HAEMATOLOGICAL STUDIES IN EXPERIMENTAL
          TUBERCULOSIS'~*

Variations in the Blood Cells of Rabbits Inoculated with
      Cultures Differing in Virulence

K. C. SMITHBURN, F. R. SABIN AND L. E. HUMMEL

AS long ago as 1896, it was observed that there were changes in the
cells of the circulating blood during tuberculous infection. Holmes
(1) stated that the characteristic changes were: a decrease in small
lymphocytes, an increase in phagocytes (neutrophiles), usually an in-
crease in large lymphocytes (probably monocytes, as these cells were not
then differentiated from lymphocytes), a decrease or disappearance
of the eosinophiles, and certain qualitative changes of disintegrative
nature involving in particular the phagocytes.  Subsequent work has
elucidated and elaborated upon Holmes's work, but the basic obser-
vations still stand. Ameth (2) and Lewis (3) observed qualitative and
quantitative changes in the neutrophilic leucocytes, which were believed
by Ameth (2) to be specific and diagnostic for tuberculosis.  The latter
was shown not to be the case by Lewis (3). In 1902 Lacaptre (4) noticed
changes in the blood cells very soon after inoculation of tubercle bacilli.
The most constant change was an increase in the number of large mono-
nuclear cells. Webb and his associates (5 to 8) later found that lymph-
ocytosis was associated with increased resistance and that lymphopenia
was evidence of advancing disease. Webb (8) believed that lymphocytes
were able to engulf tubercle bacilli.  But the identification of the mon-
ocyte by Schilliig (9) and by Sabin (lo), and subsequent studies of their
phagocytic properties by Sabin and Doan (ll), Lurie (12), Gottlieb (13)
and others have shown that it is this cell and not the lymphocyte.which
has the capacity to engulf tubercle bacilli.  However, the work of Webb
on the relation of lymphocytes to resistance in tuberculosis was con-
firmed and extended by Murphy and his coworkers (14 to 18).  It has
also been indicated (39,43) that the state of maturity of the lymphocyte

' prmM in part at a session of the Pathological Section at the 331-d annual meeting of
the National ~ubefiulosis Associition, Milwaukee, Wiionsin, June 2,1937.
' From the ~hatories of The RockefeUer Institute for Medical Research, New York City.
                       673


674          SMITHBURN, SABIN AN-D IIUMMEL

may be important; induced lymphocytosis does not invariably result in
enhanced resistance (39).

The identification of the monocyte (9,10) made possible studies of the
r8le of this cell in tuberculosis. Cunningham, Sabm, Sugiyama and
Kindwall (19) observed that with advancing disease the monocytes in-
crease in number, while the lymphocytes decrease, so that there is an
elevation of the monocyte-lymphocyte ratio.  Studies by Sabin, Doan
and Cunningham (20) further emphasized the significance of the mono-
cyte-lymphocyte ratio. These experimental studies have received ample
confkmation in both the laboratory and clinic (21 to 26,35).

Recently Medlar (27,28) has studied the blood cells in clinical tuber-
culosis; he has stressed the r8le of the neutrophilic granulocytes and dis-
cussed the relation between the blood picture and the evolution of lesions
in the tissues.  The part played by each of the blood cells was discussed
and three types of leucocytic patterns were observed in his patients.
These were described as the septic, hyperplastic and nonseptic types
(28). Crawford (29) devised a simple calculator for determining the
leucocytic index, which takes into account the neutrophile-lymphocyte
ratio and abnormal values for monocytes and total white cell count.
This index was used by Medlar in extensive clinical studies (30 to 32).
Sweany (26) has employed both the lymphocyte-monocyte and the
neutrophile-lymphocyte ratios, together with erythrocyte sedimentation
and certain chemical findings, and has devised charts which express
graphically the course of events.

As result of these earlier studies, it is generally agreed that carefully
performed total and differential leucocyte counts are of considerable
prognostic value in experimental and clinical tuberculosis.  It is believed
that the results to be described in the following paragraphs throw new
light on the r6le of the various blood cells in reflecting the course of
tuberculous infection.

MATERIALS AND METHODS

The animals used in this study and the experimental procedures, except
blood counts, were described in the first paper (36) and will be repeated
here only briefly.

Forty normal, young adult, New Zealand red rabbits were used.
Studies of the blood were made in the period from September to Decem-
ber, 1935. The animals were then divided into four groups of ten each
with litter mates equally distributed. Animals of merent age, sex


IL4EhtA~OLOG'yc)~ EXF'ERIMENTAT. TUBERCULOSIS     675

and weight were so distributed as to make the four groups as nearly alike
as possible.

Four lines of cultures, each derived from the bovine strain 39, and
grown for five successive generations at pH values of 6.0, 6.4, 6.8 and
7.2, were used.. .DifIerences in pathogenic properties of these bacteria
were known to exist, as determined from results of a prior experiment.
Each of the cultures was inoculated intravenously into one group of ten
rabbits. 8 A standard dose of 0.01 mgm. (moist weight, about 500,000
bacteria) was used throughout.  Before and after inoculation, all animals
were kept under similar conditions.  None was subjected to any thera-
peutic procedure.

Following inoculation, counts of the blood cells were done on each
rabbit once weekly until death from tuberculosis occurred, or until the
last remaining animals were sacrificed 6 months following inoculation.
Haemoglobin determinations were done by the Newcomer method;
all were read by one disinterested person to whom the identity of the
individual animals wss unknown.  Standardized blood diluting pipettes
and standardized double-ruled Levy-Hauser counting chambers were
employed for the blood counts.  Differential leucocyte counts were done
by the authors, using the supravital technique; identity of the rabbits
was unknown to the individuals making the counts. As routine, 100
cells were counted on each of two coverslip preparations; when there were
appreciable differences between the two counts, as many as 1,000 cells
were counted. Total erythrocyte and leucocyte counts were done by
competent technicians experienced in this work.

Statistical analyses of the blood studies were made following the ter-
mination of the experiment.  Data for each group of ten animals were
treated separately.  The 75 counts done on each group prior to inocula-
tion served as specific controls.  Counts following inoculation were
analyzed by months, that is, all the counts on one group during the first
month after inoculation were treated together, etc.  Values for the ab-
solute number of each of the blood cells were used. Employing the .
methods and formulae outlined by Davenport and Ekas (33), the mean,
standard deviation and probable error of the mean were determined for
each type of blood cell in each group of animals for each time interval,
namely, control period, first month after inoculation, second month,
etc. The signiscance of changes in the various blood cells was deter-
mined by calculating the deviation from the control mean, determining
the probable error of this deviation, and dividing the former by the latter.


This quotient expresses the deviation as a multiple Of its probable error.
The statistical significance (probability) of various values of such quo-
tients was taken from Pearl's (34) table 40.  From this table (or Daven-
port and Ekas's (33) table 11, p. 178), it may be seen that deviations
having the value of PE X 3.8 occur by chance only once in 96 times, and
those having the value PE X 3.9 occur by chance only once in 117 times.
Therefore the value PE X 3.85 was taken as the limit for normal varia-
tion; and deviations of order greater than this have a probability of 100
to 1, or more, of being significant.  Deviations of lesser order were con-
sidered not to be of certain significance.  The shaded areas on charts
1 to 5 show the magnitude only beyond which deviations of the mean
counts were considered to be certainly sign&ant. Comparisons of
the deviations in any one type of cell in one group of rabbits with those
in a group inoculated with a merent culture were thus made, not on the
basis of a common normal value, but on the basis of the normal value
obtained in each particular group.  It should be mentioned, however,
that differences in the mean normal values among the four groups were
not of high order.

The monocyte-lymphocyte ratios, proposed by Cunningham, Sabin,
Sugiyama and Kindwall (19), and the neutrophile-lymphocyte ratios,
proposed by Crawford (29), were calculated for each individual count;
these data were thereafter treated in precisely the same manner as those
for the blood cells.                                    .
The data included 300 counts done before inoculation and 609 COUPES
done after inoculation. The grouping of data during the disease period
was such that the mean values represented not less than seven and not
more than forty-five counts.

RESULTS

The marked differences in virulence of the four cultures used for in-
oculation of the four groups of animals (36) made possible a study of the
blood cells in various grades of tuberculous infection from mild to severe.
It was shown (36) that the culture grown at pH 6.0 was markedly atten-
uated (virulence +), that grown at pH 6.4 was moderately attenuated
(virulence ++), that grown at pH 7.2 was slightly attenuated (tiru-
lence + + +), and that grown at pH 6.8 was fully virulent (+ + + +)a
Hereafter, in order to facilitate discussion, the four groups of animals
will be referred to by numbers 1 to 4, corresponding to the virulence of


HAEIKATOLOGY OF EXPERIWZNTAL TUBERCULOSIS     677

the culture, group 1 being that inoculated with the least virulent culture,
etc. Among the four groups of animals, each type of blood cell at some,
time in the disease showed significant change from the normal. In
presenting the data, each type of cell and the two cellular ratios will be
discussed separately. Finally, the efficiency of each cell in showing sig-
n3-t change will be discussed.
Haemoglobi~: Each of the four groups of animals showed a significant
fall in haemoglobin during the first month after inoculation, as shown on
chart 1. The magnitude of this fall was greatest in those inoculated
with the most virulent culture and least in those receiving the most
attenuated culture. During the second month of the disease, only the
animals of groups 3 and 4 showed significantly low values for haemo-
globin. Paradoxically, however, the surviving animals of group 4
did not show abnormal values for haemoglobin during the third month.
This may have been due to the fact that only seven counts were obtained
and five of these were on the one animal of the group which survived
more than 72 days. During the third and fourth months, only the ani-
mals of group 3 showed abnormally low haemoglobii. But in the
fifth and sixth months, those of groups 1 and 2 also showed low values.
Every significant deviation of haemoglobin from the normal was in the
direction of a decrease. Of the nineteen means determined according to
group per month, twelve were significantly low, giving an efficiency of
63.15 per cent.

Red blood cells: During the first month of the disease, only the rabbits
of groups 4 and 2 showed marked decrease in the red blood cells, as
shown on chart 1. During the second and third months, none of the
groups showed abnormal values for red blood cells. In the fourth
month, those of group 3 showed a decrease. In the fifth and sixth
months, the rabbits of group 2 had red blood cell counts which were
significantly low. The rabbits receiving the most attenuated culture
(group 1) showed significantly low erythrocytes only in the sixth month.
Roughly then, the anaemia was proportional to the severity of disease;
but of nineteen mean values after inoculation only six were significantly
-low, giving an efficiency of only 31.57 per cent. All significant deviations
were in the direction of a decrease, however.

TotuZ Zeucocytes: In the fist and second months following inoculation,
only the animals of group 4, those inoculated with the most virulent
bacteria, showed a significant change in total leucocyte count; this was
a decrease. In the third month, none of the groups showed significant


678          SlKITHBURN, SABIN AND HUMMEL

deviation from the normal mean values. In the fourth month, groups
1 and 2 showed values which were abnormally high. Only group 2
exhibited an abnormal level for leucocytes in the fifth month, namely, a
rise. In the sixth month, groups 1 and 2 showed significant elevation.

             t 5

        "0

                      2   3   4   5   6

CHART 1.' Monthly mean deviations of haemoglobin and erythrocytes expressed as
multiples of their probable error.

The decreases, recorded on chart 2, occurring in the animals inoculated
with the more virulent organisms, exemplify the leucopenic phase de-
scribed by Sabin, Doan and Cunningham (20).  Of the nineteen mean

* Deviations in charts 1 to 5 inclusive are shown as multiples of the probable error of the
deviations. The odds against occurrence by chance are small in the case of all points falling
in the shaded aone and their significance is not certain.  But the odds against occurrence
by chance are high in the case of deviations falling outside the shaded zone; such deviations
are considered significant.  The horizontal line in the centre of the shaded zone represents
the normal value; the direction of each deviation from the normal is shown.  The +ole
number and decimal at the end of each curve indicate the pH of the medium on which the
cuhure was grown for inoculation of the animals represented by that curve.  The curves
diier in length according to the survival time of the rabbits represented.


HAEMATOLOCY OF EXPERIMENTAL TUBERCULOSIS     679

values in the four groups, seven deviated significantly from the normal,
so that the efficiency was 36.84 per cent. Of these seven significant de-
viations, two were below the normal level and five were above.  It was
interesting that group 3, receiving the culture grown at pH 7.2 (virulence
+ + +), at no time showed deviations of high order (chart 2).
NeutrojMes: Only the animals of group 4 showed abnormal values for
neutrophiles in the first two months following inoculation. This change,
a sharp decrease (chart 2), corresponded to the anaemia-leucopenia
phase of the disease as demonstrated in table 1.  In the third month

10'    I    I 1    1    I   I J

CHART 2. Monthly mean deviations of white blood cells and neutrophiles expressed as
multiples of their probable error.

this group of animals exhibited normal values for neutrophiles, as did
those of groups 3 and 1.  However, the rabbits of group 2 showed an
increase in neutrophiles at this time and thereafter the values were
always significantly high in this group.  In the fourth month the animals
of group 3 showed an increase in neutrophiles.  The group 1 which re-
ceived the most attenuated culture exhibited only one abnormal monthly
mean value, a rise in the sixth month.  Of the nineteen mean values
after inocuIation in the four groups, eight showed significant deviation
from the normal, giving an efficiency of 42.10 per cent.  Two of these


680       SMITHBURN, SABIN `AND HtMMEL

eight significant deviations were in the direction of a decrease (in ani-
mals inoculated with highly virulent organisms) and six were in the op-
posite direction. It was interest&g and probably important that the
increases in neutrophiles which did occur, did not take place until the
third month in any of the groups, despite the fact that the disease was
sufficiently established in three of the four groups that deaths were
occurring., Terminal neutrophilic leucocytoses were noted in certain
individual animals but were not of sufficient magnitude to cause signif-
icant elevation of the mean lfor the groups.  In the group receiving the
most virulent microiirganisms, in which most of the deaths occurred in
60 days or less, even individual counts taken just prior to death exhibited
neutropoenia, although their lesions exhibited the most extensive casea-
tion.

Bu.@iEes: The counts of basophiles preceding inoculation in these
groups of animals were lower than those reported by Pearce and Casey
(37) and by Thomas (40). It was shown by Sabin, Miller, Smithburn,
Thomas and Hummel (38) that the basophiles do not reach maximum
levels until the time of maturity.  Although all animals in the present
experiment were of mature age when inoculated, at the midpoint of the
period before inoculation twenty-four, or 60 per cent, were between 4.2
and 5.9 months old. Higher values might, therefore, have been obtained
had the base-line counts been made later,  However, the mean of the
final preinoculation counts of basophiles in the forty animals was 631, a
figure still below that obtained by the investigators mentioned; at this
time each animal was at least 6 months old.

No significant change in basophiles occurred in the animals receiving
the most virulent culture, at any time. But in the first month, the
other three groups each exhibited an increase.  In the rabbits of group
2 the rise extended through the second and third months.  No other
groups exhibited significant deviation in the second month. In the
third month, those of group 3 again showed a significant rise, but in the
fourth month there was a fall of equal magnitude. In the fourth,
fifth and sixth months, the rabbits of group 1 again showed a rise in base-
philes, while those of group 2 exhibited no significant deviation from the
normal. Thus there were deviations of apparent signifkance in three
of the four groups, some above and some below the normal. These
changes were neither related to the virulence of the infecting agent, nor
to the resistance of the host. Ten of the nineteen values following in-


EAEMATOLOGY OF EXPERIMENTAL TUBERCULOSIS     681

oculation showed significant deviations from the normal. Of these,
nine were in the direction of an increase, while one was in the opposite
direction, as shown on chart 3. It is interesting to note that, during the
leucopenic phase, the animals receiving the most virulent microor-
ganisms did not exhibit a decrease in basophiles, although there was a
decrease in all other cellular elements originating in the bone marrow.
It is also notable that none of the apparently significant elevations of
the basophile counts (see table 1) exceeded the normal values obtained
by Pearce and Casey (37).

t 1o PMB. @fkkncy 52.6370    I

t

1o, `ME. etfieiency 42~072
,

&UtT 3. Monthly mean deviations of basophiles and eosinophiles expressed as multiples
of &+xobable error.

Eosimphiles: Chart 3 shows that the ten animals of group 1 at no
time showed significant deviations from the normal count of eosinophiles.
Group 2 showed a significant decline in the first and fifth months only.
Those of group 4 showed a significant decline in the first and second
months, but not in the third. Group 3 showed significantly low eosino-
philes in each of 4 monthly periods following inoculation. Thus, of
eight significant deviations, all were downward and all were in the three


682         SMITHBURN, SABIN AND -L

groups receiving the more virulenE inoculum.  The decline was not de-
pendent on the leucopenic phase, as may be seen in group 3 ; these ani-
mals had no leucopenia (see charts 2 and 3).  (The eosinophiles do not
disappear entirely, as previously reported by some workers, but are
present in such small numbers during severe tuberculosis that often many
cells must be counted to find a single eosinophilic leucocyte.)
Lymphoqies: None of the four groups of animals exhibited abnormal
values for lymphocytes in the first month after inoculation, as can be

t 2. Mono. efficienoy 78.95 70
                p6.4

Monks  1   2   3   4   5   6

CJ~ART 4. Monthly mean deviations of lymphocytes and monoqtes express& as multiples
of their probable error.

seen from chart 4. A significant decline in circulating lymphocytes
occurred in each group during the second month. The lymphopenia
was marked in each case, but most profound in group 4; it persisted in
every group throughout the disease, except in group 1.  The latter ani-
mals exhibited a fall in lymphocytes, and then a rise, so that by the sixth
month the mean value was not significantly low.  From chart 4 it may
be seen that the curves for lymphocytes more faithfully reflected the


HAEMATOLOGY OF EXPERIMENTAL TUBERCULOSIS     683

course of the disease than any of the other blood cells, that is, the magni-
tude of change most closely paralleled the severity of the infection.
However, as will be shown, they were less sensitive than the monocytes
in that the latter showed abnormal values earlier.  Fourteen of the nine-
teen mean monthly values for lymphocytes following inoculation were
significantly low, giving an efficiency of 73.68 per cent for these cells.

Mo~~cytes: The rabbits of group 4 at no time showed significant
deviations from the normal counts of monocytes, as shown on chart 4.
The absolute values were at alI times above the base-line mean (as shown
in table 1) but the deviation was not statistically significant.  The ani-
mals of group 1 exhibited a mean value for monocytes in the second
month which was within the normal range.  All other monthly mean
values in all the groups showed significant deviations in the direction of
an increase.  Thus, in three of the four groups there was a signifkant
rise in the first month after inoculation, and in two groups this rise per-
sisted throughout the disease.  Fifteen of the nineteen monthly mean
values for monocytes showed sign&ant elevation, giving an efficiency
of 78.95 per cent.  Thus these cells were more sensitive than any others
in reflecting the course of events; but the fact remains that, in the ani-
mals exhibiting the most severe infection, the circulating monocytes did
not show an increase of high order.

iVeutro#iZe-lymphocyte ratio: The animals of group 4 at no time
showed significant deviation from the mean base-line value for the neu-
trophile-lymphocyte ratio.  In this group there was a rise in the second
and third months (table 1) but it proved not to be statistically significant.
Group 3 showed significant elevation of the neutrophile-lymphocyte
ratio only in the fourth and final month of disease.  Again the mean val-
ues were elevated in the second and third months, but the rise was not
significant as the probable error of the deviation was very high.  The
two groups receiving the more attenuated cultures showed signikant
elevation of the neutrophile-lymphocyte ratio during the second to sixth
months following inoculation. This elevation was in large part due to
the decrease in lymphocytes, since nine of the ten correspondmg values
for lymphocytes were significantly low, whereas only five of the ten cor-
responding values for neutrophiles were significantly elevated.  Eleven
of nineteen mean values showed significant elevation, so that the e5-
ciency of the neutrophile-lymphocyte ratio wa\ 57.89 per cent. But
it is important to note that only one of seven mean values following in-


684         SMITHBURN, SABIN AND EUMI@L

oculation showed significant elevation from the normal in the two groups
of animals inoculated with the two more virulent cultures.  Deviations
of the neutrophile-lymphocyte ratio are shown on chart 5.

Momcyte-Lymphocyte ratio: Statistically significant deviations from
the normal monocyte-lymphocyte ratio occurred in all the counts follow-
ing inoculation except in that for the first month in group 4 and that
for the second month in group 1.  Thus, of nineteen mean values at all
stages of the disease, seventeen were significantly elevated.  The e5-

t20x t?&io Efliciency - 89.47 s
          I

CHART 5. Monthly mean deviations of the monocyte-lymphocyte and neutrophik-lympho-
cyte ratios expressed as multiples of their probable error.

ciency of this ratio was therefore 89.47 per cent, and it was more sensitive
in detecting the presence of infection than the neutrophile-lymphocyte
ratio, the haemoglobin, or any of the blood cells considered separately.
Furthermore, although the values given on chart 5 show no parallelism
with the severity of infection, the actual mean values for the index given
in table 1 do show greater change in the groups receiving the more viru-
lent micro8rganismq and progressively higher mean values in each group.
Table 1 also shows that the mean values for the monocyte-lymphocyte


      HAEbUTOLOGY OF EXPERIMENTAL TUBERaoSIS     685

                      TABLE 1
&fan -al and mmn monthly Q~stinoGurorion wlwfw euch :y& of blood cdl, for the monocyta-
    lym#ocyk and n6uiroQhib-hfn#oVk ratios, ad for haanwglobin in
            each of the four groutb of animals

Haemoglobin . . . . . . . . I

Red blood cells.. . . . . . I

Whitebloodds ..... I

Neutrophiks. ........ I

Bawphiles ........... 1

Eoainophilu ..........

Lymphocytes, . . . . . . . i

IMOm. . . . . . . . . . . i

-

c

--

-

6.0
6.4
6.8
7.2

---
79.6 78.6 67.5
78.2   76.5   61.1
77.3
72.0 61.0

6.0
6.4
6.8
7.2

Msa- nasr   SEWND
IML YoNTa   MONTE

---
82.2   77.2 81.3
79.9   71.7 78.4
78.9   69.7 72.1
80.5   74.9 75.0

5,814 5,611 5,887
5,597 5,152 5,641
5,683 5,123 5,431
5,691 5,526 5,478

5,818 5,742 5,567
5,530 5,329 5,054
5,211
5,307 4,990

6.0  10,030 11,064 9,139  9,433 11,997 11,137
6.4   9,470 10,132 9,592  10,380 12,380 11,88Cf
6.8  10,268 8,654 7,083  9,778
7.2   9,969 11,155 9,166  10,851 12,185

6.0   4,345 4,850 4,461  4,287 4,941 4,895
6.4   4,003 3,727 4,661  5,181 5,942 6,591
6.8   4,417 3,094 2,931  5,324
7.2   4,687 4,540 4,071  4,776 6,224

6.0    537   785 643
6.4    440 641   885
6.8     590   578 772
7.2     586   880 93s

6.0     154   175 180
6.4    122 65 81
6.8    147 64 45
7.2     156   102 81

642 870   845
709   7.57 406
485
889   351

150   155 192
84 78 68
142
100 67

6.0   4,108 3,996 2,864  2,945 3,165 3,066
6.4   3,975 4,062 2,460  2,654 2,616 2,222
6.8   3,971 3,434 2,030  1,948
7.2   3,469 3,732 2,472  2,244 1,927

6.0    872 1,230 963  1,393 2,067 2,114
6.4    914 1,619 1,479  1,753 2,954 2,575
6.8   1,123 1,463 1,251  1,879
7.2   1,058 1,890 3,584  2,815 3,552

sum
moluln

71.1
60.8

5,493
4,365

11,988
13,705

5,484
7,068

925
318

210
77

3,538
3,006

2,401
3,221


686     SMITIIBUBN, SABIN AND IIUMJKBL

TABLE l-Con&&d

                INOCV-
                 LAnm                         xouxra rare snrm
                                             MONIN  YONTX  YONTX

               =pK
                ---       -----.
                                      1.63 1.87   1.90
Neutrophile-lyplpho-                            2.45   3.77 2.47
cyteratio... . . . .                            3.63

                                    0.712 0.820 0.853
Monocyte-lymphocyte                          1.307 1.387 I.113
ratio . . . . . . . . . . . . . . .                      I.384 I.938

Note: Statisticaily significant deviations from the normal appear in italics. Vahres
shown in ordinary type proved not to be statisticaBy significant deviations from the normal.

TABLE 2

.!?..icncies of blood cells and cellular rorios in showing abnolmol v&es in hbbxlos~

Ratio monocytes-lymphocytes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    89.47
Monocytes........................................................    78.95
Lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    73.68
Haemoglobm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    63.15
Ratio neutrophika-lymphocytes.. . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . .    57.89
Baaophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    52.632
Eosinophiles.......................................................    42.10
Neutrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    42.10**
Totalwhitecells................................................... 36.84**
Erythrocytes . . . . . . . . . . . . . . . . . . . . . . _ . _ . . _ . . . . . . . . . . . . . . . . _ . . . .    31.57

o Data open to question as base-line values were lower than those of Casey and Pearce,
and of Thomas. Animals may have shown rise in basophiles due to tiuence of state of
maturity.

o * Leas reliable than might be indicated, since some values were high and others low,
indicating that both determinations are subject to variation from a variety of causes, such as
tuberculosis of bone marrow, secondary infections, etc.

ratio were invariably elevated following inoculation, whereas only sixteen
of the values for the neutrophile-lymphocyte ratio were elevated.  The
greater efkiency of the monocyte-lymphocyte ratio is therefore borne
out by the mean values without resort to statistical formulae.

Table 2 lists in order the efficiency values for each of the blood cells,
the haemoglobin, and the two cellular ratios in showing abnormal changes
in tuberculosis. These values were obtained by dividing nineteen, the


HAEMATOLOGY OF EXPERIMENTAL TUBERCULOSIS     687

,vhole number of mean counts of each type of Cell following inoculation,
by the number of these counts showing significant deviation from the
nomal mean, and expressing the result as per cent.

DISCUSSION

There are certain fundamental ditIerences between experimental tuber-
culosis and pulmonary tuberculosis in man. Fortunately the human
being is apparently more resistant .than the usual experimental animal.
Many humans recover, but inoculation of animals with virulent micro-
organisms invariably results in death.  Moreover, the disease in experi-
mental animals usually becomes generalized in contrast to that in the
human, which most frequently involves the lungs and lymph nodes
draining them.  Furthermore, after intravenous inoculation of virulent
microorganisms, experimental animals invariably exhibit lesions in the
bone marrow.  Such lesions in man occur with less regularity.  In view
of these facts, it is our opinion that the experimental disease in our two
groups of animals receiving the more attenuated microiirganisms repre-
sented more closely the usual picture in man than did the two groups
inoculated with the more virulent microorganisms.  The latter have a
clinical parallel only in the most rapidly advancing cases of pulmonary
tuberculosis, or in patients with acute miliary tuberculosis.  It is there-
fore clear that our experiments include individuals in which any test
showing, with some regularity, a significant deviation from the normal
may be considered a sensitive test.  By the same token, any test showing
significant deviation from the normal in all four of these groups of ani-
mals may be considered highly efficient and reliable.  And if the devia-
tions be consistent in one direction, either above or below the normal,
it may be considered that intercurrent factors are less liiely to exert an
influence than if deviations occur in both directions from the normal.
Therefore, the monocyte-lymphocyte ratio may be seen to be more
sensitive and reliable as a detector for tuberculous infection than the
neutrophile-lymphocyte ratio, the haemoglobm, or any of the blood cells
alone. It exhibits statistically significant deviations more frequently
and consistently than any of the above, and always in the direction of an
increase over the normal.  Minimal tuberculosis is capable of influencing
the ratio significantly, and the magnitude of the change is roughly pro-
portional to the severity of disease.

The efficiency of the monocyte-lymphocyte ratio in tuberculosis is due


688         SMITHBURN, SABIN AND EU?d?dEL

to the fact that the lymphocytes and monocytes are the most sensitive
of all the blood cells to tuberculosis, and that the changes in these cells
are in opposite direction. While the monocyte proves to be the more
sensitive of the two in that it shows change earlier, the decline in lym-
phocytes more closely parallels the decline of the individual resistance
(therefore the advance of the disease) than any of the other cells.  The
fact that the monocyte-lymphocyte ratio has greater efficiency than the
neutrophile-lymphocyte ratio is to be expected, since the denominator
in the two is the same and the numerator of the one ratio, the monocyte,
has greater efficiency than that of the other, the neutrophile.

In regard to the rble of the neutrophile in tuberculosis, we are in agree-
ment with Medlar, that the general trend, especially in advanced disease,
is for the neutrophiles to increase.  It is likewise true that, in the acute
tuberculous lesions (41,42) the neutrophile plays a more important part
than in chronic or retrogressive lesions.  However, in certain instances,
namely, early, severe infection, the neutrophiles may diminish in number,
which renders their efficiency less than if the change were in the opposite
direction. This early decline in neutrophiles appears not to be caused
wholly by lesions in the bone marrow, since all the cells having origin
there are not equally afIected. There may be neutropenia without
anaemia, as shown by the mean values for neutrophiles and for erythro-
cytes in the group inoculated with the culture grown at pH 6.8 during
the second month (table 1). Moreover, our results indicate that an
increase in neutrophiles occurs only with well established or advanced
disease, whereas the changes in monocytes occur earlier.

It seems probable, for reasons stated before, that the elevated values
we have obtained for basophiles are less significant than is apparent.
It appears likely that the basophiles are little changed in tuberculosis.

The decline in number of eosinophiles cannot be explained due to lack
of knowledge of the function of these cells.  While not a sensitive indi-
cator, the decline is quite characteristic in advanced tuberculosis; it
does not occur in very mild tuberculosis.

Our results indicate that the anaemia in tuberculosis is of the micro-
cytic type, since the decline in haemoglobin is proportionally greater
than the decline in erythrocytes. Also the haemoglobin is more sensi-
tive than the erythrocytes, abnormal values being obtained when the
number of erythrocytes was in the normal range.  The lesions to be found
in the bone marrow seem wholly inadequate to account for the anaemia
and leucopenia which occur in severe infection.  It seems more likely


HAEMATOLOGY OF EXPERIMENTAL TUBERCULOSIS     689

that the anaemia and leucopenia are due to toxic effects of products of
the microiirganisms, as indicated by Sabm, Doan and Cunningham (20).
The report of Boissevain, Forster and Good (44) records results at
variance with most recent studies of the blood in tuberculosis.  It seems
likely that their failure to confirm earlier reports is due to three facts,
namely, their series of cases was very heavily weighted in favor of those
with far advanced disease (1,171 of 1,569 counts); the questionnaire
method of ascertaining the ultimate condition might be open to serious
question; and the number of counts per individual was so small that little
information could reasonably have been expected. (Their report in-
cludes statistical analyses of 1,569 counts of the blood from 431 patients,
an average of less than four per patient, taken over a period of 10 years.)
We believe, as do others (28), that if blood studies in tuberculosis are to
be of value they must be repeated at relatively frequent intervals.
The results which we have obtained indicate that studies of the blood
cells are of great value in tuberculosis. While the blood picture can
hardly be said to be pathognomonic, it should be of considerable aid in
diagnosis. But perhaps its greatest value is in prognosis, since the quan-
titative changes in the cells are progressive with advancing disease.
We have laid no stress on qualitative changes which we believe to be of
equal importance. Profound morphological and physiological changes
are known to occur among the monocytes (19, 20) and neutrophiles
(1, 2, 3). Wiseman and Doan (43) have also demonstrated qualitative
changes in the lymphocytes during tuberculous infection.  Since each
change in the blood cells is an expression of some particular phase of the
pathological picture, it seems that careful consideration of each quantita-
tive and qualitative change is more likely to be informative than any
blood index in which the identity of such changes is hidden by an arith-
metical formula.

Carefully controlled studies of the blood cells were made in groups of
rabbits subjected to tuberculous infection of four grades of severity.
The most significant changes noted were as follows.
1. The monocyte-lymphocyte ratio is a more sensitive indicator of
tuberculous infection than any of the blood cells alone or the neutro-
phile-lymphocyte ratio.

2. Of the individual cells, the monocyte is most sensitive in that it is
first to show significant change, but the lymphocyte is the more reliable


690          SMITHBURN, SABIN AND HUM?kfEL

for prognosis, as the degree of decline is roughly proportional to the
severity of the infection.
3. When anaemia occurs in tuberculosis, it is of the microcytic type.
The haemoglobin is more sensitive to tuberculous infection than are the
erythrocytes.
4. There is a tendency, especially with advanced disease, toward a
rise in neutrophiles and a decline in eosinophiles.
5. Thk basophilic granulocytes are probably little affected in tuber-
culosis.

6. In very acute tuberculous infection the blood cells may fail to show
the changes which are so characteristic of more slowly progressive dis-
ease. In such instances the decline in lymphocytes may be the only
significant quantitative change.

BIBLIOGRAPHY

(1) HOLMES, A. M.: Med. Rec., 1896, SO, 325; Ibid., 1897, 52, 369; J. Amer. Med. Assn.,
    1897, 29, 828.

(2) ARNETX, J.: Die Lungenschwindsucht, etc., Leipzig, J. A. Barth, 1905.
(3) LEWIS, M. R.: Bull. Johns Hopkins Hosp., 1911,22,428.
(4) LACAP~RE, G.: Thhe de Paris, 1902, no. 18.
(5) WEBB, G. B., AND WILLIAMS, W. W.: Colorado Med., 1909, 6, 165.
(6) WEBB, G. B., AND WILLIAMS, W. W.: Nat. Assn. Study and Prev. Tuberc., Tr. 5th
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(7) WEBB, G. B., WILLLIMS, W. W., AND BASINGER, A. F.: Ibid., Tr. 6th Ann. Meeting,
    1910, p. 279.

(8) WEBB, G. B.: Bull. Johns Hopkins Hosp., 1912,23, 231.
(9) SCEILLING, V.: Ztschr. klin. Med., Berlin, 1919,&?, 377.
(10) SABIN, F. R.: Bull. Johns Hopkins Hosp., 1923,34,277.
(11) SABIN, F. R., AND DOAN, C. A.: Proc. Nat. Acad. Sci., 1927,13,552.
(12) LIJRIE, M. B.: J. Exp. Med., 1932,55, 31.
(13) G-B, R.: Amer. Rev. Tuberc., 1932,25, 172.
(14) MWPEY, J. B., AND ELLIS, A. W. M.: J. Exp. Med., 1914,20,397.
(15) Moapw, J. B., AND S~oaaa, E.: Ibid., 1919,29, 1.
(16) NAKAHARA, W.: Ibid., 1919, 29, 17.
(17) MUBPBY, J. B., AND STURU, E.: Ibid., 1919, 29, 35.
(18) MXJRPEY, J. B.: The lymphocyte in resistance to tissue grafting, malignant disease
      and tuberculous infections. An experimental study, Monograph of. The Rocke-
    feller Institute for Medical Research, New York, 1926, no. 21, p. 131.
(19) CUNNINGEAK, R. S., SABIN, F. R., SUGIYAMA, S., AND KINDWALL, J. A.: Bull. Johns
    Hopkins Hosp., 1925, 37, 231.

(20) SABIN, F. R., DOAN, C. A., AND CUNNINGHAM, R. S.: Tr. Nat. Tuberc. Ass% 19262
    22nd Ann. Meeting, p. 252.

(21) Momuss, W. H., AND TAN, S. H.: Amer. Rev. Tuberc., 1927, 16, 729.
(22) CUNNINGHAM, R. S., AND TOMPKINS, E. H.: Ibid., 1928,17,204.
(23) CAMP, W., LUTON, F. H., TOMPKINS, E. H., AND CUNNINGFLU, R. S.: Ibid., 1928~
     18, 462.


HAEMATOLOGY OF EXI'ERIMENTAL TUBERCULOSIS     691

(24) ROOKUS, P. M.: New England J. Med., 1928,198,740.
(25) BLACKPAN, K. D., AND D-On, L. K.: Amer. J. Dis. Child., 1929,37,233.
(26) SWEANY, H. c., Sl-X, I., AND ckiNE=YKB, w.: Amer. Rev. Tuberc., 1937,35, 129.
(27) MEDLAE, E. M.: Amer. J. Path., 1926,2, 275.
(28) &~EDLAK, E. M.: Amer. Rev. Tuberc., 1929, 20, 312.
(29) CEAWFORD, A. M.: Ibid., 1935, 3X, 611.
(30) -LAB, E. M.: Ibid., 1935, 31, 621.
(31) MEDLAR, E. M.: Ibid., 1935,31,628.
(32) MEDLAE, E. M.: Ibid., 1935, 32, 642.
(33) DAVENPORT, C. B., AND EKAS, M. P.: Statistical methods in biology, medicine and

    psychology, John Wiley and Sons, Inc., New York, 1936, ed. 4.
(34) PEAat, R.: Introduction to medical biometry and statistics, W. B. Saunders company,

Philadelphia, 1923, p. 218.

(35) FLINN, J. W., AND Fm, R. S.: Amer. Rev. Tuberc., 1929,20,347.
(36) SNITHEIJBN, K. C.: Amer. Rev. Tuberc., 1937,36,637.
(37) PEARCE, L., AND CASEY, A. E.: J. Exp. Med., 1930,51,83.
(38) Smq F. R., -, F. R., SMITEBURN, K. C., Ta0x.q R. M., AND HUMMEL, L. E.:

Ibid., 1936, 64,97.

(39) SMITEB~~W, K. C.: Ibid., 1932, 56, 173.
(40) THOMAS, R. M.: Data included in table 4 of ref. 38.
(41) MEDLAR, E. M., AND SAW-TO, K. T.: Amer. Reu. Tuberc,, 1933,28,62.
(42) SYITEBURN, K. C.: Ibid., 1937,36, 659.
(43) WI~EMAN, B. K., AND DOAN, C. A.: Ibid., 1934,30,33.
(44) BOISSEVAIN, C. H., Foasr~a, A. M., AND GOOD, B. D.: Ibid., 1936,34,477.