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The hematological effects resulting from injection of radioactive phosphorus (P32) into albino rats.

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THE HEMATOLOGICAL EFFECTS RESULTIKG FRO31
INJECTION O F RADIOACTIVE PHOSPHORrS
(P3')' I N T O ALBINO RATS
JOHR' S T E P H E N S LSTT-4 AND RORALD E. WAGGENER'
Depavtment of A n a t o m y , University of X e b r a s k a , College of Medicine, Omaha
TWENTY FIGURES
Although much work has been reported concerning the
hematological effects from external irradiation relatively few
studies have been made concerning such effects from internally administered radioactive phosphorus. The most comprehensive reports have been those of Koletsky and Christie
('50) and Grad and Stevens ('SO), but it has not been determined with certainty just what correlation can be made between the affected bone marrow and the cells of the peripheral
blood, particularly during the period of maximum depression.
We undertook this study to attempt to clarify the problem
of the relative sensitivity to internal irradiation of the various developmental stages of the blood cells and relation of
these effects to the peripheral blood picture. I n earlier exusing
periments reported (Latta, Vose and Rosenlof, 'X!),
dosage of .25 and 1pc of P3'/gm body weight, changes in
the bone marrow were encountered which involved both myeloid and erythroid series, but of too mild degree to be reflected
in the blood or to produce a leucopenia and depression in
the reticulocyte count. I n the present studies larger doses
of P3*were used to further clarify the problem of the effects
of internal irradiation.
lP3*was obtained through the courtesy of the Oak Ridge National Laboratory
i n the form of sodium acid phosphate. It was assayed with a dipping type Geiger
counter against standards of the Pilational Bureau of Standards.
Predoctoral fellow, Atomic Energy Commission.
357
358
J O H N S. LSTTA A N D RONALD E. WAGGENER
MATERIALS AND METHODS
The animals used in these experiments were from an inbred
albino rat colony maintained by the department for many
years, on which the normal range of hematological values
had been previously established. They were fed a standard
diet of Purina Fox Checlwrs supplemented by corn and oats.
F o r experimental purposes 6 series of 7 animals each, from
4 to G months old, mere selected. Before injection values for
total erythrocyte and leucocyte counts, hemoglobin levels,
differential and reticulocyte counts were determined. I n some
series sedimentation rates, hematocrit values and icterus indice3 were also determined. In all cases periods of 11 days
were allowed for recovery from the blood loss before intraperitoneal injection of the isotope. The amount of isotope
was determined on the basis of 4.5 ye P""/gm of body weight.
Following injection animals were sacrificed after one, two,
three, 5, 7, 10, and 15 days. I n all cases the same blood determinations were made as before injection, and in addition
marrow and spleen imprints, and sections made of marrow,
spleen, and lymphatic nodes were prepared. Blood for examination and tissues for sectioning mere taken before anesthetic death occurred.
Another series of 6 rats of comparable age mas completely
studied to furnish further controls, particularly of the hemopoietic organs. Ferrocyanide-HCl stains to determine hemosiderin were made on spleen sections from another series of
1 2 nornial, and from 7 treated rats.
Haring used 6 experimental series of 7 animals each, comparihon was made between the average and range of blood
value* lxfore injection in the 42 animals and the corresponding values in 6 animals after each day of sacrifice following
injection (with two exceptions). Findings in the blood-forming organs of experimental animals were compared with
corresponding findings in the control series and with those
pi-cviouhlp reported for this colony.
359
HEMATOLOGICAL E F F E C T S O F
Periplieral blood
Erythrocytes. €'reinjection
erythrocyte counts on all experimental animals gave an average value of 8,800,000/mm3
with a range in the 6 groups of 8,100,000 to 9,300,000/mm~'.
After injection of 4.5 pc/gm of body weight total erythrocyte counts of the 6 animals (4 after 15 days) sacrificed a t
each of the stated intervals were averaged (table 1). It can
be seen that a gradual decrease began on the first day post
injection and was significant by the third day. At 15 days
after injection severe anemia had developed. These findings
are contrary to those of Koletsky and Christie ('50), who
TABLE 1
E.B.C. expressed 0s mi.llions/mnP after 4 5 pc P"/gm body weight
~ _ _
~-
~
DAY
1
2
B O . ASIX.%LS
RAIOE
-
AVERAGF.
-
8.6
r r
8.2
r
I
.a-9.8
5
i.0-8.7
6.4-9.0
5.0-9.4
c
,
5.6-8.8
7.6
10
1.5
3.4-8.0
6.5
1.7-7.3
4.1
>
i.6
7.G
noted a latent period of one week following the same dosage
with Pst2.Counts in each experimental animal were converted
to percentages of preinjection counts of each animal and
averages for those on each day post injection day computed.
T h e n these percentage values were plotted against days of
irradiation the resulting curve indicated a gradual but perceptihle decrease during the first week and a rapid decrease
during the second week. After treatment started wide variations in individual counts were obtained but the trend is unmistakable (fig. 1).
Rrticitlocyte counts were done on 35 animals before injection, and the average value determined to be 4.870, with a
ranye from 2.9 to 5.9%. Following irradiation there was an
360
J O H N S. LhTTA A S D ROXALD E. WAGGENER
immediate decrease in these values which continued throughout the period of observation the range being between 0.3
and 0.9%.
Hemoglobin determinations in the 42 animals before injections averaged 14gm/100ml blood, ~ ~ r i tah range froin 12.4
to 14.6 gm. After injection of P32these values remained within
the preinjection range during the first meek, although the
average values after the second day post injectioii steadily
NORMAL
100
O/o
90
80
70
60
50
40
1 2 3
DAYS
5
7
10
15
Fig. 1 The average percentage of normal values of erythrocyte counts
days indicated following injection of 4.5 pc P32/g~11
of body weiglit.
011
the
TSBLE 2
Hemoglobin expressed as gm/lOO m1 blood after 4.5 pc P / g m body weight
DAY
1
2
3
5
7
10
15
NO. ANIMALS
RAXGE
AVERAGE
11.6-14.5
13.2
11.8-14.9
10.4-14.0
13.7
12.5
12.4
10.8-13.6
10.4-13.1
6.0-12.5
12.3
3.2- 9.1
5.4
10.2
361
HEhIATOLOGICAL EFFECTS O F P:’2
decreased. During the second week the values sharply declined to 5.4gm (average of three animals), about 40% of
the preinjection balue (table 2).
Henzutocrit values likewise steadily decreased from a preinjection level of 50.7 cms/lOO em3 of blood to 23 cm3/100 em3
after 15 days post injection. Percentages of normal values
plotted against days of irradiation are presented graphically
(fig. 2 ) .
O/o
NORMAL
1 2 3
5
7
10
15
DAYS
Fig. 2 The average percentage of normal heniatocrit values, determined on
the days indicated following injection of 4.5 PC PZ3/gm of body weight.
L w c o c y t e cozcnts
The average number of leucocytes/mm3 of blood in the
1 2 animals 11 days before injection was 10,300 with a range
from 8,400 to 12,400. I n the 6 animals sacrificed after one
day the average total count had sharply decreased to 4,600/
mm3. At the end of two days the average had further decreased to 2,600/mm3 and after 5 days to l,200/mm3. A very
low level (600/mm3) n7as reached after 10 days irradiation
and maintained through the 15th day. Average total leuco-
362
J O H X S. LATTA AND RONALD E. WAGGENER
cyte counts plotted against days of irradiation give an esponential type of curve (fig. 3 ) .
Neutropldes. The preinjection average of neutrophilic
leucocytes of the 42 animals was found to be 22%, ranging
froni 1 7 to 31%. Absolute numbers averaged 2,400 iiiin”,
ranging froni 1,900 to 3,100/mm3. After injection of PS2
absolute numbers decreased sharply during the first three
days to less than 1,000/inni~5
(938) and again decreased rapidly
CELLS /MM3
10,600
8,600
6,600
4,600
2,600
600
1 2 3
5
7
15
10
DAYS
Fig. 3 The arerage of total leileoeyte counts/rnm”
lowing injection of 4.5 PC P’”/gm of body weight.
011
the days indicated fol-
between the 5th and 7th days to an average of 2OO,m1ii:~,
readied a minimum number of 147/mm3 after 10 days, and
increased slightly between 10 and 15 days (fig. 4).
The preinjection ratio of segmenters to younger neutrophilic forms was 6.3/1. After injection the value of this
ratio was distinctly increased during the first 5 days, the
highest value being 10.2/1 after two days. After 5 days the
ratio declined to nearly normal values. (Compare with findings of Kagaz and Van Baaren, ’53, in dogs after s-irradation.)
363
HEMATOLOGICAL EFFECTS O F P”’
L y m p h o c y t e s . The preinjection average of blood lymphocytes of the experimental groups was found to be 74%, ranging from 65 to 83%. Absolute numbers averaged 7,7O0/nim3,
ranging from 5,500 to 9,300/mm3.
After injection a remarkable decrease in lymphocytes occurred during the first day ( 2,000,/mm3)),which continued
through the second and third days ( 800/mm3). Following
CELLS / MM3
00
‘
1 2 3
DAYS
5
7
10
15
Fig. 4 Average of counts on neutropliiles/mm3 011 the days indicated following
iiijeetioii of 4.5 fie P”/gm of body weight.
this the average decrease continued more slowly to an average level of 384/mm3 after 10 days, which was also observed
after 15 days (fig. 5).
Eosirzophiles a i d basophiles were present in variable numbers in animals killed a t various intervals post injection but
these fluctuations were all within the range of preinjection
values and were not considered significant.
O t h e r fiwdings
After injection of P32large hypersegmental neutrophiles
were frequently encountered in the blood. Late in the experimental period “ b l a ~ t i c ’and
~ bilobed lymphocytes were seen
occasionally. One of the most striking late changes was the
appearance in the blood of some animals of single or clumped
364
J O H N S. LATT.1 B N D RONALD E. WAGGENER
reticular stem cells. The number of smudge forms and the
amount of cellular debris increased throughout the experimental period.
Although platelet counts, bleeding and coagulation times
were not determined, it was obvious that platelets were reduced and the bleeding time prolonged. No variations from
CELLS
8000
7000
6000
5000
4000
3000
2000
1000
0
1 2 3
5
7
10
I5
DAYS
Fig. 5 Average of counts of lymphocytes/mm3 on the days indicated following
injection of 4.5 pc P"/gm of body weight.
the normal range of values for erythrocyte fragility (7 animals) or sedimentation rate (14 animals) were found in the
experimental animals so tested.
Icterus index in a series of 8 normal animals ranged from
two to 5 clinical units. I n a series of 15 experimental animals
the icterus index ranged from 4 to 7 clinical units. This slight
increase was considered of doubtful significance.
HEhlhTOLOGICAL EFFECTS OF P"2
365
IIemoyoietic orgaas
Boize marrow
Geiieral. Both marrow imprints and sections from experimental animals showed a progressive depletion of cells,
marked even after one day of irradiation. After 5 days
severe cell depletion made fat and supporting tissue very
prominent. Much nuclear pyknosis, fragmentation and cellular debris were noted. Sinuses were r e r y prominent and
hemorrhage into marrow spaces was often seen. Hemosiderin
laden macrophages were present. I n animals after 10 days
of irradiation the marrow was almost devoid of free cells
but after 15 days some recovery was noted (figs. 9-12).
D if er ential
coumts
Erythroblasts. I n counts made on inarrow imprints from
the 6 normal rats erythroblastic elements averaged 43.3'j/c,
ranging from 35 to 48.470, which is in essential agreement
with previously reported values from our colony and others.
Of these elements, hasochromic erythroblasts averaged 1.670,
ranging from 1.8 to 8.5%. Polychromic erythroblasts averaged l6.9%, ranging from 13.6 to 19.8%. Normochromic
erythroblasts averaged 22.9%, ranging from 15.2 to 25.270.
These values are all close to those previously reported for
this colony.
injection the average percentage value of erythroAfter PtiZ
blasts dropped immediately to 17% after one day and 167;
after two days. A proportionate increase was found after
5 days to an average value of 33% but another drop in average to 1 7 % occurred after 10 days. An average percentage
increase to normal percentage range (46%) was found after
15 days. When average values of the various stages are
plotted against days of irradiation it is obvious that the
fluctuations above noted involved the polychromic and particularly the normochromic erythroblast s, basochromic erythroblasts showing little change (fig. 6). The lowest value for
polpchromic cells was reached after one day (6.574 ) and for
norinochromic cells after two days (5.5%). The percentage
366
J O H N S. L A T T A AND R O S A L D E. WAGGENER
values for both types iiicreased soiiiemhat after 5 days and
had again decreased after 10 days. After 15 days the percentage of normochromic cells had markedly increased, that
of basochroiiiic cells slightly increased, but that of polychroiiiic
cells reinaiiied a t a low value. (See fig. 13.)
During the earlier stages when the percentage of erythroblasts was so greatly diminished, the individual cells showed
%
30
NORhlCCHROMlC E R Y T H R O B L A S T S
POLYCHROUIC E R Y T H R O B U S T S
B ~ S O C H R O H I CE R Y T H R O B L a S T S
-- --
25
20
15
10
5
0
1 2 3
5
7
10
15
DAYS
Fig. 6 Arerage percentage of the various developmeutal stages of erythroblasts in femoral bone niarrow on the days indicated following injeetioii of 4.5 pc
P32”/1 of body weight.
evidence of marked damage and modified staining reaction.
Fragmenting erythroblasts v7ei-e frequently seen in imprints
and sections.
Neutropkilic series. Cells of this series in the marrow from
the 6 normal rats averaged 39.870, ranging from 32.8 to 47.276.
The range includes values for this colony reported earlier,
but the average is somemhat lower than given in other reports. I n this series myelocptcs averaged 2.2%, ranging
from 0.7 t o 3.70/0, which are lower values than previously
H E M A T O L O G I C A L E F F E C T S O F P3'
367
reported. Values of juveniles averaged 7.3C/o, ranging from
4.7 to 9.6%. Normal values for staff forins was 15.1c/c.,ranging from 12.5 to 217.. The percentage of segmented neutrophiles averaged 15.2c/,, with a range froin 15 to 24.5%. These
values agree fairly well with those previously reported.
During the first two days following injection of P32there
was a sharp increase in the percentage of iieutrophilic cells
in the marrow, froin a normal value of 40% to a maximum
30
25
20
15
10
5
0
DAYS
Fig. 7 Average percentage of the various developmental stages of neutrophilic
leucocytes in femoral bone marrov 011 the days iiidieated following injection of
4.5 pc P3'/gm of body weight.
of almost 70% (fig. 14). After this rise the percentage
dropped to the lowest value of about 15% after 10 days,
then increased to about 33% on the last day of observation.
These fluctuations in the total percentage counts were due
chiefly to variations in percentages of staff and segmented
forms, there being only a slight decrease in percentage of
juveniles and practically no deviation in myelocytes. (See
fig. 7.) The percentage of staff forms began to fall before
that of segmented cells but both had reached minimum values
365
J O H S S. LATTA A S D BOKALD E. WAGGEXEK.
after 10 days ancl both had increased after 15 days post injection.
Eosiizophilic cincl basophilic series showed little change
following injection. Eosinophilic cells in the marrow of our
normal animals averaged 8.4cjc, of which 3.8% were staff
forms, and there were between 1 and 2% each of myelocytes,
juveniles and segnienters. Rasophiles in our normal series
averaged l.ac/,, fairly evenly d i ~ i d c dlwtween the n i * i o u s
M /E
611
511
41 I
3/ I
2/ I
1/1
0.51 I
o/o
1 2 3
5
7
10
15
DAYS
Fig. S The average mlars for myeloid-ei?.tliroid ratios
folloniiig iiijection of 4.5 p c P,'/gm of body weight.
011
the days iiidiwtcd
foi*ms. Both marrow eosinophiles and basophilcs had decreased about 1% after 10 days following injection of the
isotope, due to decrease in the percentage of staff forms.
Although these decreases were similar to those reported f o r
neutrophiles, the values are so low that we a r e not justified
iii attaching significance to them.
3lycloid-erythroic7 ratios in the normal series of this study
averaged 1.02/1 which is lower than previously found in this
colony and lower than most values given by other authors
for the albino rat. In any case this ratio was markedly in-
HEMA'l'OL0C;IChL EFFECTS O F 1""
369
creased by one day aiid reached a maxiiiiuni average of 5.1/1
after two days post injection. This was followed lq- a sliarp
decrease after 5 days aiid a more gradual decline during the
remaindcr of the experimental period, ~eachiiigan average
of O.S5/1 after 15 days. ( S e e fig. 8.)
Ot hcr f i r d i n g s
The nornially sinall pei~ceiitugcof heiiiocytoblasts (0.4% )
fluctuated very little after iiijectioii of I?,
but the proportion
of fixed reticular cells iiicrewsed from a noriiial value of 0.1%
to 3.2% after '7 days. After this time their percentage relnt i d y decreased as the populatioii of free cells increahed.
dlthough the percen t q y of proiiiyelocytes averaged only
1.1%in iioriiial niarrows P2 injection was followed by a
slow decrease to an avci.ag:.e value of 0.4% after 7 days, with
a return to original avei.agc value after 3 5 days. *!qaiii
megakaryocytes averaged only (Lac/, in 11oriiial iiiarrow hut
were difficult to find in n i a i * ~ ~ m
of s aiiimals after '7 claps of
treatment, most of those eiicoiuitercd showed vaiyiiig dcgrees
of degenerative change, iiicludiiig cytoplasmic vacuolation,
nuclear pykiiosis and disintegi*ation. (Scc figs. ltj-16.)
The percentagc of lymlhocytes in the iiiarrow dropped
sharply during the first t h i w days post injection from a
nornial average of S.77(> to 3'/c, after which tlicir values remained fairly steady. However, the 1)ercciitage of plasina
cells, originally averaging 0.5 %, inciaea sed through the first
10 days post injection to a value of 7.5% after which the
peiwiitage declined to nearly normal level.
Degcnerating or disiiitcgra ting cells were seldom f ouiid in
ilornial marrows but greatly iiicreased in iiunibers in the
esperimental animals to a maximum average of 59% of cells
after 10 days. Their reduction during the nest 5 days to
2 4 9 of cells was both relative and absolute, coiisiderinp the
increased total cell population noted after 13 days.
Spleen
Compared with spleens from normal aniiiials those from
aniiiials after one day yost injectioii showed considerable
370
J O H S S. LATTA A S D ROSALD E. WAGC:E;SI<R
cellular depletion of the red pulp with large numbers of
lymphocytes showing nuclear pyknosis and fragmenting cytoplasm. bIuch cellular debris was present. Similar, though
less extensive changes mere present in the white pulp. After
two days practically no free lymphocytes were found in the
red pulp and the white pulp had become more severely damaged. Pigment laden iiiacrophages and extracellular pigment
had noticeably incrclased. These evidences of damage progressively incrcasetl throughout the experimental period.
TdELE 3
NO. O F HEXIOSIDERIN CON-
ANIMAL
T A I X I S G CELLS/H.P.F.'
El (Range 51-171)
170
238
260
396
444
4i1
61-2
S o r m a l (12 r a t s )
1 clay post inj.
2 days post inj.
3 days post inj.
5 days post inj.
7 days post inj.
10 days post inj.
13 claps post inj.
These counts were iiiade in representative areas of the red pulp of the spleens
remored a t indicated tiiiies follomiiig iii,jection t o determine more accurately the
increase i n the amount of hemosiderin deposition.
After 13 days both red and white pulp were markedly depopulated and most of the lymphocytes present had pylmotic
nuclei. Large deposits of pigment, both in macrophages and
estracellularly, were present in the red pulp and in remaining
reaction centers of the white pulp. Signs of regeneration
wei-e in the form of a few siiiall foci of erythropoiesis, not
d
present in earlier s t a oes.
Special stains f o r iron on spleens from 1 2 normal and 7
experimental animals identified the accumulated pigment as
hemosiderin. I n typical areas of the red pulp the number of
pigment laden iiiacrophages was approximated to give a
rough estimate of the rapidity and quantity of the pigment
deposition as shotni in table 3. (See also figs. 17-18.)
s
HEMATOLOGICAL EFFECTS O F PS2
371
Lynaph modes
After one day post injection much cellular debris had accumulated in the lymphatic tissue and many lymphocytes
with pyknotic nuclei were noted. After another day still
more debris was present, niuch of it within macrophages.
There were areas of complete destruction present, the only
cells present containing pylrnotic nuclei. The shrinkage in
volume of lymphatic tissue resulted in prominent sinuses,
adjacent to which the greatest damage was seen. This damage became progressively more severe through the entire
period of observation. After 10 and 15 days the sinuses were
markedly widened, and lymphocytes greatly reduced in number (figs. 19-20). Great numbers of macrophages were
present, most of which contained lymphocytic debris or hemosiderin o r both. Even though there was so much evidence
of destruction some reduced mitotic activity was usually
seen. Plasma cells were found in increasing numbers.
UISCUSSION
Erythrocytes: procluctioiz, delivery and destruction
From the observations above reported it is evident that
several factors are involved in the rapid production of severe
anemia. The life of a n erythrocyte in the peripheral blood
of the rat has been determined to be about 68 days, measured
with C14 labeled glycine (Berlin, Meyer and Lazarus, '51)
and to be about 100 days, as measured with deuterium labeled
hemin (Ponticorvo, Rittenberg and Rloch, '49). If delivery
had been completely stopped and normal rate of destruction
continued the erythrocyte count should drop from 1 to 1.4%
per day, to 91 to 93% of original value after 7 days. I n our
experimental animals the average value was 87% of normal
value after 7 days and, although reticnlocyte values decreased
sharply, they indicated some delivery during this period.
After 15 days our observed average value was 54% of normal average as compared with an estimated value with normal
rate of destruction and no delivery, of 79% of normal average.
Our findings of progressively increased deposition of hemo-
372
J O H N S. LATTA A N D RONALD E. WAGGENER
siderin in the niacrophages, particularly in the spleen and
the small increase in icterus index indicate increased destruction of cells. Dunlap ('48) has reported increased excretion
of urobilinogeii in the feces and bilirubin in the urine in such
treated animals. It follows that decreased delivery plus increased rate of destruction contributed to the anemia. The
slight increase in icterus index may be associated with the
known efficiency of the r a t liver in eliminating bile pigiiients.
Although Warren ('43) felt that chaiiges encountered after
irradiation were due chiefly to damage to liemopoietic tissues,
some change must have occurred in circulating erythrocytes
to iiicrease the rate of destruction. However, we could demoiistrate no change in fragility of unwashed red cells in salt
solutions, which is in accord with previous results we have
obtained and with the findings of several other investigators
after external irradiation (Taylor, Witherbee and Nurphy,
'19 ; Minot and Spurling, '24 ; Schoenholz and IIirscli, '29).
However, increased erythrocyte fragility has been reported
after using radium (Goulston, '32) and x rays (Holthuseii,
'24). Coincident with the great increase in macrophages
there may have been an increased erythrophagocytosis which
we did not find, although Tsuzuki ('26) and Clarkson, Mayiieord arid Parson ( '38) after x-irradiation could demonstrate
no increased phagocytosis of bacteria or neutral dyes.
Our experimental animals showed a parallel decrease in
erythrocyte count aiid hemoglobin, and hematocrit values declined to about half normal values after 15 days, indicating
some hemodilution had occurred. S o marked hemorrhagic
tendencies were noted in our animals, hut we did find eonsiderable pooling of erythrocytes in the hone niai'row, spleen
and adrenals in earlier stages of the experiment.
Hemoglobin values following 7 days post injection were
progressively lower than the corresponding values for
erythrocytes. This indicates progressive hypochromic anemia which surely was not due to iron deficiency with
such massive accuniulatioii of iron in macrophages of
the spleen (fig. 18) lymphatic nodes and other organs.
3i3
HEMATOLOGICAL EFFECTS OF P"'
During the experimental period in those animals tested,
the mean corpuscular volume remained within normal limits
but from 10 to 15 days after injection of PS2 the values
for mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were slightly l o ~ e r . (See table 4.) As
only two animals were so tested after 15 days these are not
statistically important but do tend to validate the erythrocytic hypochromia. One must assume some defect in transport or utilization of iron or defect in the manufacture of
the protein portion of the hemoglobin molecule.
TABLE 4
Average chattges i n ntean eorpiiscitlar colrrirte (M.C.77.), n i v ( r n c~orpcisculni.keinoglobin (M.C.H.), and mean corpuscular hemoglobin concentration
(M.C.H.C.) on the indicated days following injection
DAY
Normal range
Normal average
1
9
I
3
5
7
10
15
S O . ANIYALS
______
U.C.H.
X.C.H.C.
11"
111.19
72.
54-62
57.5
14-17
15.9
84-29
54.7
50.0
56.5
54.0
51.3
60.0
56.0
15.3
31.c.v.
___
16.9
16.4
16.3
16.2
15.7
13.8
_-
27.6
28.1
31.1
29.1
30.2
31.6
26.7
23.5
The initial sharp decrease in reticulocyte percentage coincides with a rapid decrease in average percentage of er;othroblastic cells in the marrow during the first two days after
injection. This represents not only a relative but an absolute
decrease in these forms as the marrow became hypoplastic.
Considerable evidence of destruction of polychromic and normochromic erythroblasts was seen. The average percentage
increase of erythroblasts between two and 5 days post injection was accounted for by more rapid destruction of iiiJ-elocytic elements during this period as the marrow became more
hypoplastic and erythroblastic destruction continued more
slowly. This relatively greater radiosensitivity of erj-thro-
374
J O H N S. LBTTA A N D RONALD E. WAGGENER
blasts is consistent with results of others (Dahl, '36; Brauer,
'40 ; Denstad, '43 ; Bloom and Bloom, '47 ; Brecker, Endicott,
Gump and Brawner, '48; Grad and Stevens, '50; Hennessy
and Huff, '50). From the 10th to the 15th days the marked
increase in the percentage of polychromic and particularly
normochromic erythroblasts represents both a relative and
actual increase as the marrow showed beginning regeneration,
though still quite hypoplastic, but little evidence of increased
delivery was noted.
~ezctrophiles:productioN, delivery and destructiom
Although the average percentage value of blood neutrophiles iiicreased from a preinjection level of 22% to a maximum of 58% two days after injection, their absolute number
sharply declined from a normal average value of 2,347/mm3
to a minimum of 147/mm3 after 10 days. This early percentage rise is accounted for by the more rapid decrease in the
number of lymphocytes, the total white count dropping precipitately from 10,300/mm3 to 3,000/mms during the first
two days post injection. Following the initial percentage
rise in neutrophiles which held through 5 days post injection,
their percentage declined sharply to a minimum value after
10 days, after which their value rose to 50% but their absolute number rose very slightly, there being no increase in
total leucocytes.
During the first two days the ratio in peripheral blood
bctween segmenters and younger forms increased from 6.3/1
to l0.2/1. This apparently was not due to delivery of segmenters from the bone marrow because there was evidence
of their retention in marrow, and because there was a sharp
decrease of total blood neutrophiles. We believe the increase
in ratio was caused by lack of delivery and maturation of
younger forms in the blood. The segmenter/younger forms
ratio gradually declined to nearly normal value by the end
of 7 days, with minor fluctuation thereafter. These we consider relatively inaccurate because of the very low total
1eucoc;vte count. It is probable that a few staff forms had
HEMATOLOGICAL EFFECTS O F P"'
375
been delivered to balance the small number of segmented
forms still present.
As previously stated the early proportionate increase of
more mature forms of neutrophiles (particularly staffs and
segmenters) in the marrow post injection indicates interference with their delivery to the blood. This has been considered to be due to endocrine control by lymphatic tissue
(Jacobson et al., '51) or other source (Johansen, '51). Following the second day a rapid decline in percentage and
actual numbers of neutrophilic myeloid elements occurred.
Considering the sharp decline in the total number of blood
neutrophiles over the first week post injection this could not
have been due to their delivery to the blood. From study
of sections the imprints of marrow, it is obvious that the
marrow was becoming continually more hypoplastic and that
the number of more mature neutrophiles constantly decreased
from the second through the 7th day post injection. The more
mature neutrophilic elements which had accumulated in the
marrow, were mostly destroyed in situ during the period
between two and 10 days after injection of the isotope, partially accounting for the great increase in unclassified degenerating cells in the marrow.
Fluctuations in neutrophilic myelocytes and juvenile forms
were not marked but they did steadily decline in total number in balance with destruction of other cell types. All neutrophilic elements had relatively and absolutely increased
after 15 days, indicating some recovery though less marked
than in the erythroid series.
Other cell types in marrow
A41though there was a remarkable percentage increase of
fixed reticular cells in the marrow during the first 10 days
post injection, this was explicable on the basis of their resistance to irradiation rather than actual increase. After
10 days the majority of the undamaged cells were classified
as either of this type or plasma cells. (See Tullis, '49, and
Liebow, Warren and DeCoursey, '49.) Their percentage decrease after 15 days is explained by beginning regeneration
3'76
J O H N S. LATT.4 A N D ROXALD E. l17AGGENER
of other cell types aiid by eliniiiiatioii of damaged cells and
debris. The perceiitages of ItemmytobZusts and promyelocytes
it must be assuiiied
being little changed after injection of
that some destruction but to a lesser degree than in the
erythroid and rieutrophilic series. Although percentages of
eosifiophiles of the blood fluctuated within normal range
their absolute nuniher decreased during the first 7 days,
proportionately with the decrease in iieutrophiles. Esamiiiatioii of the marrow indicated a coiistaiit depletion in balance
with destruction of other cell types. Siiiiilar o1)serratioiis
were made in respect to basoyl/iles.
Little quantitative change iii iiiegdiaryocytes occurred duriiig the experimental period. But persisteiitlp low percentage
values in increasingly hypoplastic marrow and histological
evidence of degenerative changes iiidica tes progressive destruction. These evidences consisted of iiidistiiict cell boundaries with increasing cytoplasiiiic vacuola tion, and iiuclear
pylmosis and fragmentation. (Figs. lt5-16.)
I t is interesting to note progressive accuiiiulatioii of plasma
cells in the niarrow through the first 10 days after injection.
This we interpret a s reaction of lymphocytes to injury. After
15 days their percentage and absolute iiumhers had decreased
as regeiieratioii of erythroid and iiiyeloid elements had begmi.
illyeloid-erythroid ratios sharply increased from the value
in our nornial animals of l.02/1 to 5.1/1 after two days partly
due to the observed iiiarrow retention of mature iieutrophiles,
but more definitely to the destruction of polychromic aiid
iiormochroinic erythroblasts referred to earlier. Although in
agreement with most findings the greater erythroblastic seiisitivity contradicts earlier findings (llilchiier and Mosse, '04 ;
Aubertin and Beaujard, '05 ; Icrause and Ziegler, '06; IJacassagne and Lavedaii, '24 ; Casati, '32 ; Halherstadter and Sinions, ' 3 3 ; TTIuiische, '38) or to those who could detect little
diff erelice in seiisitivity (Lacassagiie and Gricouroff, '41 ;
Koletsky and Christie, '50). The initial increase in ratio was
followed by a logarithmic type of decrease to reach .85/1
after 10 days. This decrease in ratio represents a more rapid
HEhIATOLOGICAL EFFECTS O F P3'
377
destruction of myeloid cells than of the remaining erythroblasts. After the 10th day degeneration of the erythroblastic
series had been just enough greater than that of the myeloid
series to change the niyeloid-erythroid ratio only slightly.
L y w p h o c y t m : production, delivery cind destruction
The rapid, marked decrease noted in the number of blood
lymphocytes following injection of
mas a good indication
of the rapid severe clamage which took place in the lymphatic
tissue. Progressively severe destruction of lymphocytes was
observed in spleens and lymph nodes of all experimental
animals. The small percentage of lymphocytes in the bone
marrow also declined a s the percentage of plasma cells increased. However, the percentage of marrow lymphocytes
leveled off from the 5th to the 10th day while the percentage
of plasma cells increased. As the marrow became more
severely hypoplastic through this period, actual numbers of
lymphocytes must have decreased throughout. The decreased
percentage in the marrow of both lymphocytes and plasma
cells hetween 10 and 15 days can be explained by the beginning regeneration of erythroid and myeloid elements.
The sharp decrease in the percentage of blood lymphocytes
froiii it norninl value of 745% to 38% after two days post
injection, was due to a more rapid aboslute reduction of
lymphocytes than of granulocytes in the blood. The average
iiumher of lymphocytes after one day had dropped from
7,700/inm3 to 2,000j1nm3, while the number of neutrophiles
h a d declined from 2,400/mm3 to 2,100/mm3. The relative
values of each type were quite similar through the 5th day,
though absolute values of both had decreased further (lyniphocytes S24/mm" and neutrophiles 938/mm3). Following
this ( 7 days) the percentage of lymphocytes had increased
because of still rapid decline in blood neutrophiles, while
delirery and loss of blood lymphocytes had apparently become balanced. After 10 days the same trend was apparent
there being 147 neutrophiles/rnm~and 384 lymphocytes/mm3.
3178
J O H E S. LATTA A N D RONALD E. WAGGENER
The greater proportionate and more rapid decrease of lyniphocytes as compared with neutrophiles is associated with
their greater sensitivity to irradiation as usually reported
(e.g., Jacobsen, Marks and Lorenz, '49 ; Koletsky and Christie,
'50 ;Wheeler, Jackson and Hahn, '51 ;Ragaz and Van Baaren,
'53). After 15 days absolute values of neutrophiles had increased but those for lymphocytes had not changed appreciably, further indicating more the rapid recovery of neutrophilic production and delivery (see Warren, '43 ; and Warren,
Slacmillan and Dixon, '50).
This continued low production and delivery of lymphocytes
tiiroughout the experimental period is in keeping with the
remarkable reduction and degeneration of lymphocytes in the
lymphatic tissue of the spleens and lymphatic nodes examined
which was progressive throughout the days of observation.
SUMMARY
Forty-two young adult albino rats from an inbred colony
were each injected with a single dose of 4.5 yc of radioactive
phosphorus (PS2)/gm of body weight 11 days after determination of their normal hemograms. Six animals were sacrificed after one, two, three, 5, 7, 10, and 15 days following
injection, when their hemograms were again determined.
Femoral bone marrow imprints and sections were prepared
from each animal and sections of spleen and lymph nodes.
Six normal rats from another group were used as controls
f o r normal marrow, spleen and lymph nodes. Hemosiderin
stains were made on spleen sections from 1 2 normal and 7
treated animals.
S f t e r injection the total erythrocyte count was progressively depressed to an average of less than 90% of normal
after 7 days and to about 50% of normal after 15 days. The
percentage and absolute numbers of erythroblasts, particularly of polychromic and normochromic stages decreased
sharply and remained low during the first 10 days. Many
erythroblasts were undergoing degeneration. After 15 days
some regeneration of normochromic erythroblasts had oc-
HEMATOLOGICAL EFFECTS O F P"
379
curred. The delivery of cells, as indicated by reticulocyte
percentages, was reduced but did not stop entirely.
The severe anemia cannot be entirely explained on the basis
of damage to erythropoietic centers. Hematocrit values mere
significantly reduced and icterus indices were slightly increased after injection of PS2.I n addition there was a marked
increase in the accumulation of hemosiderin in the splenic
macropliages.
The total leucocyte count was quickly depressed to an
average of about 40% of normal after one day post injection.
A minimum average value of about 20% of normal vas
reached after 5 days, with little following change until after
15 days when slight recovery was noted.
Differential counts of blood showed that the number of
lymphocytes decreased more rapidly and more markedly
(from a normal, average value of 7,700/mm3 to 342/mni3
after 15 days) than neutrophiles, which were also severely
depressed (from a normal average value of 2,400/iiini3 to
147/mm3 after 10 days).
The early decrease in blood neutrophiles coincided with
retention of large numbers of these cells in the marrow,
accompanying the destruction of erythroblasts. After three
days progressive marked destruction particularly of later
stages of marrow neutrophiles occurred until after 10 days,
along with continued damage to erythroblasts. Some recovery
of both erythroid and myeloid elements was noted after 15
days.
The marked reduction of blood lymphocytes coincided with
their marked destruction and reduction in the lymph nodes
which occurred after one day post injection and was progressive throughout the experimental period. Similar progressively severe damage was encountered in spleens froin
experimental animals, first in the red pulp and later in the
lymphatic tissue of the white pulp. No evidence of recovery
was seen in any lymphatic tissue during the experimental
period. Lymphocytes practically disappeared from the bone
380
JOHN S. LATTA ASL) ROSALU E. WAGGESER
marrow, most of which probably became modified a s plasma
cells in reaction to the irradiation.
The radioresistant reticular cells became more and more
proniiiient in all hemopoietic tissues following injection as
progressive hypoplasia occurred. blacrophage production
markedly increased most of which contained debris of fragmented cells or deposits of hcmosiderin or both.
LITERATURE CITED
ATBCRTIX,C., SND E. BEAU.JARD
1905 L4ctions des iayons X sur le sang et les
organes Irematopoietiques. C . R. d e la Soc. de Riol., 5 8 : 217-219.
BERLIS,S. I., L. Af. METER A N D M. LAZARUS1951 Life spau of the r a t red
blood cell as determined by glycine-2-C-14. h i . J. Ph? siol., ZG5 :
565-567.
DLOOJI,M. A., AND W. ELOON1947 The radiosensitivity of erythroblasts. J .
Lab. and Clin. hfed., 32: 654-659.
RRATER,R. 1940 Untersucliungen uber die Einwirkung unterscliiedlich verabfolgter Roentgenstrahlung auf das Knoelienniark und seine Zellelemente,
zugleich ein Eeitrag zum Zeitfaktorprohlem. Strajilentherap., G 7 :
424-501.
BRECHER,G., K. M. EXDICOTT,
H. GVMP A N D H. P. RRAWNER1948 Effects of
X-ray on lymphoid and hemopoietir tissues of albino rats. Blood, 3:
1259-1274.
CASSTI, A. 1932 Esperimentelle Untersucliungen uber die Rontgen\t irkung auf
das Knochenmark. Stralilentherap., 43 : 588-588.
C'LIRKSON,J. R., Mr. V. MAYNEORD
AND 12. I). PARSON
1938 The cffeet of total
body X-irradiation on the blood and lymphoid tissue of tnnior hearing
animals. J. Path. and Bact., 4 6 : 221-235.
I ~ H LB.
, 1936 De l'effect des rayons X sur os longs en deT-elopnient ct sur
la formation de cal. Jacoh Dybwad, Oslo.
])EXSTAD, T. 1943 The radiosensitivity of tlie bone marrow. S c t a Radiol. Suppl.
52.
Dr XLAP, C. E . 1948 I n : Pathology, cdited by \V. A. D. Anderson. C. V. Mosby
Co., St. Louis.
GOULSTON,D. 1932 The increase iii tlie fragility of the red blood corpuscles
a f t e r exposure to radium. Rr. J. Rxdiol. N. S. 5: 7iJ-ii9.
GRAD,R., A N D C. E. STEVENS1930 Histological changes produced by a single
large injection of radioactive phosphorus (P") in albino rats and in
C,H mice. Cancer Research, 20: 289-296.
HALBERSTADTFR,
L., IND A. SIMONS1933 Handbuch der Allgemeinen Hematologie. Edited by H. Hirschfeld and A. Hittrnair, Urban and Schwarzenberg, Berlin, 1: 1420-1430.
ESSEY, T. G., AND R. L. IIUFF 19.50 lkpression of tracer ion uptake curre
i n r a t erythrocytes following total body X-irradiation. Proe. SOC.Esp.
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HEMATOLOGICAL EFFECTS OF P32
381
HOLTHUSEN,
H. 1924 Die Wirkung der Rontgenstrahlen in biologischer Hinsicht.
Strahlentherap., 18: 241-262.
JACOBSON,
L. O., E. K. MARKSAND E. LORENZ1949 The hematological effects
of ionizing radiation. Radio]., 52: 371-395.
JACOBSON,
L. O., E. MARKS, E. SIMMONS,
M. J. ROBSOXAND E. GASTON 1951
Further studies on factors influencing recovery from radiation injury.
Proc. 3rd Internat. Congress of the Internat. SOC.of Hemat. Grune
and Stratton, New York.
JOHANSEN,
C. 1951 A new theory of splenic marrow inhibition. Proc. 3rd
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A., AND G. GRICOUROFF1941 Actions des radiations sur les tissus.
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1924 Les modifications histologiques du sang
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J. S., J. L. VOSE A N D R. C. ROSENLOF 1952 Correlation of changes in
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L., D. RITTENBERG
AND K. BLOCH 1949 Utilization of acetate f o r
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382
JOHN 8. LATTA A N D RONALD E. WAUGENER
WARREN,S., J. C. MACMILLANA N D F. J. DIXON 1950 Effects of jnternal
irradiation of mice with P-32. I. Spleen, lymph nodes, thymus, bone
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WHEELER,B., M. JACKSON
AND P. HAHN 1951 Hematology of the dog following
intravenous administration of radioactive colloidal gold. Am. J.
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EXPLAXATION O F PLATES
I n all cases illustrated the rats had been injected with 4.5 pc of P3'/gn1 of body
weight. Pnless otherwise noted the stain used was Delafield's hematoxylin Azur
11-EOS.
PLATE 1
EXPLANATION OF FIGURES
9
Section of femoral bone niarrow after two days following injection, showing
beginning hypoplasia and replacement with f a t cells. X 102.
1.0
As above after 7 days following injection, showing extensive hypoplasia and
degeneration. X 102.
11 As above after 1 0 days, showing extreme depopulation of the inarrow and
replacement with f a t cells. X 102.
12
As above after 15 days, shooing beginning recovery of the cell population in
the marrow. X 102.
13
Imprint from femoral hone marrow a f t e r 15 days, showing the high percentage of normochromic erythroblasts in the regenerating marrow. WrightGiemsa. X 288.
14 Imprint from femoral bone marrow, showing the accumulation of large numbers of staff and segmented neutropliiles two days following injection. WrightGiemsa. X 288.
IIEMATOLOGICAL EFFECTS OF Pa
J O H N S. LA’CTA A N D RONALD E. WAGUENER
PLATE 2
EXPLANATION O F FIGURES
15 Megakaryocyte in an imprint from femoral marrow t n o days after injection.
Note the nuclear pyknosis, vaeuolated cytoplasm and indistinct cell boundary.
X 1136.
16
Another rnegaltaryocyte from an imprint of marrow 15 days after injection,
showing complete karyolysis, marked cytoplasmic vacuolation and indistinct
cell boundary. X 1136.
17
Section of spleen removed 7 days after injection, showing extensive deposition
of hemosiderin in the red pulp and in the reactioii centers of the nodules.
Ferrocyanide-HC1 stain. X 7 2 .
IS
Similar section of spleen removed 15 days after injection, showing massive
deposition of hemosiderin in the red pulp and in the waction centers. Ferrocyanide-HC1 stain. X 72.
19
Section of a lymph node removed 7 days after injection showing the extensive
damage illustrated by nuclear pjknosis fragmentation and debris of lymphocytes. Lymphatic tissue is much reduced and the widened sinuses well filled
with macrophages containing cellular debris and/or hemosiderin. x 102.
20
Section of another node 15 days a f t e r injection, showing the great decrease
in lymphatic tissue and markedly widened sinuses containing macrophages
laden with debris and hemosiderin. Most of the lyinpliocytes show degenerative changes. X 102.
384
H E M A T O L O G I C A L E F F E C T S O F P"
J O H N S. U T T A AND RONALD E. WAGGENED
385
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