CELL PARTITION

1336 solution of a mixture of waterat sufficient concentration, two or more immiscible but equilibrated phases separate out, each rich in one component polymer. This separation phenomenon is little affected by the presence of solutes or particles, and phases may be controlled with regard to pH and tonicity. Charged species, as either solutes or suspended particles, distribute (partition) between the phases, or between one phase and the interface between phases, according to relationships formulated by Albertsson.3The partition coefficient, K, will vary with the surface charge on the particles, and because of the exponential nature of the relationship, it may vary considerably in an appropriate buffer and polymer system. The partition principle has found many applications, especially through the work of Walter and his colleagues : for instance, peripheral-blood leucocytes may be effectively separated into subpopulations by repeated partitions in an automatic counter-current distribution apparatus.4 We have used an established polymer system to detect changes in surface charge caused by antigeninduced lymphocyte supernatants on guineapig oilinduced peritoneal-exudate cells, and we have applied this to cancer diagnosis. In

Preliminary

Communication

CELL PARTITION A

Simple Test for Lymphocyte Sensitisation J. J. SMITH

J.

P. DICKINSON

University Department of Radiotherapy, Regional Radiotherapy Centre, Cookridge Hospital, Leeds LS16 6QB An established method of investigating total surface charge on particulate materials has been applied to the detection of changes in surface charge on macrophages which are induced by supernatants from the interaction of sensitised lymphocytes and appropriate antigen. The method was studied as an alternative to the established but problematical macrophage-electrophoretic-mobility (M.E.M.) test for detecting such changes. This preliminary investigation suggests that the results from subjects with and without malignant disease differ significantly.

Summary

INTRODUCTION

IN 1970 Field and Casparyl proposed a diagnostic test for cancer. This test required the observation of the electrophoretic mobility of a macrophage subpopulation (M.E.M. test) of guineapig oil-induced peritoneal-exudate cells which had been exposed to a mixture of myelin basic protein and peripheral-blood lymphocytes from a test subject. Several drawbacks to the test have become apparent. Although an operator may be trained in 4 mo to obtain accurate results,2 our own experience, and that of others, is that good results may not be obtained for many months.

a

PATIENTS

patients admitted for treatment of malignant disthe Regional Radiotherapy Centre, Cookridge Hospital, Leeds, took part in the study. They had received no therapy for 4 wk before the test. Patients attending Airedale General Hospital with non-malignant conditions and laboratory staff acted as controls. 14

ease to

METHODSS

:

1.

Carter, J. N., Eastman, C. J., Corcoran, J. M., Lazarus, L. Lancet, 1974, ii, 972. 2. Bermudez, F., Surks, M. I., Oppenheimer, J. H. J. clin. Endocr. Metab. 1975, 41, 27. 3. Burger, A., Nicod, P., Suter, P., Vallotton, M. B., Vagenakis, A., Braverman, L. Lancet, 1976, i, 653. 4. Burr, W. A., Griffiths, R: S., Black, E. G., Hoffenberg, R., Meinhold, H., Wenzel, K. W. ibid. 1975, ii, 1277. 5. Brandt, M., Kehlet, H., Hansen, J. M., Skovsted, L. ibid. 1976, i, 491. 6. Chopra, I. J., Williams, D. E., Orgiazzi, J., Solomon, D. H. J. clin. Endocr. Metab. 1975, 41, 911. 7. Burr, W. A., Ramsden, D. B., Griffiths, R. S., Black, E. G., Hoffenberg, R., Meinhold, H., Wenzel, K. W. Lancet, 1976, ii, 58. 8. Brandt, M., Kehlet, H., Binder, C., Hagen, C., McNeilly, A. S. Clin. Endocr. 1976, 5, 107. 9. Engquist, A., Brandt, M. R., Fernandes, A., Kehlet, H. Unpublished. 10. Nistrup Madsen, S., Brandt, M. R., Engquist, A., Badawi, I., Kehlet, H. Un-

published. 11. Siersbæk-Nielsen, K. Acta med. Scand. 1967, 181, 327. 12. Skovsted, L. Unpublished. 13. Hansen, H. H. Ugeskr. Lœg. 1964, 126, 1471. 14. Kehlet, H., Binder, C., Engbæk, C. Acta endocr., Copenh, 1974, 75, 119. 15. Oyama, T., Matsuki, A., Kudo, T. Anæsthesia, 1972, 27, 2. 16. Kirby, R., Clark, F., Johnston, I. D. A. Clin. Endocr. 1973, 2, 89. 17. Oyama, T., Shibata, S., Matsuki, A. Anesth. Analg. curr. Res. 1969, 48, 1. 18. MacDonald, R. G., Chapman, C., Franklin, H. Br. J. Anœsth. 1976, 48, 225. 19. Oyama, T., Maeda, A., Jin, J., Satone, T., Kudo, M. ibid. 1975, 47, 837. 20. Harland, W. A., Horton, P. W., Strang, R., Fotzgerald, B., Richards, J. R., Holloway, K. B. ibid. 1974, 46, 818. 21. Chopra, I. J., Chopra, U., Smith, S. R., Reza, M., Solomon, D. H. J. clin Endocr. Metab. 1975, 41, 1043. 22. Wilmore, D. W., Long, J. M., Mason, A. D., Pruitt, B. J. Jr. Surgery Gynec. Obstet. 1976, 142, 257. 23. Duick, D. S., Warren, D. W., Nocoloff, J. T., Otis, C. L., Croxson, M. S. J. clin. Endocr. Metab. 1974, 39, 1151. 24. Wandall, J. H. Acta chir. scand. 1974, 140, 171. 25. Sullivan, P. R. C., Bollinger, J. A., Reichlin, S. J. clin. Invest. 1973, 52, 83

(abstr.).

general, when

soluble polymers is made

Lymphocytes from the patients and controls were prepared from fresh venous blood-samples placed in siliconised universal bottles with 10 i.u. preservative-free heparin/ml of blood. Blood was layered on ’Ficoll-Paque’ (Pharmacia) and centrifuged at 500g at the interface for 20 min. The cells collected from the interface were washed twice in Dulbecco ’PBS-A’ and twice in ’TC 199’ (Wellcome Reagents Ltd). Lymphocytes (2 x 106 to 4 x 106 cells) were then incubated in TC 199 for 90 mm at 37°C, alone or with common tumour antigen’ (c.T.A.). Cells were removed by centrifugation, and the supernatants were used immediately or stored frozen at -10°C. A factor (M.s.F,) standard preparation of was made by incubation of lymphocytes with concanavahn-A, 6 as described by Preece and Lights Peritoneal exudates (principally macrophages) were raised by intraperitoneal injection of 20rnl sterile mineral oil (’Marcol 82’, Esso Research, Abingdon, Berks.) into Hartley strain guineapigs and harvested 4-8 days later by peritoneal lavage with Hanks’ balanced salt solution (B.s.s.). The macrophages were washed twice in Hanks’ B.s.s. and twice in TC 199 and given 400r (cobalt-60 radiation) before use. Samples of macrophages (2 x 106 to 4 x 106 cells) were incubated in siliconised screw-cap tubes in TC 199 at 37°C for 90 min, alone or with the addition of lymphocyte supernatants or M.s.F. The macrophages were then partitioned in the twophase aqueous polymer system described by Walter et al.’ The system contains 5% w/v dextran T 500 (Pharmacia), 4CC w/v polyethylene glycol 6000 British Drug Houses), NaCI 0.03 moJ;1, sodium phosphate 0 09 mol/1 at pH 6.8: on standing two phases of approximately equal volume separate out. Each sample of macrophages was washed once and resuspended in 2.0 ml of the upper phase, and a portion was removed for counting. An equal volume of lower phase was added, and the tubes were inverted a few times to allow mixing. The phases

macrophage-slowing

1337 DISCUSSION

These preliminary results indicate the potential of the cell-partition technique in the diagnosis of malignant disease. A double-blind trial, designed to confirm or contradict the preliminary data presented here, and to assess the reliability and accuracy of the assay system, is in progress and will be reported separately. Meanwhile, attention is drawn to this technique as a possible simple

alternative

to

the

M.E.M. test.

This work was made possible by a generous grant from the Yorkshire Cancer Research Campaign to Prof. C. A. Joslin, under whose direction it was carried out. We are grateful to all clinical colleagues who provided access to their patients, and particularly to Dr N. Cowley for selecting and reviewing patients. Requests for reprints should be sent to J. P. D. REFERENCES

Field, E. J., Caspary, E. A. Lancet, 1970, ii, 1337. Pritchard, J. A. V., Sutherland, W. H., Deeley, T. J. ibid. 1976, i, 637. Albertsson, P.-A. Partition of Cell Particles and Macromolecules; p. 15. Stockholm, 1971. 4. Walter, H., Krob, E. J., Ascher, G. S. Exp. Cell Res. 1969, 55, 279. 5. Dickinson, J. P., Smith, J. J., Dyson, J. E. D. Biochem. Soc. Trans. 1976, 4, 125. 6. Preece, A. W., Light, P. A. Clin. exp. Immun. 1974, 18, 543. 1. 2. 3.

Lymphocyte measured

response to common tumour antigen (C.T.A.), the relative change in partition of indicator cells.

as

then allowed to settle for 20 min with the tubes vertical. A part of the top phase was removed and samples taken and tounted. All cell-counts were performed using a model F Coulter counter with a 100 fLm aperture; any contaminating erythrocytes were lysed with ’Zaponin’ (Coulter Electronics,

were

Reviews of Books

Inc.). For particles of total

charge q and surface area A, the distribution or partition coefficient, K, of the particles between bulk phase 1 (total number of particles Np) and the interface between bulk phases 1 and 2 (total number of particles, Ni) is given by4: _ K

Np

ÀA+qV

Ni - exp

where k is Boltzmann’s constant, T temperature, andand V are system constants; V is the electrostatic potential across the interface, and a is a function of the three relevant interfacial tensions (between particles and phase 1; particles and phase 2; and phase 1 and phase 2). For each experiment the partition ratio, K’, is calculated. It is related to K: K’= .

c p2 Npp l+-i NP+N " Cpt

K

Where Cpl and Cp2 are the concentrations of particles in the bulk phase before and after equilibration with the complementary (in this case lower) phase. It is important that no volume

change occurs during re-equilibration. The response-ratio (R.R.) for a particular antigen or preparation is the ratio of K’for macrophages treated with antito K’ for macrophages untreated treated with antigen alone. In practice, only those macrophage preparations which gave an R.R. M.S.F. <,0-90 were considered acceptable. From preparations which gave an R.R. M.S.F. >0.90 all results were discounted. We believe that the likely cause of insensitivity to M.S.F. is some prior sensitisation of the guineapig.

gen-stimulated supernatant or

RESULTS

The response-ratios caused by supernatants from c.T.A. stimulation of lymphocytes from cancer-bearing patients and control subjects were calculated (see figure). As a group, cancer patients gave a significantly lower R.R. C.T.A. (077+0.16, n=14, mean ±S.D.) than control

subjects (1.00±0.1, n=9, P<0.001).

Diseases of the Thyroid DAVID EvERED, M.R.C.P., Royal Victoria Infirmary, Newcastle upon Tyne. London: Pitman. 1976. Pp. 182. £6. account of thyroid physiology, inand disease. Dr Evered emphasises the physiological basis of diagnosis and treatment very clearly in bringing us up to date with T.R.H. and T.S.H. No longer should the mass of hypothyroid patients in the wake of Nature’s ravages, radioiodine therapy, or surgery be given a "conventional" dose of thyroxine, but a dose which reduces serum-T.S.H. to normal, and is usually smaller. The N.H.S. purse is not likely to be a beneficiary, however, since the test is expensive and thyroxine is cheap. There is a well-balanced account of the treatment of thyrotoxicosis and some of its clinical manifestations, hypothyroidism, overt and subclinical, and autoimmune thyroid disease. Few pathologists would concur with frozen-section diagnosis of tumours on biopsy material taken from solitary nodules at operation. The other subjects covered include goitre, with a very full and interesting account of its epidemiology ; and there is a useful chapter on drugs which affect thyroid function. Dr Evered has written a comprehensive and readable book which can be recommended to physicians and surgeons who care for the thyroid. As a work of reference it can be recommended to a wider readership.

THIS is

a

concise, modern

vestigation,

Bronchial Asthma Mechanisms and Therapeutics. Edited by EnxLE B. WEISS, M.D., University of Massachusetts Medical School, and MAURICE S. SEGAL, M.D., Tufts University School of Medicine, Boston.’Boston : Little, Brown. London: Quest. 1976. Pp. 1168.$50; £34.

THIS large reference book aims to bring together in one volume the fundamentals of progress in bronchial asthma in many diverse areas. This it does in seventy-three chapters by 109 authors. The book is divided into seven sections covering

mechanisms, pharmacology, physiology, pathology, aetiology and environment, diagnosis, and treatment. On the whole the chapters are well written by experts and tend to be fairly condensed accounts. For example, chapter 24 on the mast cell is 16 pages long, 6 of which are excellent electron photomicrographs. This leaves 10 pages of text followed by 255 references. On occasion the endeavour to leave nothing out