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The effect of irradiation on human lymphocytes
THE
FA C ULTY VOL. V
THE
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A P R I L , I954
EFFECT
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OF I R R A D I A T I O N
ON H U M A N
No. 4
LYMPHOCYTES
BY J. G. HUMBLE, M.R.C.S., L.R.C.P., W. H. W. JAYNE, F.R.C.S., R. J. V. PULVERTAFT, O.B.E., M.D., F.R.C.P., AND C. W. WILSON, M.Sc., Ph.D., F.Inst.P. WESTMINSTER MEDICAL SCHOOL~ LONDONj S.x~V.I
MANY mammalian cells may be maintained alive on serum-agar, or on serum-agar diluted with physiological salt solutions. The period for which they survive varies greatly, within hours, days, or weeks ; for example, well-differentiated columnar epithelial cells survive only for hours, erythroblastic cells for days, while poorly differentiated malignant cells survive for weeks. Some further details of this technique have been given in previous communications (Pulvertaft, 1952 ; Pulvertaft, Wilson, and Jayne, 1953) and a technique basically similar has been employed in embryological studies by Wolff and Haffen (1952). Success with many tissues is unpredictable; but certain tissues invariably yield thriving preparations. Foremost among these are lymph-glands and bone-marrow, and in both cases both normal and pathological material are equally suitable.
TECHNIQUE Three per cent agar is prepared in distilled water. We use N O B L E agar (B.D.H.) which is specially purified for microbiological assay, but in fact have found it no better for our purposes than bacteriological agars. This is melted down and added in the proportion of one part to three of a mixture of equal parts of human serum and physiological salt mixture. Here, again, we find no particular merits in different sera or different salt mixtures. In fact we use normal human pooled sera from pregnant women, forwarded for routine Wassermann tests, and Hanks' salt mixture because it can be autoclaved and lasts for six months. Opaque or hmmoglobinized sera are of course discarded. Enough penicillin is added to give io units per c.c. of the final product. It will be seen that our objective has been to provide as far as possible solidified human serum, diluted with a physiological salt solution rich in glucose. We would prefer to have used the essential amino-acids and trace substances, such as vitamins, but up to the present such mixtures have not succeeded in our hands. Small cubes of agar are cut from a poured plate of this medium ; we use a platinum spade since many metals injure cells ; stainless steel, however, seems harmless. T h e cubes are mounted on sterilized microscope slides, and at the end of each side a pillar of soft paraffin is placed, just higher than the agar surface. The lymph-glands are selected from material removed in human gastrectomies ; we will confine our description in this case to the examination of this material. After removing fat the gland is bisected, and placed in a Petri dish. It is then crushed with a scalpel, and moistened slightly with the serum-salt solution mixture. A creamy pulp is soon prepared. This pulp is touched with a platinum wire, which is then applied to the agar cube. It is important not to inoculate too richly ; if this is done the cells survive for a shorter period. z6
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A clean sterile cover-slip is placed on the four paraffin pillars, and pressed gently down until the agar is seen to flatten slightly. If too large a gap is left between cover-slip and agar the cells will often remain in clumps, and be too thick for cytological study ; if too much pressure is applied the agar will crack. A space filled with fluid about 3 ° ~z deep exists even when the agar is flattened, and in this space the cells move. The edges of the cover-slip are then filled in with hard paraffin, care being taken to secure a tight seal. The preparations are placed in an incubator at 37 ° C., and examined after about an hour. We use a microscope mounted in a perspex incubator, but small mechanical stage incubators will serve for short examinations ; their temperature is, however, very uncertain. In any case it is essential to examine warm preparations. We have used throughout the phase-contrast microscope of Cooke, Troughton, and Simms, and in particular the × 45 fluorite immersion lens, with a mercury green filter and solid source lamp. We have found no entirely satisfactory lamp on the British market.
NORMAL APPEARANCES For a generation subjugated to fixed and stained preparations of cells it always comes as a surprise to discover that all living cells are in constant motion, and that the spherical form associated both in smear and section with all leucocytes and many ' fixed ' tissue ceils is representative only of the cell in a state of defence. Craigie, Lind, Hayward, and Begg (1951) and Craigie (1952) have drawn attention to cellular pleomorphism by introducing the term ' paramorph ' for a certain aspect of cellular form to which they attribute importance in the study of malignancy. It is, however, essential to recognize that all cells respond to certain stimuli, most if not all potentially nocuous, by changes in form. T h e simplest stimulus for study is lowering of temperature. When cells from lymph-glands prepared as above are examined at room temperature, and with a heat filter in the light beam, they are all found to be motionless and spherical. If sufficiently chilled the cytoplasm is condensed down on the nucleus so as to be invisible ; and thus all the cytoplasmic organoids, such as mitochondria and lipoid granules, are directly applied to the nucleus, and cannot be separately identified. This characteristic might well be borne in mind when studies are made of the ultraviolet light absorption of nuclei ; in many cases the absorption obtained will include that of the cytoplasm and its varied content. Not only do individual cells adopt spherical form when submitted to cold and trauma, but so also do cellular aggregates ; groups of lymphocytes, for example, clump together in tight spheres. This spherical form, presenting the smallest surface area, is obviously suitable for limiting the diffusion of crystalloids and gases in both directions, and is likely to minimize metabolic processes. Certain cells, e.g., well-differentiated columnar epithelial cells, while retracting their cytoplasm, retain nuclear form ; but all the cells found in lymph-glands, except secondary deposits of well-differentiated carcinoma, when first examined on agar are seen to be spheres. As soon as the heat filter is removed from the light beam cytoplasmic motion begins. Of course, in an incubator motion is visible as soon as the preparation is examined. Lymph-glands from gastrectomy cases are likely to be functionally active, since they drain, if the surgical diagnosis is correct, an inflamed area. Thus many different cells are seen ; lymphocytes, polymorphs, eosinophils, and plasma cells are soon recognized as their form is familiar from the study of stained preparations. Certain cells, however, cannot in our opinion be placed in categories likely to be acceptable to all cytologists. We shall refer to them as endothelial cells, well recognizing that more than one type of cell is involved and that in diseases of the reticulo-endothelial system cells are present which are not represented in normal lymph-glands.
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In certain cases we have examined lymph-glands invaded by secondary deposits of carcinoma. These will not be discussed in detail,; but a remarkable and consistent feature of carcinomatous cells, wherever found, is that they tend to remain attached to each other. The groups retract into spheres when chilled, and open out into sheets when warmed ; the cytoplasm of cells expands and retracts rhythmically. But they retain in general cohesion; only occasionally does a cell become detached and wander off, and only for a short distance. The cytoplasmic waves, however, cause the masses of cells to move very slowly and erratically. This cohesion is in marked distinction to the incessant to-and-from and centrifugal motion of the normal cells from lymph-glands. There is no such cell which does not show movements of translation, deformation, and internal re-arrangement of organoids ; and in many cases the nucleus shares independently in these changes, as its nucleolus changes form. All the cells of lymphglands move on the agar, not, as certain other cells move, on the cover-slip. The commonest cell found is the lymphocyte. Its nucleus has a characteristic ' watered silk ' appearance, since it has many areas which are dark with phase contrast. It moves characteristically in the shape of a pear, trailing its stalk behind it. T h e leading edge of the cytoplasm is clear and is protruded now on one side, now on the other, so that its progress is waddling, but not apparently purposeless. The lymphocyte consistently moves alongside cellular aggregates, particularly of malignant cells, and constantly tends to get caught up on or even inside the other cells. The trailed stalk of the lymphocyte contains all the organoids, which are minute and very dark to phase contrast ; when first inoculated they are optically spherical. But in the course of days appearances change greatly. The cytoplasm is greatly increased in area, and very long filamentous mitochondria are seen. Such cells recall clearly the drawings in text-books of cytology representing ideal cells ; but they appear in our preparation to be effete and unhealthy survivors. Typical lymphocytes have never been seen in mitosis. In common with all other cells they tend to extrude and loose small portions of their cytoplasm. Polymorphs move as flattened sheets when applied to the agar ; until applied to it they move with a fine pointed gimlet-like process. Their many fine granules oscillate violently at the leading edge; the trailing edge granules are motionless. When dead they become spherical and all the granules oscillate. " Eosinophils are readily recognizable by their large granules (Fig. 26o A). T h e y are the first cells to move on warming, and react first to fresh environments, e.g., transfusion of fresh fluids under the cover-slip. They have never been seen to phagocytose bacteria or anything else. Plasma cells in these slide preparations retain the eccentric position of their nuclei. T h e y show mitochondria in the form of fine bacillary rods arranged in a V form, of which the nucleus forms the base. They have never been seen to show movements of translation and survive poorly. The remaining cells, which we have called endothelial cells, form a group which as yet we are not prepared to classify (Fig. 260 B, C). One type is common ; it has a nucleus many times the volume of a lymphocyte, often reniform, and this nucleus has usually three spherical dark areas or nucleoli. It moves relatively sluggishly, and its volume in its cytoplasm is rich in organoids, which are very dark. This cell is scarce in resting lymph-glands, but is present in large numbers in glands draining inflamed areas. This cell contrasts clearly with another, often seen when carbon pigment is present, for the carbon is in an extremely tenuous cell, with the most pallid of nuclei which spreads out its cytoplasm on being warmed without showing movements of progression ; and this cell quickly dies. When first examined ' active ' lymph-glands show many cells in mitosis. These are sensitive to light, and can only be examined intermittently ; but if so examined divide successfully. Sometimes for several days mitosis continues. The cells Which mitose do not form lymphocytes, but appear to belong to the endothelial class.
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F o r at m o s t 7 2 h o u r s t h e s e s l i d e p r e p a r a t i o n s m a i n t a i n v e r y m u c h t h e a p p e a r a n c e s e e n w h e n they are first set up, but sooner or later they change greatly. The lymphocytes show spontaneously all t h e a p p e a r a n c e s w h i c h d e v e l o p s p e e d i l y f o l l o w i n g i r r a d i a t i o n ; t h e p o l y m o r p h s d e v e l o p v a c u o l e s and larger and larger granules ; the eosinophils become enormous and burst ; the ' endothelial '
A
B
C
D
E
F
G
H
KEY
A, Eosinophil. B, Mitosis in cell of reticulo-endothelial series. C, Reticulo-endothelial cell with lymphocytes. D, Lymphoeytes immediately after inoculation. The remaining illustrations are all of the same field after I78 r radiation. E, Lymphocytes 3o minutes after radiation. F, Four hours after radiation. Early cyst formation. G, Six hours after radiation. Three well-formed cysts, bl Eleven hours after radiation. Much debris and degeneration. Key Cyst with well-marked gradule. Fig. 26o.-=Living cultures, phase contrast. ( × 65o.)
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cells cease to move; elongated fibroblastic cells are found, and others with enormous spherical inclusions of uniform size. Meanwhile debris accumulates, and after about seven days, as a rule, little more motion is seen. Bone-marrow and malignant-cell preparations are much more hardy and may survive a month or more ; but the lymph-gland has at most 7z hours during which it maintains its appearances on inoculation. This, however, contrasts very favourably with the two hours during which preparations sealed up without agar survive. This method is admirably adapted to the examination of living cells, but is not, in its present form at least, in any way to be compared with tissue culture as a method of obtaining or maintaining a line of cells which reproduce themselves. The soft agar bed prevents the rupture of cells, which can thus be safely flattened. The relatively large volume of serum agar maintains an adequate reserve of oxygen and of the nutrient material. It is of course true that cells do not normally exist between glass and agar surfaces ; many extraordinary features of cytoplasmic behaviour which we have noted may be due to this fact. It is true, too, that phase contrast, while avoiding the pitfalls of older histological methods, introduces optical artefacts, for indeed what we see is merely that aspect of the whole which our limited equipment reveals, and the cytoplasmic organoids which are recognizable may be only a few of those present, but not optically differentiated. It is possible that the interference microscope might succeed in the further analysis of cytoplasmic structures ; at present there is plenty of work for those willing to study their amazing variety by phase contrast. All experiments on these slide cultures have been recorded by cinemicrography, using the timelapse technique. In this way motion or change of any kind is accelerated at will ; in the case of preparation of lymph-glands, where the cells move rapidly, the acceleration has been of the order of sixteen to forty times that of the actual motion. The ease with which the isolated cells of lymph-glands could be kept alive suggested the possibility of the analysis of the effect of gamma radiation on them, since all authorities are agreed on the importance of the lymphocyte in particular as an indicator of radiation damage. Early studies of this were reported by Senn (i9o 3 a, b) and Heineke (i9o4, I9o 5 a, b). Lazarus-Barlow (i92o) found, after 15 hours' treatment of animals with 5 g. of radium bromide, that very few lymphocytes remained in the blood, lymph-glands, or spleen. Lacassagne and Fricouroff (i927) noted cessation of migration of lymphocytes from fragments of spleen, thymus, and lymph-gland, in hanging-drop preparations after the application of X rays. Schrek (i946 , I947 a, b, i948 ) gave a very complete study of thymic cells treated with iooo r from X rays and subsequently examined by dark-ground illumination. More recently Trowell (r952) has reported on the changes found following the radiation of isolated lymph-glands maintained on cotton-wool. While in most cases animal and not human lymph-glands, as in our case, have been studied by other authors, it is clear that their results are very similar to ours. As, however, abnormal lymphglands, including those from the reticuloses, can be readily examined by these means, this technique may well have wide applications. RESULTS OF IRRADIATION
Slide preparations from 20 human lymph-glands were submitted to radiation, 17 from gastrectomy cases, 2 from the axilla, and ~ from the mesocolon ; none of these were invaded by malignant ceils. Six preparations were made from each, and incubated overnight at 37 ° C. so that the cells might spread out as far as possible ; if cells are irradiated before they spread out and become motile they Often fail to do so. Varying doses of radiation were employed, from 3° r to 712 r, irradiation being always at 37° C. ; some preparations were then incubated for further periods and examined intermittently, while others were continuously examined and photographic records taken by time-lapse cinemicrography. Twenty-four hours after irradiation 5o0 cells were counted in an attempt to estimate the degree of
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lethal action; but this was found to be quite inaccurate as so much debris existed, and so many cells had completely disintegrated. The dosage required to produce significant changes when compared with controls was surprisingly small. In some cases this resulted from 3° r only; 60 r consistently produced up to 80 per cent of damage. The first changes were noted after a lapse of six hours following radiation, which is in agreement with the latent interval found by Trowell, although Schrek noticed them somewhat earlier. The nucleus becomes darker by phase contrast, and motility is lessened. In the dark areas a cyst develops which corresponds to the primary vacuole of Schrek ; and cytoplasmic detail disappears. Bursting
/
( L
©) L
I
I
0
1
2
I
B CM
Fig, 2 6 I . - - R a d i u m applicator.
of these cysts was not noted, but the vacuolated cells gradually disintegrated, so that the agar was covered by cellular debris and cell ghosts. Not all the lymphocytes were equally sensitive; on the contrary, fully formed cysts were visible and the majority of the lymphocytes were converted to debris while a few moved and appeared exactly like the controls. The large cells of the endothelioid type were far more resistant to radiation than the lymphocytes, and indeed with the dosages used in these experiments neither they nor the polymorphs or eosinophils showed differences from the controls. The changes in the lymphocytes following irradiation are not specific ; they can all be noted, occurring at varying rates, in preparations kept without radiation for seven to nine days, which is the extreme period of survival of the lymphocyte by this technique. Irradiation accelerated the normal rate and stages of death and disintegration (Fig. 260 D-H).
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R A D I U M G A M M A R A Y S O U R C E F O R I R R A D I A T I O N OF C U L T U R E S
A special radium gamma ray source was constructed for the irradiation of the cultures, using several small radium sources which happened to be free. This was designed so that (a) it could be safely handled for short periods and (b) it provided an adequate dose-rate at the cultures which was relatively uniform over a small area. Details of its construction are shown in Fig. 261 A. T h e individual radium sources used were a glass tube containing 2I'35 mg. of radium elements enclosed in a cylindrical leaden container with I - m m . thick wails (a in Fig. 26I A) and 4 × I-mg. radium needles of 8 mm. active length and o. 5 mm. Pt. filtration (b in Fig. 26I A). These five sources were mounted together in a thick cylinder of lead as illustrated, T h e front, emitting face of the source was made of i - r a m , thick brass sheet in order to keep secondary beta radiation at a m i n i m u m (Wilson, i945). F o r irradiation of the cultures, the culture slide was placed between perspex supports of known thickness and the source was placed on the supports (Fig. 261 8). U n d e r these conditions the distance between the front face of the brass secondary filter and the culture was ~ mm. T h e dose-rate at a culture was determined by measurements made with an aerion condenser ionization chamber having a depth of air volume of only i mm. (Wilson, i944). I n order to take Table / . - - A G E
GROUPS
D I S T R I B U T I O N OF A G E G R O U P S I N D E C A D E S SITES
ioo
II 20
Abdomen
2I 3O
31 -4 o
II
II
51
5o
6o
6I 70
5
I
2
8
9
9
6 5
4I
Chest
I
3
Neck
I
2
5
i6
Head
I
m
5
6
5
8
Limbs
5
14
6
I
I
I
Total
6
14
24
23
24
35
:2:Z
71
8o
81 9O
I
I
I
I
account of any secondary radiations from the glass cover-slips carrying the cultures, the wall of the condenser chamber which received the radiation was of thin graphited paper and a glass cover-slip was laid upon this during the measurements. Dose-rate measurements were made at 0"5 mm., I'3 mm., and t'6 mm. from the source, and by interpolation on the curve from these three observations the dose-rate at a culture was found to be 356 r/hr. T h e ionization chamber was calibrated by thickening its front wall up to 3 mm. of aerion and exposing it to an accurately known gamma ray dose-rate. CLINICAL O B S E R V A T I O N S
T h e effects of radiation depend on a n u m b e r of factors ; in the following section, however, the relationship between the part of human patients irradiated and the effect on circulating leucocytes and lymphocytes will be studied. P a t i e n t s a n d M e t h o d s . - - T h e types of case studied in this paper comprise five groups of 3 ° patients. These groups represent irradiation of the abdomen, the chest, the neck, the skull, and the limbs. T h e patients in each group were selected only in that, in no case, were patients with obvious generalized metastases present included and the t u m o u r at the site of irradiation was small (as in the larynx) or had been previously removed (as in the breast and testis). T h e y are referred to below as groups I, II, I I I , IV, V.
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Group I comprised 3 ° males undergoing routine post-operative irradiation with X rays generated at 2oo or 250 kv. for seminoma or teratoma testis. In each the primary tumour had been excised. The irradiation was given as described by Prosser (I95O). These patients received two separate courses of treatment, the first to the lower abdomen and pelvis, followed 6-8 weeks later by a further course to the upper abdomen. Group II comprises 3 ° women undergoing post-operative X-ray therapy; 20o or 250 kv. following radical excision of the breast for carcinoma. The method employed was that of multiple tangential fields. Group III consists of 28 males and 2 females undergoing radium teletherapy (io-g. source) for carcinoma of the larynx and lower pharynx. The methods used are described by Wilson (1951) . Abdomen
Thorax
BEFORE TREATMENT Neck
Skull
Limbs
7"-
20-
I
.£-Yl_ over 9001 6001 3001 0-3000over 9001 6001 3001 0-3000 over 90016001 3001 0-3000over 9001 6001 3001 0-3000over 9001 6001 3001 0-3000 12,000 12,000 9000 6000 12,000 12,0009000 6000 12,000 12,000 9000 6000 12,000 12,000 9000 6000 12,000 12,0009000 6000 -
AFTER TREATMENT
! D
20"i I
I ....
over 90016001 3001 0-3000 over 90016001 3001 0-3000 over 90016001 3001 0-3000 over 90016001 30010-3000 over 90016001 3001 0-3000 12,000 12,00090006000 12,000 12,0009000 6000 12,000 12,0009000 6000 12,000 12,00090006000 12,000 12,0009000 6000
Fig.
262.--The
effect o f a t h e r a p e u t i c c o u r s e o f i r r a d i a t i o n o n t h e t o t a l l e u c o c y t e c o u n t .
Group I V consists of 14 male and i6 female patients treated for a variety of ailments located in the skull, viz., carcinoma of the superior nasopharynx io cases, brain tumours 13 cases, other conditions (all malignant) 7 cases. These patients were treated by a variety of methods, teletherapy using 4 g- and io g. of radium, X-rays of 2oo and 250 kv. and by the 2-MeV. Van de Graaf linear accelerator. Group V consists of 17 male and 13 female patients suffering from lesions of the limbs not involving the trunk. They comprise 19 cases of osteogenie sarcoma, 4 cases of synovioma and other soft-tissue tumours, 6 cases of osteoclastoma, and I case of Ewing's tumour. These again were treated by a variety of methods as in Group IV. Groups IV and V are by no means as homogeneous as Groups I - I I I as regards age and sex. The age and sex of each group are recorded in Table L The blood-counts were made on capillary blood by experienced technicians and by medical men using the same techniques, especially as regard to the method of performing the differential white count. These counts are ' routine ' in that special efforts to obtain increased accuracy beyond
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z35
a single estimation of white-cell count and a differential count of IOO cells were not employed. T h e y are, however, free from 'unconscious bias' (Briggs and Macmillan, i95i ). It is clear that such estimations are subject to considerable degrees of latitude and so deductions from small changes in total leucocyte count and total count of lymphocytes cannot be made. Instead, the figures of each group of patients have been placed in the following ranges : leucocytes 0-3000 per e.mm., 31oo=6ooo, 6zoo-9ooo , 9ioo-12,ooo, and more than zz,ooo. The lymphocytes were similarly grouped : o-2oo, 2oi-6oo, 6 o i - i o o o , IOOi and upwards. The effect of a therapeutic course of irradiation in each group of patients is seen in Figs. 26z and 263. It is seen that lymphopenia and leukopenia are produced in the following order : (I) abdomen, (2) thorax, (3) neck, (4)
Thorax
Abdomen Pelvis
25A
BEFORETREATMENT Neck
Head
Limbs
J
1 0 ~ over 600 200 0 1000 1000 600 200
.__ over 600 200 0 1000 1000 600 200
25
over 600 200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
over 600200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
over 600 200 0 I000 I000 600 200
AFTERTREATMENT
,0. I _
600 200 0 1000 1000 600 200
over
over 600 200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
Fig. 2 6 3 . - - T h e effect of a the r a pe utic course of irradiation on th e total lymphocyte count.
skull, and (5) limbs. Furthermore, when the second course of irradiation follows in group I (the interval being 6-8 weeks) then a further and even greater fall of leucocyte count and of lymphocytes occurs (Fig. 264). The pre- and post-treatment total leucocyte and total lymphocyte counts of the whole I5o cases as grouped in rising intervals of 400 per c.mm. are shown in Fig. 265. It will be seen that there is an overall fall of lymphocytes following therapy, but by comparison with Fig. 263 the effect is most marked when the trunk is irradiated. W h e n the cases were further analysed it was seen that not all the cases show a fall of lymphocytes, and that in fact certain cases in each group show a rise in total lymphocyte count. As these cases received the same treatment as the others it is evident that individual reaction must play a considerable part in the production of the results. These effects are summarized in Table H.
DISCUSSION It is difficult to compare the present series with those previously reported. Kornblum, Boerner, and Henderson (1938) in a study of Ioo cases noted the production of leukopenia and
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BEFORE TREATMENT SECOND COURSE
FIRST COURSE
Leucocytes
Abdomen Lym p h o c y t e s
Leucocytes
Abdomen Lym phocytes
20-
9001 6001 3001 0-3000 12,000 12,000 9000 6000
over
over 6000 200 0 1000 1000 600 200
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 1000 1000 600 200
AFTER TREATMENT 20
.__[ over 9001 6001 3001 0-3000 12,000 12,00090006000
over 600 200 0 I000 I000 600 200
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 1000 1000 600 200
Fig. 2 6 4 . - - T h e effect of a second course of therapy on G r o u p i patients.
BEFORE IRRADIATION Total Leucocytes Total Lymphocytes
AFTER I R R A D I A T I O N
.m over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 I000 1000 600 200
Fig. 265.--All cases (I5O).
.
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
.
.
.
over 600 200 0 I000 1000 600 200
T h e effect of a therapeutic course of irradiation in I5o patients.
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lymphopenia, but were unable to correlate the changes seen in the blood with the site irradiated or the amount of irradiation administered. Goodfellow (i936) studied 6I cases, comprising 3 ° cases of carcinoma of the breast, 13 cases of malignant disease in structures of the neck, and I8 other cases, and was of the opinion that the lymphocyte fall was greater in t h e neck cases. His cases were all treated by radium needle implantation or by surface application of radium. All authorities Table
H.--THE
SITE
B E H A V I O U R OF T H E L Y M P H O C Y T E C O U N T NO CHANGE
RISE
~bdomen Zhest Neck Head Limb
5 6 9 I5
3
2I
I
Fotal
56
Percentage
37"3
THE SITE
I
FALL
150
TREATED CASES
FALL TO LESS THAN 200 C.MM.
22
23 21
I5 8
89 3"3
RISE
14
59"3
B E H A V I O U R OF T H E L E U C O C Y T E C O U N T
N o CHANGE
IN
FALL
9"3 IN
15o
TREATED C A S E S
FALL TO LESS THAN 3000 C.MM.
a.bdomen F.hest Neck Head Limb
13 I6 16 13 9
4 3 4 5
13 13 II 13 16
total
67
I7
66
7
Percentage
44"6
II" 3
44
4'6
I
2
4 I O O
discussing the mechanisms by which these changes are produced, have failed to provide a uniform and consistent explanation. T h e following points may be made, however : - I. It is unlikely that destruction of circulating lymphocytes can be the cause, for in all the cases studied in this paper the circulating lymphocytes must be heavily irradiated yet the fall in skull and limb is minimal. 2. In irradiation of the abdomen, chest, and the neck a considerable amount of lymph-node tissue is irradiated directly and destroyed or profoundly modified by the treatment. This is not so in the skull and the limbs. 3. Patients who have had two separate areas of the abdomen irradiated show a much greater response than do other patients in this series. It is therefore suggested that the lymphopenia following irradiation is due to destruction of lymphocyte-forming tissue. This will explain a finding common to this series and also that of G o o d f e l l o w - - a fall of lymphocytes during the early part of a course of therapy, followed by a later rise even though therapy continues. Here it is postulated that other lymphocyte-forming tissues hypertrophy 'to take over the function of the destroyed tissue and so maintain the status quo in the blood-stream. Both the red bone-marrow and the lymphocyte-forming tissues are much more capable of response to a destructive process such as irradiation than is commonly realized. In the case of the marrow Denstad ( i 9 4 i ) has claimed to show that destruction of one area of red marrow is rapidly compensated for by hypercellularity
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of other areas. It will be noted that lymphopenia and leukopenia as induced by irradiation are not per se dangerous to the patient so long as he retains the ability to hypertrophy the remaining u n damaged lymphopoietic and leucopoietic tissues. When no such areas remain as a result of'previous irradiation or by the concomitant effects of such drugs as the ' chlorethylamines ', urethane, or triethylene melanine, then the leucopenia and lymphopenia remain permanently, witnesses of the poverty of the resources remaining. C O N C L U S I O N S
i. T h e response to therapeutic irradiation of five groups of 3° cases each has been studied. 2. Those groups represent the abdomen , thorax, neck, skull, and the limbs. 3. T h e constant change in the blood of these patients as noted by other authors, viz., lymphopenia and leukopenia, is confirmed. 4. T h e areas showing these changes in order of severity are the abdomen, chest, neck, the skull, and the limbs. 5. A second course to a different area of the abdomen 6-8 weeks following the first course is seen to produce a more serious effect. 6. It is suggested that these effects are produced by destruction or modification of the structures producing the lymphoid cells. 7. Permanent leucopenia and lymphopenia reflects the destruction of large areas of tissue. Our thanks are due to the Medical and Surgical Staff of Westminster Hospital for their generous co-operation: to the department of Medical Photography; and to Mr. J. Haynes for technical assistance. W. H. W. Jayne is working with a full time grant from the Imperial Cancer Research F u n d . T h e expenses of this research were covered by a generous grant from the Governors' Discretionary Fund, Westminster Hospital, and the work of one of the authors (C.W.W. is supported financially by the British Empire Cancer Campaign. REFERENCES BRIGGS, R., and MACMILLAN,R. L. (I95I), Brit. ff. clin. Path., I, 283, 290. CRAIGIE, J. (I952), ft. Path. Bact., 64, 25I. -- - LIND, P. E., HAYWARD,M. E., and BEGG,A. M. (i95i), Ibid., 63, I77. DENSTAD, T. (I94I), Acta radiol., Stockh., suppl., 52. GOODFELLOW, D. R. (I936), Brit. J. Radiol., 9, 695. HEINEKE, H. (I9o4), Miinch. reed. Wschr., 5x, 785. - - - - (I9o5, a), Mitt. Grenzgeb. ]Fled. Chir., x4, 2t. - - - - (I9o5, b), Dtsch. Z. Chit., 78, I96. KORNBLUM, K., BOERNER, F., and HENDERSON, S. G. (I938), Amer. ft. Roegentol., 39, 235. LACASSAaNE,A., and FmCOUROEF,G. (I927), C. R. Soc. Biol. Paris, 96, 862. LAZARUS-BARLOW,W. S. (I92O), Proc. R. Soc. Med., 14, I. PROSSER, T. M. (I95O), Brit. J. Surg., 38, 473. PDLVERTAFT,R. J. V. (I952), Med. Biol., 3, 198. -- - WILSON, C. W., and JAYNE, H. (I953), Nature, Lond., I7I , ii57. SCrIaEK, R. (I946), J. cell. comp. Physiol., 2 8 , 277. - - - - (1947, a), Ibid., 3o, 2o3. - - - - (1947, b), Proc. Soc. exp. Biol., N . Y . , 64, 38I. -- - (I948), Amer. J. Path., 24, lO55. SENN, N. (I9o3, a), N . Y . med. J., 77, 665. - - - - (19o3, b), N . Y . Med. Rec., 64, 28i. TROWELL, O. A. (I952), J. Path. Bact., 64, 687. WILSON, C. W. (I944), Brit. J. Radiol., I7, 86. - - - - (I945), Radium Therapy--Its Physical Aspects. London : Chapman & Hall. (I95I), J. Radiol., 24, 374. WOLFF, E., and HAFEEN, K. (I952), Tex. Rep. Biol. Med., IO, 463.
FA C ULTY VOL. V
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No. 4
LYMPHOCYTES
BY J. G. HUMBLE, M.R.C.S., L.R.C.P., W. H. W. JAYNE, F.R.C.S., R. J. V. PULVERTAFT, O.B.E., M.D., F.R.C.P., AND C. W. WILSON, M.Sc., Ph.D., F.Inst.P. WESTMINSTER MEDICAL SCHOOL~ LONDONj S.x~V.I
MANY mammalian cells may be maintained alive on serum-agar, or on serum-agar diluted with physiological salt solutions. The period for which they survive varies greatly, within hours, days, or weeks ; for example, well-differentiated columnar epithelial cells survive only for hours, erythroblastic cells for days, while poorly differentiated malignant cells survive for weeks. Some further details of this technique have been given in previous communications (Pulvertaft, 1952 ; Pulvertaft, Wilson, and Jayne, 1953) and a technique basically similar has been employed in embryological studies by Wolff and Haffen (1952). Success with many tissues is unpredictable; but certain tissues invariably yield thriving preparations. Foremost among these are lymph-glands and bone-marrow, and in both cases both normal and pathological material are equally suitable.
TECHNIQUE Three per cent agar is prepared in distilled water. We use N O B L E agar (B.D.H.) which is specially purified for microbiological assay, but in fact have found it no better for our purposes than bacteriological agars. This is melted down and added in the proportion of one part to three of a mixture of equal parts of human serum and physiological salt mixture. Here, again, we find no particular merits in different sera or different salt mixtures. In fact we use normal human pooled sera from pregnant women, forwarded for routine Wassermann tests, and Hanks' salt mixture because it can be autoclaved and lasts for six months. Opaque or hmmoglobinized sera are of course discarded. Enough penicillin is added to give io units per c.c. of the final product. It will be seen that our objective has been to provide as far as possible solidified human serum, diluted with a physiological salt solution rich in glucose. We would prefer to have used the essential amino-acids and trace substances, such as vitamins, but up to the present such mixtures have not succeeded in our hands. Small cubes of agar are cut from a poured plate of this medium ; we use a platinum spade since many metals injure cells ; stainless steel, however, seems harmless. T h e cubes are mounted on sterilized microscope slides, and at the end of each side a pillar of soft paraffin is placed, just higher than the agar surface. The lymph-glands are selected from material removed in human gastrectomies ; we will confine our description in this case to the examination of this material. After removing fat the gland is bisected, and placed in a Petri dish. It is then crushed with a scalpel, and moistened slightly with the serum-salt solution mixture. A creamy pulp is soon prepared. This pulp is touched with a platinum wire, which is then applied to the agar cube. It is important not to inoculate too richly ; if this is done the cells survive for a shorter period. z6
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A clean sterile cover-slip is placed on the four paraffin pillars, and pressed gently down until the agar is seen to flatten slightly. If too large a gap is left between cover-slip and agar the cells will often remain in clumps, and be too thick for cytological study ; if too much pressure is applied the agar will crack. A space filled with fluid about 3 ° ~z deep exists even when the agar is flattened, and in this space the cells move. The edges of the cover-slip are then filled in with hard paraffin, care being taken to secure a tight seal. The preparations are placed in an incubator at 37 ° C., and examined after about an hour. We use a microscope mounted in a perspex incubator, but small mechanical stage incubators will serve for short examinations ; their temperature is, however, very uncertain. In any case it is essential to examine warm preparations. We have used throughout the phase-contrast microscope of Cooke, Troughton, and Simms, and in particular the × 45 fluorite immersion lens, with a mercury green filter and solid source lamp. We have found no entirely satisfactory lamp on the British market.
NORMAL APPEARANCES For a generation subjugated to fixed and stained preparations of cells it always comes as a surprise to discover that all living cells are in constant motion, and that the spherical form associated both in smear and section with all leucocytes and many ' fixed ' tissue ceils is representative only of the cell in a state of defence. Craigie, Lind, Hayward, and Begg (1951) and Craigie (1952) have drawn attention to cellular pleomorphism by introducing the term ' paramorph ' for a certain aspect of cellular form to which they attribute importance in the study of malignancy. It is, however, essential to recognize that all cells respond to certain stimuli, most if not all potentially nocuous, by changes in form. T h e simplest stimulus for study is lowering of temperature. When cells from lymph-glands prepared as above are examined at room temperature, and with a heat filter in the light beam, they are all found to be motionless and spherical. If sufficiently chilled the cytoplasm is condensed down on the nucleus so as to be invisible ; and thus all the cytoplasmic organoids, such as mitochondria and lipoid granules, are directly applied to the nucleus, and cannot be separately identified. This characteristic might well be borne in mind when studies are made of the ultraviolet light absorption of nuclei ; in many cases the absorption obtained will include that of the cytoplasm and its varied content. Not only do individual cells adopt spherical form when submitted to cold and trauma, but so also do cellular aggregates ; groups of lymphocytes, for example, clump together in tight spheres. This spherical form, presenting the smallest surface area, is obviously suitable for limiting the diffusion of crystalloids and gases in both directions, and is likely to minimize metabolic processes. Certain cells, e.g., well-differentiated columnar epithelial cells, while retracting their cytoplasm, retain nuclear form ; but all the cells found in lymph-glands, except secondary deposits of well-differentiated carcinoma, when first examined on agar are seen to be spheres. As soon as the heat filter is removed from the light beam cytoplasmic motion begins. Of course, in an incubator motion is visible as soon as the preparation is examined. Lymph-glands from gastrectomy cases are likely to be functionally active, since they drain, if the surgical diagnosis is correct, an inflamed area. Thus many different cells are seen ; lymphocytes, polymorphs, eosinophils, and plasma cells are soon recognized as their form is familiar from the study of stained preparations. Certain cells, however, cannot in our opinion be placed in categories likely to be acceptable to all cytologists. We shall refer to them as endothelial cells, well recognizing that more than one type of cell is involved and that in diseases of the reticulo-endothelial system cells are present which are not represented in normal lymph-glands.
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In certain cases we have examined lymph-glands invaded by secondary deposits of carcinoma. These will not be discussed in detail,; but a remarkable and consistent feature of carcinomatous cells, wherever found, is that they tend to remain attached to each other. The groups retract into spheres when chilled, and open out into sheets when warmed ; the cytoplasm of cells expands and retracts rhythmically. But they retain in general cohesion; only occasionally does a cell become detached and wander off, and only for a short distance. The cytoplasmic waves, however, cause the masses of cells to move very slowly and erratically. This cohesion is in marked distinction to the incessant to-and-from and centrifugal motion of the normal cells from lymph-glands. There is no such cell which does not show movements of translation, deformation, and internal re-arrangement of organoids ; and in many cases the nucleus shares independently in these changes, as its nucleolus changes form. All the cells of lymphglands move on the agar, not, as certain other cells move, on the cover-slip. The commonest cell found is the lymphocyte. Its nucleus has a characteristic ' watered silk ' appearance, since it has many areas which are dark with phase contrast. It moves characteristically in the shape of a pear, trailing its stalk behind it. T h e leading edge of the cytoplasm is clear and is protruded now on one side, now on the other, so that its progress is waddling, but not apparently purposeless. The lymphocyte consistently moves alongside cellular aggregates, particularly of malignant cells, and constantly tends to get caught up on or even inside the other cells. The trailed stalk of the lymphocyte contains all the organoids, which are minute and very dark to phase contrast ; when first inoculated they are optically spherical. But in the course of days appearances change greatly. The cytoplasm is greatly increased in area, and very long filamentous mitochondria are seen. Such cells recall clearly the drawings in text-books of cytology representing ideal cells ; but they appear in our preparation to be effete and unhealthy survivors. Typical lymphocytes have never been seen in mitosis. In common with all other cells they tend to extrude and loose small portions of their cytoplasm. Polymorphs move as flattened sheets when applied to the agar ; until applied to it they move with a fine pointed gimlet-like process. Their many fine granules oscillate violently at the leading edge; the trailing edge granules are motionless. When dead they become spherical and all the granules oscillate. " Eosinophils are readily recognizable by their large granules (Fig. 26o A). T h e y are the first cells to move on warming, and react first to fresh environments, e.g., transfusion of fresh fluids under the cover-slip. They have never been seen to phagocytose bacteria or anything else. Plasma cells in these slide preparations retain the eccentric position of their nuclei. T h e y show mitochondria in the form of fine bacillary rods arranged in a V form, of which the nucleus forms the base. They have never been seen to show movements of translation and survive poorly. The remaining cells, which we have called endothelial cells, form a group which as yet we are not prepared to classify (Fig. 260 B, C). One type is common ; it has a nucleus many times the volume of a lymphocyte, often reniform, and this nucleus has usually three spherical dark areas or nucleoli. It moves relatively sluggishly, and its volume in its cytoplasm is rich in organoids, which are very dark. This cell is scarce in resting lymph-glands, but is present in large numbers in glands draining inflamed areas. This cell contrasts clearly with another, often seen when carbon pigment is present, for the carbon is in an extremely tenuous cell, with the most pallid of nuclei which spreads out its cytoplasm on being warmed without showing movements of progression ; and this cell quickly dies. When first examined ' active ' lymph-glands show many cells in mitosis. These are sensitive to light, and can only be examined intermittently ; but if so examined divide successfully. Sometimes for several days mitosis continues. The cells Which mitose do not form lymphocytes, but appear to belong to the endothelial class.
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F o r at m o s t 7 2 h o u r s t h e s e s l i d e p r e p a r a t i o n s m a i n t a i n v e r y m u c h t h e a p p e a r a n c e s e e n w h e n they are first set up, but sooner or later they change greatly. The lymphocytes show spontaneously all t h e a p p e a r a n c e s w h i c h d e v e l o p s p e e d i l y f o l l o w i n g i r r a d i a t i o n ; t h e p o l y m o r p h s d e v e l o p v a c u o l e s and larger and larger granules ; the eosinophils become enormous and burst ; the ' endothelial '
A
B
C
D
E
F
G
H
KEY
A, Eosinophil. B, Mitosis in cell of reticulo-endothelial series. C, Reticulo-endothelial cell with lymphocytes. D, Lymphoeytes immediately after inoculation. The remaining illustrations are all of the same field after I78 r radiation. E, Lymphocytes 3o minutes after radiation. F, Four hours after radiation. Early cyst formation. G, Six hours after radiation. Three well-formed cysts, bl Eleven hours after radiation. Much debris and degeneration. Key Cyst with well-marked gradule. Fig. 26o.-=Living cultures, phase contrast. ( × 65o.)
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cells cease to move; elongated fibroblastic cells are found, and others with enormous spherical inclusions of uniform size. Meanwhile debris accumulates, and after about seven days, as a rule, little more motion is seen. Bone-marrow and malignant-cell preparations are much more hardy and may survive a month or more ; but the lymph-gland has at most 7z hours during which it maintains its appearances on inoculation. This, however, contrasts very favourably with the two hours during which preparations sealed up without agar survive. This method is admirably adapted to the examination of living cells, but is not, in its present form at least, in any way to be compared with tissue culture as a method of obtaining or maintaining a line of cells which reproduce themselves. The soft agar bed prevents the rupture of cells, which can thus be safely flattened. The relatively large volume of serum agar maintains an adequate reserve of oxygen and of the nutrient material. It is of course true that cells do not normally exist between glass and agar surfaces ; many extraordinary features of cytoplasmic behaviour which we have noted may be due to this fact. It is true, too, that phase contrast, while avoiding the pitfalls of older histological methods, introduces optical artefacts, for indeed what we see is merely that aspect of the whole which our limited equipment reveals, and the cytoplasmic organoids which are recognizable may be only a few of those present, but not optically differentiated. It is possible that the interference microscope might succeed in the further analysis of cytoplasmic structures ; at present there is plenty of work for those willing to study their amazing variety by phase contrast. All experiments on these slide cultures have been recorded by cinemicrography, using the timelapse technique. In this way motion or change of any kind is accelerated at will ; in the case of preparation of lymph-glands, where the cells move rapidly, the acceleration has been of the order of sixteen to forty times that of the actual motion. The ease with which the isolated cells of lymph-glands could be kept alive suggested the possibility of the analysis of the effect of gamma radiation on them, since all authorities are agreed on the importance of the lymphocyte in particular as an indicator of radiation damage. Early studies of this were reported by Senn (i9o 3 a, b) and Heineke (i9o4, I9o 5 a, b). Lazarus-Barlow (i92o) found, after 15 hours' treatment of animals with 5 g. of radium bromide, that very few lymphocytes remained in the blood, lymph-glands, or spleen. Lacassagne and Fricouroff (i927) noted cessation of migration of lymphocytes from fragments of spleen, thymus, and lymph-gland, in hanging-drop preparations after the application of X rays. Schrek (i946 , I947 a, b, i948 ) gave a very complete study of thymic cells treated with iooo r from X rays and subsequently examined by dark-ground illumination. More recently Trowell (r952) has reported on the changes found following the radiation of isolated lymph-glands maintained on cotton-wool. While in most cases animal and not human lymph-glands, as in our case, have been studied by other authors, it is clear that their results are very similar to ours. As, however, abnormal lymphglands, including those from the reticuloses, can be readily examined by these means, this technique may well have wide applications. RESULTS OF IRRADIATION
Slide preparations from 20 human lymph-glands were submitted to radiation, 17 from gastrectomy cases, 2 from the axilla, and ~ from the mesocolon ; none of these were invaded by malignant ceils. Six preparations were made from each, and incubated overnight at 37 ° C. so that the cells might spread out as far as possible ; if cells are irradiated before they spread out and become motile they Often fail to do so. Varying doses of radiation were employed, from 3° r to 712 r, irradiation being always at 37° C. ; some preparations were then incubated for further periods and examined intermittently, while others were continuously examined and photographic records taken by time-lapse cinemicrography. Twenty-four hours after irradiation 5o0 cells were counted in an attempt to estimate the degree of
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lethal action; but this was found to be quite inaccurate as so much debris existed, and so many cells had completely disintegrated. The dosage required to produce significant changes when compared with controls was surprisingly small. In some cases this resulted from 3° r only; 60 r consistently produced up to 80 per cent of damage. The first changes were noted after a lapse of six hours following radiation, which is in agreement with the latent interval found by Trowell, although Schrek noticed them somewhat earlier. The nucleus becomes darker by phase contrast, and motility is lessened. In the dark areas a cyst develops which corresponds to the primary vacuole of Schrek ; and cytoplasmic detail disappears. Bursting
/
( L
©) L
I
I
0
1
2
I
B CM
Fig, 2 6 I . - - R a d i u m applicator.
of these cysts was not noted, but the vacuolated cells gradually disintegrated, so that the agar was covered by cellular debris and cell ghosts. Not all the lymphocytes were equally sensitive; on the contrary, fully formed cysts were visible and the majority of the lymphocytes were converted to debris while a few moved and appeared exactly like the controls. The large cells of the endothelioid type were far more resistant to radiation than the lymphocytes, and indeed with the dosages used in these experiments neither they nor the polymorphs or eosinophils showed differences from the controls. The changes in the lymphocytes following irradiation are not specific ; they can all be noted, occurring at varying rates, in preparations kept without radiation for seven to nine days, which is the extreme period of survival of the lymphocyte by this technique. Irradiation accelerated the normal rate and stages of death and disintegration (Fig. 260 D-H).
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R A D I U M G A M M A R A Y S O U R C E F O R I R R A D I A T I O N OF C U L T U R E S
A special radium gamma ray source was constructed for the irradiation of the cultures, using several small radium sources which happened to be free. This was designed so that (a) it could be safely handled for short periods and (b) it provided an adequate dose-rate at the cultures which was relatively uniform over a small area. Details of its construction are shown in Fig. 261 A. T h e individual radium sources used were a glass tube containing 2I'35 mg. of radium elements enclosed in a cylindrical leaden container with I - m m . thick wails (a in Fig. 26I A) and 4 × I-mg. radium needles of 8 mm. active length and o. 5 mm. Pt. filtration (b in Fig. 26I A). These five sources were mounted together in a thick cylinder of lead as illustrated, T h e front, emitting face of the source was made of i - r a m , thick brass sheet in order to keep secondary beta radiation at a m i n i m u m (Wilson, i945). F o r irradiation of the cultures, the culture slide was placed between perspex supports of known thickness and the source was placed on the supports (Fig. 261 8). U n d e r these conditions the distance between the front face of the brass secondary filter and the culture was ~ mm. T h e dose-rate at a culture was determined by measurements made with an aerion condenser ionization chamber having a depth of air volume of only i mm. (Wilson, i944). I n order to take Table / . - - A G E
GROUPS
D I S T R I B U T I O N OF A G E G R O U P S I N D E C A D E S SITES
ioo
II 20
Abdomen
2I 3O
31 -4 o
II
II
51
5o
6o
6I 70
5
I
2
8
9
9
6 5
4I
Chest
I
3
Neck
I
2
5
i6
Head
I
m
5
6
5
8
Limbs
5
14
6
I
I
I
Total
6
14
24
23
24
35
:2:Z
71
8o
81 9O
I
I
I
I
account of any secondary radiations from the glass cover-slips carrying the cultures, the wall of the condenser chamber which received the radiation was of thin graphited paper and a glass cover-slip was laid upon this during the measurements. Dose-rate measurements were made at 0"5 mm., I'3 mm., and t'6 mm. from the source, and by interpolation on the curve from these three observations the dose-rate at a culture was found to be 356 r/hr. T h e ionization chamber was calibrated by thickening its front wall up to 3 mm. of aerion and exposing it to an accurately known gamma ray dose-rate. CLINICAL O B S E R V A T I O N S
T h e effects of radiation depend on a n u m b e r of factors ; in the following section, however, the relationship between the part of human patients irradiated and the effect on circulating leucocytes and lymphocytes will be studied. P a t i e n t s a n d M e t h o d s . - - T h e types of case studied in this paper comprise five groups of 3 ° patients. These groups represent irradiation of the abdomen, the chest, the neck, the skull, and the limbs. T h e patients in each group were selected only in that, in no case, were patients with obvious generalized metastases present included and the t u m o u r at the site of irradiation was small (as in the larynx) or had been previously removed (as in the breast and testis). T h e y are referred to below as groups I, II, I I I , IV, V.
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Group I comprised 3 ° males undergoing routine post-operative irradiation with X rays generated at 2oo or 250 kv. for seminoma or teratoma testis. In each the primary tumour had been excised. The irradiation was given as described by Prosser (I95O). These patients received two separate courses of treatment, the first to the lower abdomen and pelvis, followed 6-8 weeks later by a further course to the upper abdomen. Group II comprises 3 ° women undergoing post-operative X-ray therapy; 20o or 250 kv. following radical excision of the breast for carcinoma. The method employed was that of multiple tangential fields. Group III consists of 28 males and 2 females undergoing radium teletherapy (io-g. source) for carcinoma of the larynx and lower pharynx. The methods used are described by Wilson (1951) . Abdomen
Thorax
BEFORE TREATMENT Neck
Skull
Limbs
7"-
20-
I
.£-Yl_ over 9001 6001 3001 0-3000over 9001 6001 3001 0-3000 over 90016001 3001 0-3000over 9001 6001 3001 0-3000over 9001 6001 3001 0-3000 12,000 12,000 9000 6000 12,000 12,0009000 6000 12,000 12,000 9000 6000 12,000 12,000 9000 6000 12,000 12,0009000 6000 -
AFTER TREATMENT
! D
20"i I
I ....
over 90016001 3001 0-3000 over 90016001 3001 0-3000 over 90016001 3001 0-3000 over 90016001 30010-3000 over 90016001 3001 0-3000 12,000 12,00090006000 12,000 12,0009000 6000 12,000 12,0009000 6000 12,000 12,00090006000 12,000 12,0009000 6000
Fig.
262.--The
effect o f a t h e r a p e u t i c c o u r s e o f i r r a d i a t i o n o n t h e t o t a l l e u c o c y t e c o u n t .
Group I V consists of 14 male and i6 female patients treated for a variety of ailments located in the skull, viz., carcinoma of the superior nasopharynx io cases, brain tumours 13 cases, other conditions (all malignant) 7 cases. These patients were treated by a variety of methods, teletherapy using 4 g- and io g. of radium, X-rays of 2oo and 250 kv. and by the 2-MeV. Van de Graaf linear accelerator. Group V consists of 17 male and 13 female patients suffering from lesions of the limbs not involving the trunk. They comprise 19 cases of osteogenie sarcoma, 4 cases of synovioma and other soft-tissue tumours, 6 cases of osteoclastoma, and I case of Ewing's tumour. These again were treated by a variety of methods as in Group IV. Groups IV and V are by no means as homogeneous as Groups I - I I I as regards age and sex. The age and sex of each group are recorded in Table L The blood-counts were made on capillary blood by experienced technicians and by medical men using the same techniques, especially as regard to the method of performing the differential white count. These counts are ' routine ' in that special efforts to obtain increased accuracy beyond
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a single estimation of white-cell count and a differential count of IOO cells were not employed. T h e y are, however, free from 'unconscious bias' (Briggs and Macmillan, i95i ). It is clear that such estimations are subject to considerable degrees of latitude and so deductions from small changes in total leucocyte count and total count of lymphocytes cannot be made. Instead, the figures of each group of patients have been placed in the following ranges : leucocytes 0-3000 per e.mm., 31oo=6ooo, 6zoo-9ooo , 9ioo-12,ooo, and more than zz,ooo. The lymphocytes were similarly grouped : o-2oo, 2oi-6oo, 6 o i - i o o o , IOOi and upwards. The effect of a therapeutic course of irradiation in each group of patients is seen in Figs. 26z and 263. It is seen that lymphopenia and leukopenia are produced in the following order : (I) abdomen, (2) thorax, (3) neck, (4)
Thorax
Abdomen Pelvis
25A
BEFORETREATMENT Neck
Head
Limbs
J
1 0 ~ over 600 200 0 1000 1000 600 200
.__ over 600 200 0 1000 1000 600 200
25
over 600 200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
over 600200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
over 600 200 0 I000 I000 600 200
AFTERTREATMENT
,0. I _
600 200 0 1000 1000 600 200
over
over 600 200 0 1000 1000 600 200
over 600 200 0 1000 1000 600 200
Fig. 2 6 3 . - - T h e effect of a the r a pe utic course of irradiation on th e total lymphocyte count.
skull, and (5) limbs. Furthermore, when the second course of irradiation follows in group I (the interval being 6-8 weeks) then a further and even greater fall of leucocyte count and of lymphocytes occurs (Fig. 264). The pre- and post-treatment total leucocyte and total lymphocyte counts of the whole I5o cases as grouped in rising intervals of 400 per c.mm. are shown in Fig. 265. It will be seen that there is an overall fall of lymphocytes following therapy, but by comparison with Fig. 263 the effect is most marked when the trunk is irradiated. W h e n the cases were further analysed it was seen that not all the cases show a fall of lymphocytes, and that in fact certain cases in each group show a rise in total lymphocyte count. As these cases received the same treatment as the others it is evident that individual reaction must play a considerable part in the production of the results. These effects are summarized in Table H.
DISCUSSION It is difficult to compare the present series with those previously reported. Kornblum, Boerner, and Henderson (1938) in a study of Ioo cases noted the production of leukopenia and
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BEFORE TREATMENT SECOND COURSE
FIRST COURSE
Leucocytes
Abdomen Lym p h o c y t e s
Leucocytes
Abdomen Lym phocytes
20-
9001 6001 3001 0-3000 12,000 12,000 9000 6000
over
over 6000 200 0 1000 1000 600 200
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 1000 1000 600 200
AFTER TREATMENT 20
.__[ over 9001 6001 3001 0-3000 12,000 12,00090006000
over 600 200 0 I000 I000 600 200
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 1000 1000 600 200
Fig. 2 6 4 . - - T h e effect of a second course of therapy on G r o u p i patients.
BEFORE IRRADIATION Total Leucocytes Total Lymphocytes
AFTER I R R A D I A T I O N
.m over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
over 600 200 0 I000 1000 600 200
Fig. 265.--All cases (I5O).
.
over 9001 6001 3001 0-3000 12,000 12,000 9000 6000
.
.
.
over 600 200 0 I000 1000 600 200
T h e effect of a therapeutic course of irradiation in I5o patients.
EFFECT
OF
IRRADIATION
ON
HUMAN
LYMPHOCYTES
237
lymphopenia, but were unable to correlate the changes seen in the blood with the site irradiated or the amount of irradiation administered. Goodfellow (i936) studied 6I cases, comprising 3 ° cases of carcinoma of the breast, 13 cases of malignant disease in structures of the neck, and I8 other cases, and was of the opinion that the lymphocyte fall was greater in t h e neck cases. His cases were all treated by radium needle implantation or by surface application of radium. All authorities Table
H.--THE
SITE
B E H A V I O U R OF T H E L Y M P H O C Y T E C O U N T NO CHANGE
RISE
~bdomen Zhest Neck Head Limb
5 6 9 I5
3
2I
I
Fotal
56
Percentage
37"3
THE SITE
I
FALL
150
TREATED CASES
FALL TO LESS THAN 200 C.MM.
22
23 21
I5 8
89 3"3
RISE
14
59"3
B E H A V I O U R OF T H E L E U C O C Y T E C O U N T
N o CHANGE
IN
FALL
9"3 IN
15o
TREATED C A S E S
FALL TO LESS THAN 3000 C.MM.
a.bdomen F.hest Neck Head Limb
13 I6 16 13 9
4 3 4 5
13 13 II 13 16
total
67
I7
66
7
Percentage
44"6
II" 3
44
4'6
I
2
4 I O O
discussing the mechanisms by which these changes are produced, have failed to provide a uniform and consistent explanation. T h e following points may be made, however : - I. It is unlikely that destruction of circulating lymphocytes can be the cause, for in all the cases studied in this paper the circulating lymphocytes must be heavily irradiated yet the fall in skull and limb is minimal. 2. In irradiation of the abdomen, chest, and the neck a considerable amount of lymph-node tissue is irradiated directly and destroyed or profoundly modified by the treatment. This is not so in the skull and the limbs. 3. Patients who have had two separate areas of the abdomen irradiated show a much greater response than do other patients in this series. It is therefore suggested that the lymphopenia following irradiation is due to destruction of lymphocyte-forming tissue. This will explain a finding common to this series and also that of G o o d f e l l o w - - a fall of lymphocytes during the early part of a course of therapy, followed by a later rise even though therapy continues. Here it is postulated that other lymphocyte-forming tissues hypertrophy 'to take over the function of the destroyed tissue and so maintain the status quo in the blood-stream. Both the red bone-marrow and the lymphocyte-forming tissues are much more capable of response to a destructive process such as irradiation than is commonly realized. In the case of the marrow Denstad ( i 9 4 i ) has claimed to show that destruction of one area of red marrow is rapidly compensated for by hypercellularity
238
JOURNAL
OF
THE
FACULTY
OF
RADIOLOGISTS
of other areas. It will be noted that lymphopenia and leukopenia as induced by irradiation are not per se dangerous to the patient so long as he retains the ability to hypertrophy the remaining u n damaged lymphopoietic and leucopoietic tissues. When no such areas remain as a result of'previous irradiation or by the concomitant effects of such drugs as the ' chlorethylamines ', urethane, or triethylene melanine, then the leucopenia and lymphopenia remain permanently, witnesses of the poverty of the resources remaining. C O N C L U S I O N S
i. T h e response to therapeutic irradiation of five groups of 3° cases each has been studied. 2. Those groups represent the abdomen , thorax, neck, skull, and the limbs. 3. T h e constant change in the blood of these patients as noted by other authors, viz., lymphopenia and leukopenia, is confirmed. 4. T h e areas showing these changes in order of severity are the abdomen, chest, neck, the skull, and the limbs. 5. A second course to a different area of the abdomen 6-8 weeks following the first course is seen to produce a more serious effect. 6. It is suggested that these effects are produced by destruction or modification of the structures producing the lymphoid cells. 7. Permanent leucopenia and lymphopenia reflects the destruction of large areas of tissue. Our thanks are due to the Medical and Surgical Staff of Westminster Hospital for their generous co-operation: to the department of Medical Photography; and to Mr. J. Haynes for technical assistance. W. H. W. Jayne is working with a full time grant from the Imperial Cancer Research F u n d . T h e expenses of this research were covered by a generous grant from the Governors' Discretionary Fund, Westminster Hospital, and the work of one of the authors (C.W.W. is supported financially by the British Empire Cancer Campaign. REFERENCES BRIGGS, R., and MACMILLAN,R. L. (I95I), Brit. ff. clin. Path., I, 283, 290. CRAIGIE, J. (I952), ft. Path. Bact., 64, 25I. -- - LIND, P. E., HAYWARD,M. E., and BEGG,A. M. (i95i), Ibid., 63, I77. DENSTAD, T. (I94I), Acta radiol., Stockh., suppl., 52. GOODFELLOW, D. R. (I936), Brit. J. Radiol., 9, 695. HEINEKE, H. (I9o4), Miinch. reed. Wschr., 5x, 785. - - - - (I9o5, a), Mitt. Grenzgeb. ]Fled. Chir., x4, 2t. - - - - (I9o5, b), Dtsch. Z. Chit., 78, I96. KORNBLUM, K., BOERNER, F., and HENDERSON, S. G. (I938), Amer. ft. Roegentol., 39, 235. LACASSAaNE,A., and FmCOUROEF,G. (I927), C. R. Soc. Biol. Paris, 96, 862. LAZARUS-BARLOW,W. S. (I92O), Proc. R. Soc. Med., 14, I. PROSSER, T. M. (I95O), Brit. J. Surg., 38, 473. PDLVERTAFT,R. J. V. (I952), Med. Biol., 3, 198. -- - WILSON, C. W., and JAYNE, H. (I953), Nature, Lond., I7I , ii57. SCrIaEK, R. (I946), J. cell. comp. Physiol., 2 8 , 277. - - - - (1947, a), Ibid., 3o, 2o3. - - - - (1947, b), Proc. Soc. exp. Biol., N . Y . , 64, 38I. -- - (I948), Amer. J. Path., 24, lO55. SENN, N. (I9o3, a), N . Y . med. J., 77, 665. - - - - (19o3, b), N . Y . Med. Rec., 64, 28i. TROWELL, O. A. (I952), J. Path. Bact., 64, 687. WILSON, C. W. (I944), Brit. J. Radiol., I7, 86. - - - - (I945), Radium Therapy--Its Physical Aspects. London : Chapman & Hall. (I95I), J. Radiol., 24, 374. WOLFF, E., and HAFEEN, K. (I952), Tex. Rep. Biol. Med., IO, 463.