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CYTOCIDAL ACTION OF COLCHICINE IN VITRO ON LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKÆMIA
868
the 82 babies, with the exception of 2 babies with pyogenic eye infections, who, however, yielded no pathogenic staphylococci at the three selected sites. Discussion
Our results showed that washing the skin of newborns with a solution of hexachlorophene reduced the numbers of the two main components of the resident flora-i.e., staphylococci and diphtheroid bacilli (more particularly
staphylococci). Although pathogenic microorganisms were found in the eyes of 2 babies,their absence at the selected sites did not allow us to draw any conclusions on the effect of hexachlorophene against pathogens. Hexachlorophene is bacteriostatic but also bactericidal
against gram-positive organisms when used routinely (Kjellander and Nygren 1962, Knights and Harvey 1964). Contamination with gram-negative organisms has been reported by Anderson (1962), and this risk is said to be reduced by adding 3% cetrimide to the hexachlorophene. Hexachlorophene is deposited in the horny layer of human skin and therefore has a cumulative effect, particularly if it is applied in vehicles such as dimethylacetamide(Stoughton 1966). Regular long-continued application is said to be most effective; our finding that the effect of hexachlorophene on the bacterial flora began after a time lag and gradually increased supports this view. Previous reports of a reduction of the pathogenic flora of the skin were based of necessity on relatively small numbers of cultures of pyogenic staphylococci. Clinical pyogenic infections were found to be fewer in infants
Preliminary Communication CYTOCIDAL ACTION OF COLCHICINE IN VITRO ON LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKÆMIA
Preliminary data show that the majority of cells in lymphocyte populations isolated from seven patients with chronic lymphocytic leukaemia (C.L.L.) were abnormal in that they were susceptible to the cytocidal action of colchicine in a concentration (10-7 M) which is at least one thousand times less than that required to affect similarly lymphocytes isolated from healthy people. This finding indicates that the in-vitro use of colchicine may be a valuable means of screening lymphocyte populations circulating in C.L.L. before, during, or Summary
after various forms of treatment. INTRODUCTION
HETEROGENEITY of circulating lymphocytes in a number of patients with chronic lymphocytic leukaemia (C.L.L.) has been demonstrated by the fractionation of separated lymphocytes on a column of polystyrene beads.1 The existence of abnormal column-adherent populations of cells and of non-adherent populations with properties more like those from healthy individuals was observed. The results were interpreted as meaning that separation of normal from abnormal cells had been partly but never completely achieved owing to the failure of some abnormal cells to adhere to polystyrene. Cells from post-column populations had a greater capacity for survival than did gross (pre-column) populations in the 3-day cultures acting as controls for those inoculated with phyto1.
Thomson, A. E. R., Robinson, 1966, ii, 200.
M. A.,
Wetherley-Mein,
G.
Lancet,
bathed in hexachlorophene when these were observed over long periods. The overall reduction of non-pathogens in hexachlorophene-treated babies in our trial clearly indicates the effect of this substance, but several questions arise. The clinical significance of suppressing the growth of non-pathogens on the skin is uncertain. If, in fact, this is associated with a diminished tendency to clinical infection, is there likely to be a rebound when the applications are stopped ? Gezon et al. (1964) reported no clinical evidence of a rebound during a follow-up period of 3 weeks after hexachlorophene had been applied for 3 weeks. In a trial of phisohex for the treatment of acne, McLean et al. (1962) reported that 3 of their 67 patients found that hexachlorophene produced erythema and pruritus. We found no objective side-effects, but this possibility must also be borne in mind. This work was supported by a grant from the William Shepherd bequest of the endowment fund of the Royal Free Hospital. We thank the consultant obstetricians and nursing staff of the Royal Free Hospital for their help with this work. Requests for reprints should be addressed to I. S. REFERENCES
Anderson, (1962) Med. J. Aust. ii, 463. Farquharson, C. D., Penney, S. F., Edwards, H. E., Barr, E. (1952) Can. med. Ass. J. 67, 247. Gezon, H. M., Donovan, J. T., Rogers, K. D., Hatch, T. F., Taylor, P. M. (1964) New Engl. J. Med. 270, 379. Gillespie, W. A., Simpson, K., Tozer, R. C. (1958) Lancet, ii, 1075. Kjellander, J., Nygren, B. (1962) Nord. Med. 67, 79. Knights, H. T., Harvey, J. (1964) N.Z. med. J. 63, 653. McLean, I. E. D., Graham, K. T., East, M. O. (1962) Practitioner, 189, 82. Sarkany, I., Gaylarde, C. (1967) Lancet, i, 589. Stoughton, R. B. (1966) Archs Derm. 94, 646. K.
haemagglutinin (P.H.A.). At the same time, the mortality in cultures of pre-column cells was regarded as surprisingly high and was inconsistent with levels reported by Schrek2 for cultures lasting 7 days. We have now found that this is due partly to a selective cytocidal action on abnormal lymphocytes of the alkaloid, colchicine (10-7 M). This had been incorporated into the culture medium to arrest P.H.A.-induced mitosis and was, consequently, also present in control cultures. The colchicine action is shown to apply to both column-non-adhesive and columnadhesive abnormal cells and permits assessment of the abnormal-cell content of lymphocyte populations in C.L.L. PATIENTS, MATERIALS, AND METHODS Seven patients, in whom the diagnosis of C.L.L. had been established for between 1 month and 91/2 years, were investigated. Four patients (cases 1-4) had received or were receiving various forms of chemotherapy and three (cases 6, 8, and 9) had not been treated. No patient had been given radiotherapy. Freshly withdrawn venous blood was subjected to the same method of fractionation as described elsewhere. 13 Wherever possible, however, the column of polystyrene beads was equilibrated at 37°C in cell-free serum-gelatin mixture (in place of the buffered balanced salt solution) before loading with cells suspended in serum-gelatin. With this modification the consistently high yields from columns of lymphocytes from healthy donors (see Results) indicated that adherence to polystyrene of normal lymphocytes in the c.L.L. populations would be largely prevented (this being the reverse situation to that obtained with a protein-free suspending medium 4). Lymphocyte cultures were set up (1000 cells per c.mm.) essentially as before, with autologous serum always being used in preparation of the culture medium and colchicine being omitted until later, when it was added in solution by micro’
2. Schrek, R. J. natn. Cancer Inst. 1964, 33, 837. 3. Thomson, A. E. R., Bull, J. M., Robinson, M. A. Br. J. Hœmat. 1966, 12, 433. 4. Thomson, A. E. R., Robinson, M. A. Rep. imp. Cancer Res. Fund, 1966, 64, 95. Published 1967.
869 CYTOCIDAL ACTION OF COLCHICINE ON LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKaeMIA
*
Cases also previously investigated by Thomson et al.l t Column was equilibrated in serum before use (see Materials and Methods). t Duration of 1-day cultures varied between 19 and 21 hours after adding colchicine, gassing with 5 % carbon dioxide, and placing in carbon-dioxide incubator. § With 10-6 M and 10-4 M colchicine, figures were 12% (4 experiments) and 13% (3 experiments), respectively. Figures in parentheses show number of experiments.
pipette to individual cultures. These were removed from the 5% carbon-dioxide incubator after 1 day (19-21 hours) and 3 days (69-71 hours), when pyknotic counts were performed on wet-fixed (susa), hasmalum-stained smears prepared by the Trowell5 procedure. The purity of the colchicine used (L. Light & Co. Ltd., batch 2976) was checked by examination of its physical properties (melting-point, and ultraviolet absorption at 350-5 and 243 m(l.). RESULTS
The results from representative experiments are summarised in the table. The mortality in pre-column C.L.L. populations (representative of lymphocytes in defibrinated blood) after 1 day in vitro and in the absence of colchicine (column a) varied, but was generally much greater than for lymphocytes isolated from healthy individuals and cultured under similar conditions. In the presence of 10-7 M-colchicine (column c) the mortality was raised substantially to a level which exceeded 80% in all except two cases. By the same criteria, lymphocytes from healthy individuals were, in contrast, comparatively unaffected by 10-7 M colchicine and were even unaffected by 10-5 M or 10-4 M colchicine. During 2 further days in vitro, few more pre-column cells died but a noticeable increase in mortality of lymphocytes from healthy individuals occurred where colchicine (10-7 M) was present. In comparison with a range in yield from polystyrene columns (pre-equilibrated in serum) of 80-100% for lymphocytes from seven healthy donors, abnormally low proportions of populations from cases 1, 2, 6, and 9 were recovered. In three of these (1, 2, and 6), the post-column cells displayed capacities for survival much nearer normal in the presence of colchicine, as judged by reductions in
(columns c and d). lymphocytes were separated from heparinised blood (and, therefore, were representative of the circulating population) no striking difference in lymphocyte mortality were recorded. This was confirmed by similar (unpublished) experiments where heparin was also used. pyknotic
count
Where
5.
Trowell,
O. A.
Expl Cell Res. 1955, 9, 258.
Recovery of cells measured by hxmocytometer exceeded 90% for almost every culture. Bearing in mind the error in the determinations (within ±10%), individual values within the set for each experiment were close enough to suggest that no greater losses of cells had occurred from cultures giving higher pyknotic counts. DISCUSSION
Colchicine was cytocidal in the concentration 10-7 M for the majority of lymphocytes in populations isolated from all the cases of C.L.L. The fact that lymphocytes from healthy individuals were resistant to at least one thousand times this concentration during the same period (1 day) in vitro shows that the sensitive lymphocytes circulating in the patients were abnormal cells (or normal cells which do not, or not to any extent, normally circulate). Their differential sensitivity strongly recommends the use of colchicine in vitro in low concentration as a means for determining the relative proportions of abnormal and normal cells in c.L.L. which circulate before, during, and after treatment. In this connection, two basic requirements should be met to obtain reliable results: the concentration of alkaloid used in a stipulated culture period should be sufficient to kill all abnormal lymphocytes and not any normal lymphocytes, and the population tested should be representative of the population circulat-
ing in vivo. Experiments with varying concentrations of colchicine6 have indicated that the use of 10--7 M colchicine for approximately 20 hours satisfies the first requirement reasonably well for the cases described here, despite the variable mortality in the absence of colchicine for the different cases. However, the second requirement is not met where defibrination is used to prevent clotting, because of accompanying losses (trapping) of lymphocytes. Nevertheless, the table indicates a crude inverse relationship between circulating-white-blood-cell count and proportion of ostensibly normal (colchicine-resistant) lymphocytes calculable from column c, with patients 3 and 8 occupying the extreme positions. The preliminary 6.
Thomson, A. E. R., Walker,
A. F.
Unpublished.
870 results showing that mortality of lymphocytes cultured without colchicine was no higher after their separation from heparinised blood imply that heparin might successfully replace defibrination in a colchicine-sensitivity test. The polystyrene column permits subclassification of colchicine-sensitive lymphocyte populations into those which are predominantly adhesive and those which are predominantly non-adhesive. For populations in the first category, the column may also permit separation of colchicine-resistant lymphocytes in partially purified form and thereby potentially make for more reasonable comparison of their properties with, lymphocytes from healthy individuals. For example, a sevenfold purification of colchicine-resistant cells was achieved by columntreatment of lymphocytes from patient 2. The column further provides a potential means of obtaining abnormal, adhesive cells in pure form for other studies. These investigations will be described more extensively elsewhere together with results from other experiments performed on the same cases. In particular, the results will be discussed in relation to clinical, haematological and
therapeutic data. We thank Prof. G. Wetherley-Mein, department of hasmatology, St. Thomas’s Hospital, London S.E.I, for providing the cases for study and for the considerable interest he has shown.
Cytochemistry Section, Division of Chemistry and Biochemistry, Imperial Cancer Research Fund, London W.C.2
A. E. R. THOMSON St. And., PH.D. Lond MARY A. ROBINSON
B.SC.
B.SC.
Lond.
Reviews of Books
Principles and Practice of Obstetric Analgesia
and
Anesthesia Vo/. Vol. of
7. Fundamental Considerations. 1.
BoNiCA, M.D., JOHN J. BONICA,
pro-
departmentof anesthesiology,University fessor Washington and chairman, Pp. Philadelphia: Publications.
School of Medicine. Oxford: Blackwell Scientific F. A. Davis Co. 1967. 837.
E9 15s.
’
Professor Bonica’s first volume (the second is to appear next is devoted to fundamental considerations for the anxsthetist working in an obstetric unit. The book deals extensively with the physiology and psychology of pregnancy, and includes detailed information on placental function and the transfer of anavsthetic and allied drugs to the foetus together with discussion of the bearing of this on the anaesthetic management of the individual case. Professor Bonica is a wholehearted advocate of the highest possible standard of obstetric anaesthesia—which, he believes, should always be in the hands of an anaesthetist who not only is expert in his specialty but also has a full background knowledge of obstetric physiology. This is an essential reference book for all obstetric and anaesthetic
year)
departments. Current
Perspectives
in
Surgery
Editors: WILLIAM S. BLAKEMORE, M.D., professor of surgery, University of Pennsylvania School of Medicine, Philadelphia; L. KRAEER FERGUSON, M.D., professor of surgery, University of Pennsylvania School of Medicine. London and New York: 1967. Pp. 317. E98.; §13-50. Harper & Row.
THIS book is one of a series on current perspectives in by contributors drawn from university medical schools in the U.S.A. Each section is followed by verbatim reports of informal panel discussions by the various authors. There is an interesting report on the location of gastrointestinal bleeding by mesenteric arteriography-a method of some marginal promise in obscure bleeding. surgery
Regional Cerebral Blood Flow Delayed Hypersensitivity J. L. TURK, director of M.R.C. Research Group on the Immunological Aspects of Dermatology, Institute of Dermatology, University of London. Amsterdam: North-Holland Publishing Co. 1967. Pp. 252. 80s. THE subject for this volume, the fourth of a series entitled Frontiers in Biology, is well chosen, since work on delayed hypersensitivity is in an interesting phase of development. Dr. Turk is well qualified to write such a book, since he has made many important original contributions. He has produced a treatise which is comprehensive, detailed, and well arranged. Having defined delayed hypersensitivity itself, Dr. Turk goes on to discuss several other topics, including
immunological unresponsiveness, homograft reactions, autoimmunity, and the possible roles of various cells and antibodies, in relation to the central theme. The book should appeal, not only to experts, but also to clinical and experimental workers with interests in immunology, hasmatology, and pathology. A
Surgeons’ Guide
to
Cardiac
Diagnosis
Part II:The Clinical Picture. DONALD N. Ross, B.SC., M.B., F.R.C.S., consultant thoracic surgeon, Guy’s Hospital, London. Berlin, Heidelberg, New York: Springer Verlag. 1967. Pp. 88. DM z6.
THIS remarkable short book covers not only the essentials in the diagnosis of congenital and acquired heart-disease that may be treated by surgery but also the principles of treatment. It defines in clear and precise terms the anatomy, haemodynamic clinical features, special investigations, differential diagnosis, and principles of treatment of each type of cardiac lesion. Where necessary, clear illustrations and excellent reproductions of electrocardiograms and X-rays are included. Inevitably explanations of complex physiological disturbances are simplified for the sake of clarity; but this detracts in no way from the practical value of the book. Although written primarily for the cardiac surgeon, this work can be read with profit by all interested in the diagnosis and surgical treatment of heartdisease. Mr. Ross is to be congratulated.
The Intra-Arterial Injection Method. KAI HfdEDT-RASMUSSEN. Copenhagen:Munksgaard. 1967. Pp. 79. Danish Kroner 25.
THE method of estimating cerebral blood-flow described in this monograph is to inject into the internal carotid or vertebral artery the diffusible, physiologically inactive, gammaemitting isotope 83krypton or 133xenon, both of which are almost completely eliminated by one passage through the lungs. An extracranial scintillation detector records the clearance curve, which resolves itself into two mono exponential curves from which, with due corrections, the flow in grey and white matter is separately calculated. The method entails very low exposure to radioactivity, and can be combined with angiography, but it does involve an injection. Whether an injection or an inhalation method, or both, will finally be in routine use remains to be seen, but this account will be of considerable value in the assessment. The resolution of the results into flows through grey and white matter is an interesting innovation. The mathematical formulas for calculating flow from the extrapolation of an exponential part of a curve are stated but not explained-the mathematical background has to be accepted, understood, or consulted elsewhere-but the clinical and anatomical details are spelled out in detail. The translation reads as though the work had been written in
English. New Editions Bedside Medicine.-2nd ed. By I. Snapper and Alvin I. Kahn. London: Heinemann Medical Books. New York: Grune & Stratton. 1967.
Pp. 824. E7; §18.75.
Textbook of Anatomy.-2nd ed. By W. Henry Hollinshead. New York and London: Harper & Row. 1967. Pp. 994. 18.75; E7 4s. Diseases of the Ear.-2nd ed. By Stuart R. Mawson. Edward Arnold. 1967. Pp. 554. E5.
London:
Techniques in Blood Grouping: Vols. I and n.-2nd ed. By Ivor Dunsford and C. Christopher Bowley. Edinburgh: Oliver & Boyd. 1967.
Pp.
534. E5 5s.
the 82 babies, with the exception of 2 babies with pyogenic eye infections, who, however, yielded no pathogenic staphylococci at the three selected sites. Discussion
Our results showed that washing the skin of newborns with a solution of hexachlorophene reduced the numbers of the two main components of the resident flora-i.e., staphylococci and diphtheroid bacilli (more particularly
staphylococci). Although pathogenic microorganisms were found in the eyes of 2 babies,their absence at the selected sites did not allow us to draw any conclusions on the effect of hexachlorophene against pathogens. Hexachlorophene is bacteriostatic but also bactericidal
against gram-positive organisms when used routinely (Kjellander and Nygren 1962, Knights and Harvey 1964). Contamination with gram-negative organisms has been reported by Anderson (1962), and this risk is said to be reduced by adding 3% cetrimide to the hexachlorophene. Hexachlorophene is deposited in the horny layer of human skin and therefore has a cumulative effect, particularly if it is applied in vehicles such as dimethylacetamide(Stoughton 1966). Regular long-continued application is said to be most effective; our finding that the effect of hexachlorophene on the bacterial flora began after a time lag and gradually increased supports this view. Previous reports of a reduction of the pathogenic flora of the skin were based of necessity on relatively small numbers of cultures of pyogenic staphylococci. Clinical pyogenic infections were found to be fewer in infants
Preliminary Communication CYTOCIDAL ACTION OF COLCHICINE IN VITRO ON LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKÆMIA
Preliminary data show that the majority of cells in lymphocyte populations isolated from seven patients with chronic lymphocytic leukaemia (C.L.L.) were abnormal in that they were susceptible to the cytocidal action of colchicine in a concentration (10-7 M) which is at least one thousand times less than that required to affect similarly lymphocytes isolated from healthy people. This finding indicates that the in-vitro use of colchicine may be a valuable means of screening lymphocyte populations circulating in C.L.L. before, during, or Summary
after various forms of treatment. INTRODUCTION
HETEROGENEITY of circulating lymphocytes in a number of patients with chronic lymphocytic leukaemia (C.L.L.) has been demonstrated by the fractionation of separated lymphocytes on a column of polystyrene beads.1 The existence of abnormal column-adherent populations of cells and of non-adherent populations with properties more like those from healthy individuals was observed. The results were interpreted as meaning that separation of normal from abnormal cells had been partly but never completely achieved owing to the failure of some abnormal cells to adhere to polystyrene. Cells from post-column populations had a greater capacity for survival than did gross (pre-column) populations in the 3-day cultures acting as controls for those inoculated with phyto1.
Thomson, A. E. R., Robinson, 1966, ii, 200.
M. A.,
Wetherley-Mein,
G.
Lancet,
bathed in hexachlorophene when these were observed over long periods. The overall reduction of non-pathogens in hexachlorophene-treated babies in our trial clearly indicates the effect of this substance, but several questions arise. The clinical significance of suppressing the growth of non-pathogens on the skin is uncertain. If, in fact, this is associated with a diminished tendency to clinical infection, is there likely to be a rebound when the applications are stopped ? Gezon et al. (1964) reported no clinical evidence of a rebound during a follow-up period of 3 weeks after hexachlorophene had been applied for 3 weeks. In a trial of phisohex for the treatment of acne, McLean et al. (1962) reported that 3 of their 67 patients found that hexachlorophene produced erythema and pruritus. We found no objective side-effects, but this possibility must also be borne in mind. This work was supported by a grant from the William Shepherd bequest of the endowment fund of the Royal Free Hospital. We thank the consultant obstetricians and nursing staff of the Royal Free Hospital for their help with this work. Requests for reprints should be addressed to I. S. REFERENCES
Anderson, (1962) Med. J. Aust. ii, 463. Farquharson, C. D., Penney, S. F., Edwards, H. E., Barr, E. (1952) Can. med. Ass. J. 67, 247. Gezon, H. M., Donovan, J. T., Rogers, K. D., Hatch, T. F., Taylor, P. M. (1964) New Engl. J. Med. 270, 379. Gillespie, W. A., Simpson, K., Tozer, R. C. (1958) Lancet, ii, 1075. Kjellander, J., Nygren, B. (1962) Nord. Med. 67, 79. Knights, H. T., Harvey, J. (1964) N.Z. med. J. 63, 653. McLean, I. E. D., Graham, K. T., East, M. O. (1962) Practitioner, 189, 82. Sarkany, I., Gaylarde, C. (1967) Lancet, i, 589. Stoughton, R. B. (1966) Archs Derm. 94, 646. K.
haemagglutinin (P.H.A.). At the same time, the mortality in cultures of pre-column cells was regarded as surprisingly high and was inconsistent with levels reported by Schrek2 for cultures lasting 7 days. We have now found that this is due partly to a selective cytocidal action on abnormal lymphocytes of the alkaloid, colchicine (10-7 M). This had been incorporated into the culture medium to arrest P.H.A.-induced mitosis and was, consequently, also present in control cultures. The colchicine action is shown to apply to both column-non-adhesive and columnadhesive abnormal cells and permits assessment of the abnormal-cell content of lymphocyte populations in C.L.L. PATIENTS, MATERIALS, AND METHODS Seven patients, in whom the diagnosis of C.L.L. had been established for between 1 month and 91/2 years, were investigated. Four patients (cases 1-4) had received or were receiving various forms of chemotherapy and three (cases 6, 8, and 9) had not been treated. No patient had been given radiotherapy. Freshly withdrawn venous blood was subjected to the same method of fractionation as described elsewhere. 13 Wherever possible, however, the column of polystyrene beads was equilibrated at 37°C in cell-free serum-gelatin mixture (in place of the buffered balanced salt solution) before loading with cells suspended in serum-gelatin. With this modification the consistently high yields from columns of lymphocytes from healthy donors (see Results) indicated that adherence to polystyrene of normal lymphocytes in the c.L.L. populations would be largely prevented (this being the reverse situation to that obtained with a protein-free suspending medium 4). Lymphocyte cultures were set up (1000 cells per c.mm.) essentially as before, with autologous serum always being used in preparation of the culture medium and colchicine being omitted until later, when it was added in solution by micro’
2. Schrek, R. J. natn. Cancer Inst. 1964, 33, 837. 3. Thomson, A. E. R., Bull, J. M., Robinson, M. A. Br. J. Hœmat. 1966, 12, 433. 4. Thomson, A. E. R., Robinson, M. A. Rep. imp. Cancer Res. Fund, 1966, 64, 95. Published 1967.
869 CYTOCIDAL ACTION OF COLCHICINE ON LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKaeMIA
*
Cases also previously investigated by Thomson et al.l t Column was equilibrated in serum before use (see Materials and Methods). t Duration of 1-day cultures varied between 19 and 21 hours after adding colchicine, gassing with 5 % carbon dioxide, and placing in carbon-dioxide incubator. § With 10-6 M and 10-4 M colchicine, figures were 12% (4 experiments) and 13% (3 experiments), respectively. Figures in parentheses show number of experiments.
pipette to individual cultures. These were removed from the 5% carbon-dioxide incubator after 1 day (19-21 hours) and 3 days (69-71 hours), when pyknotic counts were performed on wet-fixed (susa), hasmalum-stained smears prepared by the Trowell5 procedure. The purity of the colchicine used (L. Light & Co. Ltd., batch 2976) was checked by examination of its physical properties (melting-point, and ultraviolet absorption at 350-5 and 243 m(l.). RESULTS
The results from representative experiments are summarised in the table. The mortality in pre-column C.L.L. populations (representative of lymphocytes in defibrinated blood) after 1 day in vitro and in the absence of colchicine (column a) varied, but was generally much greater than for lymphocytes isolated from healthy individuals and cultured under similar conditions. In the presence of 10-7 M-colchicine (column c) the mortality was raised substantially to a level which exceeded 80% in all except two cases. By the same criteria, lymphocytes from healthy individuals were, in contrast, comparatively unaffected by 10-7 M colchicine and were even unaffected by 10-5 M or 10-4 M colchicine. During 2 further days in vitro, few more pre-column cells died but a noticeable increase in mortality of lymphocytes from healthy individuals occurred where colchicine (10-7 M) was present. In comparison with a range in yield from polystyrene columns (pre-equilibrated in serum) of 80-100% for lymphocytes from seven healthy donors, abnormally low proportions of populations from cases 1, 2, 6, and 9 were recovered. In three of these (1, 2, and 6), the post-column cells displayed capacities for survival much nearer normal in the presence of colchicine, as judged by reductions in
(columns c and d). lymphocytes were separated from heparinised blood (and, therefore, were representative of the circulating population) no striking difference in lymphocyte mortality were recorded. This was confirmed by similar (unpublished) experiments where heparin was also used. pyknotic
count
Where
5.
Trowell,
O. A.
Expl Cell Res. 1955, 9, 258.
Recovery of cells measured by hxmocytometer exceeded 90% for almost every culture. Bearing in mind the error in the determinations (within ±10%), individual values within the set for each experiment were close enough to suggest that no greater losses of cells had occurred from cultures giving higher pyknotic counts. DISCUSSION
Colchicine was cytocidal in the concentration 10-7 M for the majority of lymphocytes in populations isolated from all the cases of C.L.L. The fact that lymphocytes from healthy individuals were resistant to at least one thousand times this concentration during the same period (1 day) in vitro shows that the sensitive lymphocytes circulating in the patients were abnormal cells (or normal cells which do not, or not to any extent, normally circulate). Their differential sensitivity strongly recommends the use of colchicine in vitro in low concentration as a means for determining the relative proportions of abnormal and normal cells in c.L.L. which circulate before, during, and after treatment. In this connection, two basic requirements should be met to obtain reliable results: the concentration of alkaloid used in a stipulated culture period should be sufficient to kill all abnormal lymphocytes and not any normal lymphocytes, and the population tested should be representative of the population circulat-
ing in vivo. Experiments with varying concentrations of colchicine6 have indicated that the use of 10--7 M colchicine for approximately 20 hours satisfies the first requirement reasonably well for the cases described here, despite the variable mortality in the absence of colchicine for the different cases. However, the second requirement is not met where defibrination is used to prevent clotting, because of accompanying losses (trapping) of lymphocytes. Nevertheless, the table indicates a crude inverse relationship between circulating-white-blood-cell count and proportion of ostensibly normal (colchicine-resistant) lymphocytes calculable from column c, with patients 3 and 8 occupying the extreme positions. The preliminary 6.
Thomson, A. E. R., Walker,
A. F.
Unpublished.
870 results showing that mortality of lymphocytes cultured without colchicine was no higher after their separation from heparinised blood imply that heparin might successfully replace defibrination in a colchicine-sensitivity test. The polystyrene column permits subclassification of colchicine-sensitive lymphocyte populations into those which are predominantly adhesive and those which are predominantly non-adhesive. For populations in the first category, the column may also permit separation of colchicine-resistant lymphocytes in partially purified form and thereby potentially make for more reasonable comparison of their properties with, lymphocytes from healthy individuals. For example, a sevenfold purification of colchicine-resistant cells was achieved by columntreatment of lymphocytes from patient 2. The column further provides a potential means of obtaining abnormal, adhesive cells in pure form for other studies. These investigations will be described more extensively elsewhere together with results from other experiments performed on the same cases. In particular, the results will be discussed in relation to clinical, haematological and
therapeutic data. We thank Prof. G. Wetherley-Mein, department of hasmatology, St. Thomas’s Hospital, London S.E.I, for providing the cases for study and for the considerable interest he has shown.
Cytochemistry Section, Division of Chemistry and Biochemistry, Imperial Cancer Research Fund, London W.C.2
A. E. R. THOMSON St. And., PH.D. Lond MARY A. ROBINSON
B.SC.
B.SC.
Lond.
Reviews of Books
Principles and Practice of Obstetric Analgesia
and
Anesthesia Vo/. Vol. of
7. Fundamental Considerations. 1.
BoNiCA, M.D., JOHN J. BONICA,
pro-
departmentof anesthesiology,University fessor Washington and chairman, Pp. Philadelphia: Publications.
School of Medicine. Oxford: Blackwell Scientific F. A. Davis Co. 1967. 837.
E9 15s.
’
Professor Bonica’s first volume (the second is to appear next is devoted to fundamental considerations for the anxsthetist working in an obstetric unit. The book deals extensively with the physiology and psychology of pregnancy, and includes detailed information on placental function and the transfer of anavsthetic and allied drugs to the foetus together with discussion of the bearing of this on the anaesthetic management of the individual case. Professor Bonica is a wholehearted advocate of the highest possible standard of obstetric anaesthesia—which, he believes, should always be in the hands of an anaesthetist who not only is expert in his specialty but also has a full background knowledge of obstetric physiology. This is an essential reference book for all obstetric and anaesthetic
year)
departments. Current
Perspectives
in
Surgery
Editors: WILLIAM S. BLAKEMORE, M.D., professor of surgery, University of Pennsylvania School of Medicine, Philadelphia; L. KRAEER FERGUSON, M.D., professor of surgery, University of Pennsylvania School of Medicine. London and New York: 1967. Pp. 317. E98.; §13-50. Harper & Row.
THIS book is one of a series on current perspectives in by contributors drawn from university medical schools in the U.S.A. Each section is followed by verbatim reports of informal panel discussions by the various authors. There is an interesting report on the location of gastrointestinal bleeding by mesenteric arteriography-a method of some marginal promise in obscure bleeding. surgery
Regional Cerebral Blood Flow Delayed Hypersensitivity J. L. TURK, director of M.R.C. Research Group on the Immunological Aspects of Dermatology, Institute of Dermatology, University of London. Amsterdam: North-Holland Publishing Co. 1967. Pp. 252. 80s. THE subject for this volume, the fourth of a series entitled Frontiers in Biology, is well chosen, since work on delayed hypersensitivity is in an interesting phase of development. Dr. Turk is well qualified to write such a book, since he has made many important original contributions. He has produced a treatise which is comprehensive, detailed, and well arranged. Having defined delayed hypersensitivity itself, Dr. Turk goes on to discuss several other topics, including
immunological unresponsiveness, homograft reactions, autoimmunity, and the possible roles of various cells and antibodies, in relation to the central theme. The book should appeal, not only to experts, but also to clinical and experimental workers with interests in immunology, hasmatology, and pathology. A
Surgeons’ Guide
to
Cardiac
Diagnosis
Part II:The Clinical Picture. DONALD N. Ross, B.SC., M.B., F.R.C.S., consultant thoracic surgeon, Guy’s Hospital, London. Berlin, Heidelberg, New York: Springer Verlag. 1967. Pp. 88. DM z6.
THIS remarkable short book covers not only the essentials in the diagnosis of congenital and acquired heart-disease that may be treated by surgery but also the principles of treatment. It defines in clear and precise terms the anatomy, haemodynamic clinical features, special investigations, differential diagnosis, and principles of treatment of each type of cardiac lesion. Where necessary, clear illustrations and excellent reproductions of electrocardiograms and X-rays are included. Inevitably explanations of complex physiological disturbances are simplified for the sake of clarity; but this detracts in no way from the practical value of the book. Although written primarily for the cardiac surgeon, this work can be read with profit by all interested in the diagnosis and surgical treatment of heartdisease. Mr. Ross is to be congratulated.
The Intra-Arterial Injection Method. KAI HfdEDT-RASMUSSEN. Copenhagen:Munksgaard. 1967. Pp. 79. Danish Kroner 25.
THE method of estimating cerebral blood-flow described in this monograph is to inject into the internal carotid or vertebral artery the diffusible, physiologically inactive, gammaemitting isotope 83krypton or 133xenon, both of which are almost completely eliminated by one passage through the lungs. An extracranial scintillation detector records the clearance curve, which resolves itself into two mono exponential curves from which, with due corrections, the flow in grey and white matter is separately calculated. The method entails very low exposure to radioactivity, and can be combined with angiography, but it does involve an injection. Whether an injection or an inhalation method, or both, will finally be in routine use remains to be seen, but this account will be of considerable value in the assessment. The resolution of the results into flows through grey and white matter is an interesting innovation. The mathematical formulas for calculating flow from the extrapolation of an exponential part of a curve are stated but not explained-the mathematical background has to be accepted, understood, or consulted elsewhere-but the clinical and anatomical details are spelled out in detail. The translation reads as though the work had been written in
English. New Editions Bedside Medicine.-2nd ed. By I. Snapper and Alvin I. Kahn. London: Heinemann Medical Books. New York: Grune & Stratton. 1967.
Pp. 824. E7; §18.75.
Textbook of Anatomy.-2nd ed. By W. Henry Hollinshead. New York and London: Harper & Row. 1967. Pp. 994. 18.75; E7 4s. Diseases of the Ear.-2nd ed. By Stuart R. Mawson. Edward Arnold. 1967. Pp. 554. E5.
London:
Techniques in Blood Grouping: Vols. I and n.-2nd ed. By Ivor Dunsford and C. Christopher Bowley. Edinburgh: Oliver & Boyd. 1967.
Pp.
534. E5 5s.