Hematological Changes in Animals with Radiation Sickness Caused by Betatron Rays

G. P. GARTANEEV

(Presented by the scientific seminar of the Physico-Technical Faculty)

The available literature data on the biological action of betatron rays concern mainly the question of treating malignant neoplasms, and only in isolated works, briefly, in the course of case histories, are hematological data sometimes mentioned. In the domestic literature there are no articles on the betatron at all.

The experiment was conducted by the Department of Pathophysiology of the Tomsk Medical Institute on the betatron of the Tomsk Order of the Red Banner of Labor Polytechnic Institute named after S. M. Kirov, with an energy of 10 MeV.

All the animals, depending on the nature of the experiment and the task set, were divided into 8 groups. Single irradiation was carried out in the following groups: No. 1 (doses from 20 to 240 r), No. 5 (dose 3600 r), No. 6 (dose 7300 r), No. 7 (dose 1824 r), and No. 8 (doses 385 and 480 r). In three groups the irradiation was repeated. In group No. 2 the dose per session was 75–180 r, the total dose 150–405 r and, in one case, 5415 r. In group No. 3 the doses per session were 100–200 r, the total doses 1000 r and, in two cases, 820 and 1650 r. In group No. 4, irradiation during the day was fractionated and reached doses of 0.5–6 r.

Group No. 1 included 5 rabbits and 2 guinea pigs; group No. 4, 5 white rats; the remaining groups consisted of guinea pigs (groups Nos. 2, 3, 5, 7 were made up of 10 pigs each; Nos. 6, 8—of 5 pigs each).

Irradiation in all cases was general, without the use of any filters, with the exception of group No. 4, where animals of the other groups placed in the beam zone served as a kind of filter (the cage with the rats was always placed by the wall, at the point where the beam fell, at a distance of 1½ m from the target).

Dosimetry was carried out with the aid of a USDD-type dosimeter.

As a result of irradiation, the animals developed a typical picture of injury in the form of acute and chronic radiation sickness with typical hematological changes.

The most pronounced changes were observed in the leukocyte count.

For all groups with single irradiation, the presence of leukocytosis in the first hours after irradiation was characteristic, with a wave-like change in the number of leukocytes. Sometimes the phase of leukocytosis was preceded by a very brief-

a temporary phase of decrease in the number of leukocytes. This was observed with particular constancy at doses of 1824–7300 r. Leukocytosis was always replaced by leukopenia, which at doses of 1824–3600 r progressed up to days 7–11 (until the death of the animal). In the case of the animal’s death after 7–11 hours, as occurred at doses of 7300 r and sometimes at 1824 and 3600 r, we observed only the phase of leukocytosis, which in the first 1–2 hours was usually preceded by a decrease in the number of leukocytes. When doses of 20–240 r were used, one further phase was observed—the phase of restoration of blood indices.

In groups with repeated irradiation, observations were not carried out during the first hours, but on subsequent days leukopenia was always noted. After irradiation was discontinued, as a rule, by the end of the second month, at doses of 820–1650 r we observed a return of the number of leukocytes to the initial values. At a dose of 5415 r, the leukocytes remained at a low level up to the 77th day of observation, when the guinea pig was transferred to the Department of Pathological Anatomy.

As the studies showed, the time and degree of manifestation of leukocytosis cannot be explained solely by the magnitude of the dose; they should also be linked with the reactivity of the organism. Thus, for example, in rabbit No. 4, at a dose of 104 r, the maximum rise occurred at the seventh hour, and the number of leukocytes was 490% of the initial value. At the same time, in rabbit No. 5, at a dose of 240 r, the peak occurred at the fifth hour, and the number of leukocytes reached 240% of the initial value.

Leukopenia was least pronounced in the case of small single doses. At doses of 1824 and 3600 r, the number of leukocytes, steadily decreasing, reached very low figures. At doses of 1824 r, in only one case was this decrease 96% of the initial value. As a rule, it was usually less pronounced, and in a number of guinea pigs the fall in the number of leukocytes was always 40–36%. At doses of 3600 r, the decrease in leukocytes in all guinea pigs was 93–98%.

Consequently, the degree of manifestation of leukopenia can be linked, on the one hand, with the dose and, on the other hand, with the reactivity of the organism, since within one and the same group different degrees of manifestation of this phenomenon are observed (Table 1).

Table 1. Dose 1824 r

This is especially noticeable at small doses, when one may observe more pronounced leukopenia at small doses and, conversely, at larger doses—less pronounced leukopenia. Thus, in rabbit No. 5, at a dose of 240 r, a decrease of 25% was observed, whereas in rabbit No. 1, at a dose of 60 r, the decrease was 44%.

Observations with repeated irradiations showed that in this case the leukopenic state develops more rapidly, especially when the reactivity of the organism is reduced. Thus, in group No. 2, under conditions of incomplete

with inadequate nutrition, over the course of 2–6 days, at total doses of 150–405 r, the number of leukocytes fell by 75–90%; at the same time, in group No. 3, with adequate nutrition, this decrease over 9–14 days, at total doses of 820–1615 r, was 53–84%.

With respect to the recovery period, when it occurred in our cases, one detail may be noted—its duration. In addition, at doses of 385 and 480 r this period was characterized by irregular fluctuations in the number of leukocytes, which were observed up to and including the day of the animal’s death.

The ratio of the formed elements of the white blood in the various periods indicated above varies. In the period of leukocytosis and in the brief period of leukocyte decline preceding it, an increase in the absolute number of neutrophils is observed. In the period preceding leukocytosis, lymphocytes always decreased in number, whereas in the period of leukocytosis this was far from always observed, and in a number of cases the leukocytosis was not neutrophilic but mixed in character. Thus, for example, in rabbits given single doses of 20–240 r, leukocytosis was due both to pseudoeosinophils and to lymphocytes. In guinea pigs, at doses of 385–7300 r, the leukocytosis was predominantly neutrophilic or purely neutrophilic in character and was accompanied by a left nuclear shift, especially at doses of 1824–7300 r.

During the period of leukopenia, the number of neutrophils fell more sharply than the number of lymphocytes. Thus, for example, in guinea pig No. 29 (dose 3600 r), by the end of the fifth day there were 750 lymphocytes in 1 mm³ and not a single neutrophil; in guinea pig No. 30 (dose 3600 r), on the fifth day there were 480 lymphocytes and 100 neutrophils in 1 mm³ (with initial values of 17,235 lymphocytes and 9632 neutrophils).

With single doses of 20–240 r in rabbits and 385–480 r in guinea pigs, and also at remote times after repeated irradiations in guinea pigs and rats, a change is observed in the relationship between neutrophils and lymphocytes toward an increase in the latter. Thus, in rat No. 4, before irradiation, lymphocytes were 62%, neutrophils 32%, whereas on the 57th day of the experiment lymphocytes comprised 92% and neutrophils 7.5%. The absolute number of leukocytes was not counted, but on examination of the smear the picture resembled a leukemoid reaction. In rabbit No. 1 (dose 60 r), the initial number of pseudoeosinophils fluctuated between 3468 and 4939 per mm³, and lymphocytes from 5304 to 7788; on day 101 there were 1760 pseudoeosinophils per mm³ and 6080 lymphocytes.

The remaining groups of leukocytes in our cases did not show any regular fluctuations, but quite often at remote times in animals of groups No. 1, No. 3, and No. 8 an increased number of eosinophils was observed.

Morphological changes are detected earliest in neutrophils or pseudoeosinophils and later in lymphocytes, eosinophils, basophils, and monocytes.

In rabbits, pseudoeosinophils appear earliest (in the first hours) with lysis of eosinophilic granularity in the protoplasm and the presence in it of coarse azurophilic granules, which may be very abundant in the later hours. Later, pseudoeosinophils appear with pale segments of the nucleus or with an entirely unstained nucleus, as well as destroyed pseudoeosinophils. In individual cells, areas of pyknosis in the nucleus may be noted, sometimes in combination with lysis. Hypersegmented forms may appear early, but are always present in small numbers.

Changes on the part of lymphocytes occur somewhat later and are not so pronounced. Usually we observed pyknosis of the nucleus, disintegration of the nucleus; phenomena of pyknosis and lysis are often observed in the very different—

...in various combinations, atypicality of the nuclear contours, increased basophilia of the protoplasm, and pseudopodia. Disturbances of the nucleus are also encountered in eosinophils, basophils, and monocytes.

Usually the morphological changes proceed in waves: now intensifying, now disappearing completely, now appearing at the most remote times. Thus, for example, in rabbit No. 1 (dose 60 r), on days 40, 47, and 79, morphological changes of the pseudoeosinophils and lymphocytes could be observed.

What has been said about the morphological change of the pseudoeosinophil nucleus will also be true with respect to the neutrophil nucleus.

The earliest changes in the neutrophil nucleus should probably be considered fragmentation and budding off of its small parts. Somewhat later, but within the first 7 hours, phenomena of chromatinolysis, pyknosis of individual areas of neutrophil nuclei—sometimes in combination with lysis—pallor of staining, and disintegration of the entire cell were observed. Morphological disturbances in lymphocytes, in the form of pyknosis of the nucleus, its lysis, the presence of a double nucleus, increased basophilia of the protoplasm, and coarsening of the azurophilic granularity in the protoplasm, appear somewhat later and are not as sharply expressed as in neutrophils.

Throughout the entire period, especially at late times, hypersegmented neutrophils were encountered in cases of both single and repeated irradiation. Several days before the animal’s death, giant neutrophils with a swollen nucleus and basophilic protoplasm, often vacuolized, appeared in the peripheral blood. These cells were observed especially often at doses of 1824 and 3600 r. Vacuolized monocytes with an atypical nucleus could also often be encountered.

The most vivid changes in blood cells of a morphological character in guinea pigs were observed in groups with single irradiation at doses of 1824, 3600 r and with repeated irradiation in group No. 2, where the morphological changes developed very rapidly. At doses of 7300 r the morphological changes were not very vivid, but the phenomenon of chromatinolysis was especially clearly manifested.

Study of bone-marrow punctates taken agonally after 7–11 hours showed depletion of the latter in lymphoid elements in cases of very severe radiation sickness (7300 r). At doses of 1824 and 3600 r and death of the animals after 5–8 days, depletion of the bone marrow in myeloid elements was observed, with the appearance of a considerable number of plasma cells and elements of the lymphoid series.

Changes in the red blood were not so sharply expressed. Thus, the number of erythrocytes and hemoglobin did not show any noticeable fluctuations at doses of 1824 and 3600 r, although at a dose of 7300 r some decrease in the number of erythrocytes could be observed. At doses up to 480 r, in the late periods a decrease in the number of erythrocytes and hemoglobin is observed. The increase of these indices in the preagonal period is explained by thickening of the blood.

Reticulocytes at doses of 1824, 3600, and 7300 r rapidly disappear from the blood and do not appear up to the moment of death; the same occurs also with repeated irradiation at doses of 100–200 r per session when the organism’s reactivity is reduced. In the event that the animal is removed from the experiment and survives (with normal reactivity), reticulocytes return to the initial and even higher values. In rats during the period of irradiation we observed a very low content of reticulocytes, but after their removal from the experiment the reticulocytes increased to high values. With small single doses (in rabbits), reticulocytes, after an initial decrease, rose above the initial figures and then returned to normal.

A change in the ESR in the last days of life toward acceleration was observed by us only in guinea pigs irradiated once with doses of 385 and 480 r.

Morphological changes in the erythrocytes were most often expressed in the appearance in the peripheral blood of erythroblasts and basophilic-granular erythrocytes, which may be encountered in the first hours after irradiation. Erythrocytes with Ehrlich and Jolly bodies were encountered rarely.