial and portal-venous streams remain functionally separate as they perfuse the liver tissue. Although anatomically the sinusoids are supplied by arterioles and portal vessels, there may be some mechanism whereby the distribution of arterial and portal-venous blood can be varied independently. Since the routes appear functionally separate, the higher flow-rates measured by the portal path may be associated with the lower oxygen-content of its blood. The 133Xe method gives no information about the absolute flow by the two routes, nor about their distribution within the liver. However, the simultaneous flow measurements
by isotope and bromsulphthalein clearance
(table III) may be used 3.
to
calculate how much liver is
Bradley, S. E. in Handbook of Physiology (edited by W. F. Hamilton and P. Dow); vol. II, section 2, p. 1387. Baltimore, 1963.
IN the culture of blood-leucocytes from cases of chronic lymphatic leukaemia Bernard et aLl reported that the addition of phytohaemagglutinin P (Difco) induced only limited cellular transformation, compared with the effect of this substance on cultures from healthy controls. Quaglino and Cowlingalso noted there was " impaired capacity for cell growth under the stimulus of P.H.A.". Similar findings have been reported by others.34 On the other hand, Elves and Wilkinson,5 in cultures of blood with phytohsemagglutinin M (Difco) from cases both of chronic and of acute lymphatic leukaemia, observed almost the same morphological changes as in normal blood cultures, except that mitoses were seen in small numbers from the outset and rapidly increased. Gunz et al.,6 Court Brown,and Nowell and Hungerford8 have also reported successful blood cultures in cytogenetic studies of chronic lymphatic leukaemia, though Nowell and Hungerford noted that small lymphocytes from the blood in chronic lymphatic leukaemia did not grow very vigorously in the presence of phytohasmagglutinin. In the present work blood leucocytes from seven cases of chronic lymphatic leukaemia (table I) and nine normal subjects (table 11) were cultured with
phytohsemagglutinin M. METHODS
added to all cultures, but no Phytohsemagglutinin antibiotics were used. In accordance with the method of Moorhead et awl. cultures had an initial cell population of the order of 103 per c.mm., and in some of the leukxmias the cell suspensions had to be diluted with autologous cell-free plasma 1. Bernard, C., Geraldes, A., Boiron, M. Lancet, 1964, i, 667. M was
Quaglino, D., Cowling, D. C. Brit. J. Hœmat. 1964, 10, 358. Nowell, P. C. Exp. Cell Res. 1960, 19, 267. 4. Schrek, R., Rabinowitz, Y. Proc. Soc. exp. Biol. N. Y. 1963, 113, 191. 5. Elves, M. W., Wilkinson, J. F. Exp. Cell Res. 1963, 30, 200. 6. Gunz, F. W., Fitzgerald, P. H., Adams, A. Brit. med. J. 1962, ii, 1097. 7. Court Brown, W. M. Lancer, 1964, i, 986. 8. Nowell, P. C., Hungerford, D. A. Ann. N.Y. Acad. Sci. 1964, 113, 654. 9. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., Hungerford, D. A. Exp. Cell Res. 1960, 20, 613. 2. 3.
564 TABLE I-RESULTS WITH LEUKAMIC BLOODS
Treatments Case 2: no treatment since 5’/8 years ago, when he was given chlorambucil and abdominal irradiation. Case 3: spinal and pelvic irradiation; no previous treatment. Cases 1, 4, and 5: receiving prednisone 10 mg. b.d. Case 1 had received chlorambucil and splenic irradiation 9 months previously, case 4 splenic irradiation 5 months previously, case 5 three courses of splenic irradiation and two courses of spinal irradiation, the last course being given 9 months previously, with blood-transfusion. Case 6: (a) received 5 mC. phosphorus-32 2 days before culture; chlorambucil and splenic irradiation 1-2 months previously; several previous courses of splenic irradiation.
(b) receiving cyclophosphamide. but had treatment, receiving splenic and cervical-node irradiation 5 months previously.
Case 7:
no
reduce the final count to this level. The cultures at 0 hours (i.e., on first setting up the cultures) and at 72 hours. Tritiated thymidine was added to parallel cultures (1 C per ml. of culture) for 1 hour before examination at 72 hours. Radioautographs of smears were prepared by coating with emulsion (Eastman Kodak NTB 3), exposing for 8 days, processing, and staining through the fixed emulsion. When examining cultures at 72 hours three criteria of growth were used: (1) the percentage of blast-cells 15 11 or
in order were
to
sampled
TABLB II-RESULTS WITH NORMAL BLOODS AT
72 HOURS’ CULTURE
At 72 hours there was a striking difference between the two sets of cultures. In the control cultures there were numerous blast-cells, a raised mitotic index, and a high proportion of cells incorporating tritiated thymidine (table 11)—nndings conspicuously absent in chronic lymphatic leukaemia blood (with one exception). In four cases of leukaemia (nos. 1, 4, 6, and 7) there were neither blast-cells nor mitoses at 72 hours, while few or no cells were incorporating thymidine (table i). Two cases (3 and 5) showed 2% and 1% of blast-cells respectively at 72 hours, and no mitoses; though in case 3 thymidine was found in 4-5% of cells. The findings in case 2 were
exceptional. DISCUSSION
in
diameter; (2) the
total percentage of cells in mitosis; the total (3) percentage of cells incorporating tritiated thymidine. By recording as blast-cells those which were 15 !l or more in diameter, a varying number of cells, which were obviously in process of transformation but had not yet attained this size, were excluded. This classification is quite arbitrary, since there is a continuous spectrum of cell sizes. The point is worthy of emphasis since it may be responsible for some of the discrepancies in the literature. more
and
RESULTS
The white-cell count varied widely in blood from patients with chronic lymphatic leukaemia. The proportion of lymphocytes and smear-cells was also variable (table i). In the blood of control subjects the total and differential white-cell counts were within normal limits. 10 11 In cases of chronic lymphatic leukaemia almost all the lymphocytes in cultures at 0 hours were small,12 and no blast-cells or mitoses were seen. The control cultures contained about 60% small lymphocytes, the remainder being medium or large. 10. 11.
Dacie, J. V. Practical Hæmatology. London, 1956. Whitby, L. E. H., Britton, C. J. C. Disorders ofthe Blood. London,
1953. 12. Bessis, M. Cytology of the Blood and Blood Forming Organs. New York, 1956.
Our results seem to accord with the view that lymphocytes in some cases of chronic lymphatic leukaemia show reduced or negligible transformation. 1-48 Accurate comparison, however, is not possible except with the results of Quaglino and Cowling,2 since none of the other authors state whether their patients were undergoing treatment. All the patients in our investigation were either undergoing treatment (irradiation and/or prednisone) or had been treated in the past (patients 2 and 7). Patient 7 had been recently treated and was in partial relapse at the time of culture. Patient 2, who had not been treated for many months, was in good remission and was the only one showing a substantial degree of transformation. At first sight this suggests as one possibility that failure to grow might be the result of cell damage caused by the treatment, rather than of any intrinsic defect in the leukasmic cells. There is also the additional complication that one may be dealing with a mixture, in varying and unknown proportions, of normal and abnormal cells. Our case 2, in remission for 5 years, may well have been one in which there were many normal cells in the blood, and this case appears in some ways to resemble case 4 of Quaglino and Cowling.22 SUMMARY
Blood from seven cases of chronic lymphatic leuksemia cultured with phytohsemagglutinin M. Comparison was made with normal blood cultures by assessment at 72 hours, using the morphological appearance of stained was
565 smears, the mitotic
index, and the technique of labelling cells in the D.N.A.-synthetic phase of the mitotic cycle by incorporation of tritiated thymidine. Six of the seven showed little or no evidence of transformation. The possible effects of treatment and the phase of the condition are discussed. It is a pleasure to acknowledge our indebtedness to Dr. R. C. Tudway and Dr. S. Curwen, of the department of radiotherapy, Bristol General Hospital, for kindly allowing us to investigate their patients. We are also indebted to Mrs. S. Powell and Miss C. Brearley for technical assistance, and to Mrs. M. Galvin for help in preparation of the manuscript. G. C. B. WINTER cases
M.B., B.D.S. Brist.
D. G. OSMOND B.SC.
M.B.,
Department of Anatomy, The Medical School,
Bristol,
Manc., F.R.C.S. D. J. MAHY
M.D., D.SC.
University of Bristol Department of Radiotherapy, Bristol General
Brist.
J. M. YOFFEY
Hospital,
M.B.
2
Brist.
CHANGES IN RENAL FUNCTION IN LATE PREGNANCY WHEN a woman in late pregnancy lies flat on her back, the capacity of the kidneys to excrete water and electrolytes is reduced.1 We know that in the non-pregnant subject renal function alters with posture, and recent work has suggested that there may also be effects peculiar to pregnancy, which result from venous obstruction by the gravid uterus. Thus measurements of pressure in the inferior vena cava indicate that there is often a considerable degree of obstruction to the flow of blood in the vein when a woman in late pregnancy lies on her back.2 The capacity of the kidney to excrete the large amounts of steroid metabolites from the placenta might be affected by such venous obstruction, and give rise to a false impression of decreased placental function. Tests of kidney function and steroid excretion were therefore made on women in late pregnancy: once in a position where maximum pressure from the uterus on the vena cava might be expected, and once in a position calculated to relieve the vein from any pressure. In the course of the experiments, hypertensive women tended to behave differently, and therefore the subjects were divided into two groups: one consisted of normotensive women, and the other of women who had a diastolic blood-pressure of between 90 and 110 mm. Hg on at least two occasions during the 2 days of the test, but who had no proteinuria or overt oedema. EXPERIMENTAL DESIGN
The experiments were designed to achieve some standardisation of the conditions under which the urine samples were 1. 2.
Pritchard, J. A., Barnes, A. C., Bright, R. H. J. clin. Invest. 1955, 34, 777. Scott, D. B., Kerr, M. G. J. Obstet. Gynœc. Brit. Cwlth, 1963, 70, 1044.
collected, and in particular to try and eliminate short-term fluctuations in the flow and composition of the urine. Women who were 37-39 weeks pregnant, and who had fasted overnight, stayed in a quiet room by themselves, drinking 100 ml. of water every 15 minutes and emptying the bladder every hour. They ate nothing, did not smoke, and movement was restricted to the least possible. During the first 2 hours, the urine-volume and urea output changed rapidly as the diuresis was established; thereafter the fluctuations became less if a steady water intake was maintained. Estimations were therefore done on the last 2 hours’ urine in a 4-hour period of such sustained diuresis. Each woman was tested twice, once lying on her side and once in a semirecumbent position. To find a position least likely to cause pressure on the vena cava, the woman was placed on her side with a cushion under her hip so that the pregnant uterus was tipped forward, away from the vena cava. The position thought most likely to cause the uterus to press upon the vena cava was sitting but leaning well back, so that the uterus would lie against the posterior abdominal wall and the presenting part be drawn toward the pelvis by gravity. The test subjects, nine normal and seven hypertensive women, were admitted to a metabolic unit, and a sustained diuresis was induced in one of the two positions, as described. Next day the test was repeated in the other position. The order in which these positions were employed was alternated from one subject to the next. An accurately timed urine sample was collected during the last 2 hours of each test, and the output of creatinine, urea, sodium, potassium, oestriol, and pregnanediol was assayed. RESULTS
A comparison between urine outputs in the two positions is shown in the figure. The 2-hour urine-volume when lying on the side (unobstructed position) is plotted against the volume produced by the same subject when in the semirecumbent (obstructed) position. Points falling below the line drawn at 45° indicate values in which the urine-volume in the side position exceeded that in the semirecumbent position. All the subjects produced more urine in the unobstructed position than in the obstructed position. The difference between the two positions was exaggerated in hypertensive subjects, who also tended to produce less urine than normotensives in both positions. The figure also compares sodium excretion in the two positions. The findings are similar to those for urine-volume.
The outputs in the urine of potassium, urea, and the steroids oestriol and pregnanediol compared in the two positions.
creatinine, were
also
The mean results are shown in table I. Changes in excretion with position apply to water and sodium only; both hypertensives and normotensives show the same effect, the change being greater in the case of hypertensives. The hypertensive group excreted slightly less pregnanediol than did the normotensives, and in the former group the excretion of pregnanediol was less when lying on the side than in the semirecumbent
position. The data
submitted
were
to a
statistical analysis, the
results of which are summarised in table 11. As regards the urine volume produced in the two positions, two conclusions may be
drawn: 1.
more urine is excreted when the side than when semirecumbent by both groups-an average of 130 ml. more in normotensives and 313 ml. more in hypertensives. 2. Significantly more urine is excreted by normotensives than by hypertensives in both positions-an average of 330 ml. more in the semirecumbent and 147 iffl. more in the lying-on-theside position.
Significantly
lying
on
As regards sodium excretion, conclusions may be drawn: Effect of posture
on
(a) urine output and (b) sodium output.
1.
two
similar
Significantly more sodium is excreted when
artery and how much by portal vein, well as the flow-rates in each. The calculation assumes that the flow-paths remain separate. The hepatic artery supplies about 30% of the liver mass and about 15% of the liver blood-flow: the portal vein supplies the remainder
supplied by hepatic
as
(table iv). SUMMARY
measured in dogs by deterthe disappearance-rate of xenon 133 from the mining liver. Flow-rates were greater when the isotope was given by the portal vein than when it was given by the hepatic artery. We conclude that portal-venous and arterial streams remain functionally separate as they perfuse the capillaries and sinusoids of the liver.
Hepatic blood-flow
TABLE IV-PROPORTION OF LIVER MASS SUPPLIED BY HEPATIC ARTERY AND PORTAL VEIN, AND PROPORTION OF LIVER BLOOD-FLOW SUPPLIED BY BACH. RESULTS IN TWO DOGS
was
We are grateful for grants from the research fund of the board of governors of the Westminster Hospital, from the British Heart Foundation, and from the Peel Medical Research Trust. Mr. Richard Robbins gave valuable technical help throughout. We are grateful to Mr. J. P. Nicholson, of the physics department, for arranging supplies of xenon 133, and to Mr. H. S. Grainger for supplies of drugs. We thank Dr. C. J. Gavey for his encouragement.
J. RUSSELL REES Cantab., M.R.C.P. V. J. REDDING
M.A., M.D.
M.B.
Departments of Cardiology and Clinical Measurement, Westminster Hospital, London, S.W.1
Lond.
RICHARD ASHFIELD M.B. Lond., M.R.C.P.
LEUCOCYTE CULTURES WITH PHYTOHÆMAGGLUTININ IN CHRONIC LYMPHATIC LEUKÆMIA
comparison of flows by the isotope method and bromsulphthalein clearance over the same period of one hour is shown in table III. In both, as might be predicted, flow-rates by dye clearance lie between flow-rates by the isotope method and closer to the values via the portal venous route. In dog no. 11, P02 measurements were made. Mean results showed hepatic-arterial P02 levels of 120 mm. Hg; portal venous P02 levels of 41 mm. Hg, and hepatic-venous Po2 levels of 24 mm. Hg. A
DISCUSSION
Current views about the intrahepatic vascular network have been reviewed by Bradley.3 The precise arrangement is still disputed, but the portal-venous and arterial systems probably meet in the sinusoids and in the capillary network of the portal tracts. The arterioles may join the terminal portal veins as they enter the sinusoids, or may join the latter directly. If there is functional mixing of the two streams, then the rates of xenon clearance should be equal, regardless of the route of administration. Our findings suggest that the main hepatic-arterial and portal-venous streams remain functionally separate as they perfuse the liver tissue. Although anatomically the sinusoids are supplied by arterioles and portal vessels, there may be some mechanism whereby the distribution of arterial and portal-venous blood can be varied independently. Since the routes appear functionally separate, the higher flow-rates measured by the portal path may be associated with the lower oxygen-content of its blood. The 133Xe method gives no information about the absolute flow by the two routes, nor about their distribution within the liver. However, the simultaneous flow measurements
by isotope and bromsulphthalein clearance
(table III) may be used 3.
to
calculate how much liver is
Bradley, S. E. in Handbook of Physiology (edited by W. F. Hamilton and P. Dow); vol. II, section 2, p. 1387. Baltimore, 1963.
IN the culture of blood-leucocytes from cases of chronic lymphatic leukaemia Bernard et aLl reported that the addition of phytohaemagglutinin P (Difco) induced only limited cellular transformation, compared with the effect of this substance on cultures from healthy controls. Quaglino and Cowlingalso noted there was " impaired capacity for cell growth under the stimulus of P.H.A.". Similar findings have been reported by others.34 On the other hand, Elves and Wilkinson,5 in cultures of blood with phytohsemagglutinin M (Difco) from cases both of chronic and of acute lymphatic leukaemia, observed almost the same morphological changes as in normal blood cultures, except that mitoses were seen in small numbers from the outset and rapidly increased. Gunz et al.,6 Court Brown,and Nowell and Hungerford8 have also reported successful blood cultures in cytogenetic studies of chronic lymphatic leukaemia, though Nowell and Hungerford noted that small lymphocytes from the blood in chronic lymphatic leukaemia did not grow very vigorously in the presence of phytohasmagglutinin. In the present work blood leucocytes from seven cases of chronic lymphatic leukaemia (table I) and nine normal subjects (table 11) were cultured with
phytohsemagglutinin M. METHODS
added to all cultures, but no Phytohsemagglutinin antibiotics were used. In accordance with the method of Moorhead et awl. cultures had an initial cell population of the order of 103 per c.mm., and in some of the leukxmias the cell suspensions had to be diluted with autologous cell-free plasma 1. Bernard, C., Geraldes, A., Boiron, M. Lancet, 1964, i, 667. M was
Quaglino, D., Cowling, D. C. Brit. J. Hœmat. 1964, 10, 358. Nowell, P. C. Exp. Cell Res. 1960, 19, 267. 4. Schrek, R., Rabinowitz, Y. Proc. Soc. exp. Biol. N. Y. 1963, 113, 191. 5. Elves, M. W., Wilkinson, J. F. Exp. Cell Res. 1963, 30, 200. 6. Gunz, F. W., Fitzgerald, P. H., Adams, A. Brit. med. J. 1962, ii, 1097. 7. Court Brown, W. M. Lancer, 1964, i, 986. 8. Nowell, P. C., Hungerford, D. A. Ann. N.Y. Acad. Sci. 1964, 113, 654. 9. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., Hungerford, D. A. Exp. Cell Res. 1960, 20, 613. 2. 3.
564 TABLE I-RESULTS WITH LEUKAMIC BLOODS
Treatments Case 2: no treatment since 5’/8 years ago, when he was given chlorambucil and abdominal irradiation. Case 3: spinal and pelvic irradiation; no previous treatment. Cases 1, 4, and 5: receiving prednisone 10 mg. b.d. Case 1 had received chlorambucil and splenic irradiation 9 months previously, case 4 splenic irradiation 5 months previously, case 5 three courses of splenic irradiation and two courses of spinal irradiation, the last course being given 9 months previously, with blood-transfusion. Case 6: (a) received 5 mC. phosphorus-32 2 days before culture; chlorambucil and splenic irradiation 1-2 months previously; several previous courses of splenic irradiation.
(b) receiving cyclophosphamide. but had treatment, receiving splenic and cervical-node irradiation 5 months previously.
Case 7:
no
reduce the final count to this level. The cultures at 0 hours (i.e., on first setting up the cultures) and at 72 hours. Tritiated thymidine was added to parallel cultures (1 C per ml. of culture) for 1 hour before examination at 72 hours. Radioautographs of smears were prepared by coating with emulsion (Eastman Kodak NTB 3), exposing for 8 days, processing, and staining through the fixed emulsion. When examining cultures at 72 hours three criteria of growth were used: (1) the percentage of blast-cells 15 11 or
in order were
to
sampled
TABLB II-RESULTS WITH NORMAL BLOODS AT
72 HOURS’ CULTURE
At 72 hours there was a striking difference between the two sets of cultures. In the control cultures there were numerous blast-cells, a raised mitotic index, and a high proportion of cells incorporating tritiated thymidine (table 11)—nndings conspicuously absent in chronic lymphatic leukaemia blood (with one exception). In four cases of leukaemia (nos. 1, 4, 6, and 7) there were neither blast-cells nor mitoses at 72 hours, while few or no cells were incorporating thymidine (table i). Two cases (3 and 5) showed 2% and 1% of blast-cells respectively at 72 hours, and no mitoses; though in case 3 thymidine was found in 4-5% of cells. The findings in case 2 were
exceptional. DISCUSSION
in
diameter; (2) the
total percentage of cells in mitosis; the total (3) percentage of cells incorporating tritiated thymidine. By recording as blast-cells those which were 15 !l or more in diameter, a varying number of cells, which were obviously in process of transformation but had not yet attained this size, were excluded. This classification is quite arbitrary, since there is a continuous spectrum of cell sizes. The point is worthy of emphasis since it may be responsible for some of the discrepancies in the literature. more
and
RESULTS
The white-cell count varied widely in blood from patients with chronic lymphatic leukaemia. The proportion of lymphocytes and smear-cells was also variable (table i). In the blood of control subjects the total and differential white-cell counts were within normal limits. 10 11 In cases of chronic lymphatic leukaemia almost all the lymphocytes in cultures at 0 hours were small,12 and no blast-cells or mitoses were seen. The control cultures contained about 60% small lymphocytes, the remainder being medium or large. 10. 11.
Dacie, J. V. Practical Hæmatology. London, 1956. Whitby, L. E. H., Britton, C. J. C. Disorders ofthe Blood. London,
1953. 12. Bessis, M. Cytology of the Blood and Blood Forming Organs. New York, 1956.
Our results seem to accord with the view that lymphocytes in some cases of chronic lymphatic leukaemia show reduced or negligible transformation. 1-48 Accurate comparison, however, is not possible except with the results of Quaglino and Cowling,2 since none of the other authors state whether their patients were undergoing treatment. All the patients in our investigation were either undergoing treatment (irradiation and/or prednisone) or had been treated in the past (patients 2 and 7). Patient 7 had been recently treated and was in partial relapse at the time of culture. Patient 2, who had not been treated for many months, was in good remission and was the only one showing a substantial degree of transformation. At first sight this suggests as one possibility that failure to grow might be the result of cell damage caused by the treatment, rather than of any intrinsic defect in the leukasmic cells. There is also the additional complication that one may be dealing with a mixture, in varying and unknown proportions, of normal and abnormal cells. Our case 2, in remission for 5 years, may well have been one in which there were many normal cells in the blood, and this case appears in some ways to resemble case 4 of Quaglino and Cowling.22 SUMMARY
Blood from seven cases of chronic lymphatic leuksemia cultured with phytohsemagglutinin M. Comparison was made with normal blood cultures by assessment at 72 hours, using the morphological appearance of stained was
565 smears, the mitotic
index, and the technique of labelling cells in the D.N.A.-synthetic phase of the mitotic cycle by incorporation of tritiated thymidine. Six of the seven showed little or no evidence of transformation. The possible effects of treatment and the phase of the condition are discussed. It is a pleasure to acknowledge our indebtedness to Dr. R. C. Tudway and Dr. S. Curwen, of the department of radiotherapy, Bristol General Hospital, for kindly allowing us to investigate their patients. We are also indebted to Mrs. S. Powell and Miss C. Brearley for technical assistance, and to Mrs. M. Galvin for help in preparation of the manuscript. G. C. B. WINTER cases
M.B., B.D.S. Brist.
D. G. OSMOND B.SC.
M.B.,
Department of Anatomy, The Medical School,
Bristol,
Manc., F.R.C.S. D. J. MAHY
M.D., D.SC.
University of Bristol Department of Radiotherapy, Bristol General
Brist.
J. M. YOFFEY
Hospital,
M.B.
2
Brist.
CHANGES IN RENAL FUNCTION IN LATE PREGNANCY WHEN a woman in late pregnancy lies flat on her back, the capacity of the kidneys to excrete water and electrolytes is reduced.1 We know that in the non-pregnant subject renal function alters with posture, and recent work has suggested that there may also be effects peculiar to pregnancy, which result from venous obstruction by the gravid uterus. Thus measurements of pressure in the inferior vena cava indicate that there is often a considerable degree of obstruction to the flow of blood in the vein when a woman in late pregnancy lies on her back.2 The capacity of the kidney to excrete the large amounts of steroid metabolites from the placenta might be affected by such venous obstruction, and give rise to a false impression of decreased placental function. Tests of kidney function and steroid excretion were therefore made on women in late pregnancy: once in a position where maximum pressure from the uterus on the vena cava might be expected, and once in a position calculated to relieve the vein from any pressure. In the course of the experiments, hypertensive women tended to behave differently, and therefore the subjects were divided into two groups: one consisted of normotensive women, and the other of women who had a diastolic blood-pressure of between 90 and 110 mm. Hg on at least two occasions during the 2 days of the test, but who had no proteinuria or overt oedema. EXPERIMENTAL DESIGN
The experiments were designed to achieve some standardisation of the conditions under which the urine samples were 1. 2.
Pritchard, J. A., Barnes, A. C., Bright, R. H. J. clin. Invest. 1955, 34, 777. Scott, D. B., Kerr, M. G. J. Obstet. Gynœc. Brit. Cwlth, 1963, 70, 1044.
collected, and in particular to try and eliminate short-term fluctuations in the flow and composition of the urine. Women who were 37-39 weeks pregnant, and who had fasted overnight, stayed in a quiet room by themselves, drinking 100 ml. of water every 15 minutes and emptying the bladder every hour. They ate nothing, did not smoke, and movement was restricted to the least possible. During the first 2 hours, the urine-volume and urea output changed rapidly as the diuresis was established; thereafter the fluctuations became less if a steady water intake was maintained. Estimations were therefore done on the last 2 hours’ urine in a 4-hour period of such sustained diuresis. Each woman was tested twice, once lying on her side and once in a semirecumbent position. To find a position least likely to cause pressure on the vena cava, the woman was placed on her side with a cushion under her hip so that the pregnant uterus was tipped forward, away from the vena cava. The position thought most likely to cause the uterus to press upon the vena cava was sitting but leaning well back, so that the uterus would lie against the posterior abdominal wall and the presenting part be drawn toward the pelvis by gravity. The test subjects, nine normal and seven hypertensive women, were admitted to a metabolic unit, and a sustained diuresis was induced in one of the two positions, as described. Next day the test was repeated in the other position. The order in which these positions were employed was alternated from one subject to the next. An accurately timed urine sample was collected during the last 2 hours of each test, and the output of creatinine, urea, sodium, potassium, oestriol, and pregnanediol was assayed. RESULTS
A comparison between urine outputs in the two positions is shown in the figure. The 2-hour urine-volume when lying on the side (unobstructed position) is plotted against the volume produced by the same subject when in the semirecumbent (obstructed) position. Points falling below the line drawn at 45° indicate values in which the urine-volume in the side position exceeded that in the semirecumbent position. All the subjects produced more urine in the unobstructed position than in the obstructed position. The difference between the two positions was exaggerated in hypertensive subjects, who also tended to produce less urine than normotensives in both positions. The figure also compares sodium excretion in the two positions. The findings are similar to those for urine-volume.
The outputs in the urine of potassium, urea, and the steroids oestriol and pregnanediol compared in the two positions.
creatinine, were
also
The mean results are shown in table I. Changes in excretion with position apply to water and sodium only; both hypertensives and normotensives show the same effect, the change being greater in the case of hypertensives. The hypertensive group excreted slightly less pregnanediol than did the normotensives, and in the former group the excretion of pregnanediol was less when lying on the side than in the semirecumbent
position. The data
submitted
were
to a
statistical analysis, the
results of which are summarised in table 11. As regards the urine volume produced in the two positions, two conclusions may be
drawn: 1.
more urine is excreted when the side than when semirecumbent by both groups-an average of 130 ml. more in normotensives and 313 ml. more in hypertensives. 2. Significantly more urine is excreted by normotensives than by hypertensives in both positions-an average of 330 ml. more in the semirecumbent and 147 iffl. more in the lying-on-theside position.
Significantly
lying
on
As regards sodium excretion, conclusions may be drawn: Effect of posture
on
(a) urine output and (b) sodium output.
1.
two
similar
Significantly more sodium is excreted when