THE OCCURRENCE OF T H E SEX CHROMATIN IN WHITE BLOOD CELLS
OF YOUNG ADULTS
I. STUDY OF NORMAL PERIPHERAL BLOOD
M. S. N. MURTHY, M.Sc, M.B., B.S., AND EMMERICH VON HAAM, M.D.
Department of Pathology, Ohio State University College of Medicine, Columbus, Ohio
In 1954, Davidson and Smith3 reported
the presence of sex chromatin in the form
of drumsticks in the polymorphonuclear
neutrophils of women (Fig. 1). This structure
was found to be present only in women and
had to be distinguished from certain other
appendages found in both sexes. Since then,
several articles 66 have been published confirming their findings and reporting on
the diagnostic usefulness of this finding in
cases of "indeterminate sex." In none of the
above studies, however, was an attempt
made to indicate the distribution of the
drumsticks in neutrophils encountered in a
routine differential count of white blood
cells. Moreover, the incidence and significance of the nuclear appendages, such as
small clubs, sessile nodules, and racket formation, have not received sufficient attention. Davidson and Smith felt that the small
clubs were "perhaps more common in the
male than in the female," that the sessile
nodules and tags were more frequent in
women, and that the rare racket forms were
more frequent in men. Sun and RakofP
found the occurrence of "small clumps,"
"rackets," and "sessile nodules" about the
same in both sexes, whereas Briggs and
Kupperman 2 noted the complete absence of
the sessile nodules in the peripheral blood
cells of men, and on that basis regarded
the chromatin-rich sessile nodule as probably characteristic of women. Kosenow and
Scupin" regard these sessile nodules as parts
of drumsticks partially covered by the lobule
of the nucleus and have devised an elaboReceived, July 26, 1957; revision received,
April 5, 1958; accepted for publication April 21.
Dr. Murthy was Assistant Instructor, Department of Pathology; his present position is Assistant Professor of Pathology, All India Institute of
Medical Science, New Delhi, India. Dr. von Haam
is Chairman, Department of Pathology.
rate method for arriving at Index Q in the
determination of sex by a study of the peripheral blood smear.
The occurrence of the sex chromatin in
many tissues of the body prompted us to
examine white blood cells for the planoconvex mass of dark-staining chromatin
closely applied to the inner aspect of the
nuclear membrane, as orginally described by
Barr and his associates.1 Therefore, a study
was undertaken in this laboratory to confirm, and if possible to extend, the original
findings.
MATERIALS AND METHODS
First, an attempt was made to standardize
a method for the study of the smears of
blood. Various procedures for fixation and
stains have been used to stain differentially
the clump of sex chromatin. As the sex chromatin is rich in desoxyribosenucleic acid,
the Feulgen technic has been the staining
method of choice. Pyronin methyl green,
cresyl violet, and cresyl echt violet have also
found favor with some workers. Papanicolaou's technic of fixation and staining has
been used especially in the study of exfoliated cells for sex chromatin. A preliminary
study to evaluate these stains and their
suitability for the smears of blood was undertaken.
Peripheral smears of blood were obtained
from 4 apparently normal men and 4
apparently normal women on coverglasses by
the "pull-slip" technic. The coverslips were
fixed immediately, i.e., when wet, in Papanicolaou's fixative of equal parts of 95 per
cent ethyl alcohol and ethyl ether. A number
of coverslips with smears of blood were air
dried. Both sets of smears were stained by
the following procedures: (1) Feulgen technic, (2) Papanicolaou's stain, (3) cresyl echt
violet, and (4) Wright's stain.
Air drying and the use of Wright's stain
216
Sept. 1958
SEX CHROMATIN IN LEUKOCYTES
proved to be the method by which chromatin
clumps and drumsticks could be most easily
distinguished. When smears were fixed in
Papanicolaou's alcohol-ether, drumsticks
were much more difficult to identify. Airdried smears stained by the Feulgen or
Papanicolaou technic were of excellent technical quality but offered no special advantage over the routine laboratory procedure
with the Wright stain. Since the last procedure is the one used routinely in most laboratories, it enables one to "sex" all the smears
of blood that pass through the clinical
laboratories.
Twenty-five healthy, apparently normal
women (students of Medical Technology at
the Ohio State University) and 25 healthy,
apparently normal men (medical students
at Ohio State University) served as subjects for the study. Smears of blood were
obtained on coverglasses and were air dried.
They were then stained with Wright's stain.
A differential count was performed on 400
leukocytes of each subject. Drumsticks,
small clubs, and sessile nodules or tags were
counted. All other white blood cells were
carefully scrutinized for any structure conforming to the criteria for sex chromatin laid
down by Barr.
The thickness of the smear proved important. In thick smears the nuclear lobes and
appendages were crowded. A drumstick
might be partially covered by a lobe of the
nucleus in the polymorphonuclear leukocyte, making it difficult to identify as a
typical drumstick. In smears that were too
thin, the nuclei were distorted and partially
crushed, resulting in the formation of
indistinct clumps of chromatin and nuclear
tags. In fields that were neither too thin nor
too thick, no difficulty was encountered in
recognizing and differentiating the various
nuclear appendages from the typical drumsticks.
RESULTS
The distribution of typical drumsticks in
the routine differential leukocyte count is
recorded in Table 1. In most cases at least 1
typical drumstick was encountered in the
first 100 leukocytes; in an occasional case
it was not seen in as many as 200 leukocytes.
217
Although 6 out of 189 eosinophils counted
in this study showed the typical drumstick,
none of the 42 basophilic leukocytes in 25
smears from the women showed this structure.
The incidence of drumsticks as percentages of the total neutrophilic leukocytes is
presented in graph 1. The lowest incidence
was 2 per cent and the highest 17.8 per cent
with an average of 6.6 per cent. These
figures are slightly higher than those presented by Davidson and Smith,' in which the
average was 1 in 38 polymorphonuclear
neutrophils.
No typical drumsticks were observed in
the blood of the 25 men students. The small
clubs seen in the polymorphonuclear leukocytes in the men sometimes resembled the
drumstick but were always smaller than
1.5yu and less deeply stained. When they were
located at the tip of the lobule of a polymorphonuclear leukocyte they could be
mistaken for the drumstick. Their incidence
in this location varied from 0.8 to 9.9 per
cent of all polymorphonuclear leukocytes,
with an average of 4.08 per cent. This frequency is definitely higher than in the
women. It is tempting to suggest that these
might be the male counterpart of the drumsticks in the women.
The chromatin-rich sessile nodules and
tags occurred in equal or slightly' greater
frequency in the men than in the women.
The paucity of racket forms made impossible
any statistical evaluation of their relative
frequency in women and men.
The study of the white blood cells other
than the segmented neutrophilic leukocytes
was rewarding. Many nonsegmented and
a few segmented leukocytes in the women
showed a planoconvex, deeply basophilic,
well-outlined mass of chromatin closely
applied to the inner aspect of the nuclear
membrane (Fig. 2). Its size as compared
with the sex chromatin in other tissues was
close to 1 or 1.5M- This we interpreted as the
sex chromatin that Barr and his associates1
described in the intermitotic nuclei of the
somatic cells of the women. In an end-on
view, it was elliptical. Occasionally it was
seen at 1 of the blunt ends of the nucleus of
the nonsegmented leukocyte. In 3 women
218
MURTHY AND VON HAAM
subjects where the incidence of the drumstick was low, i.e., 3.9 per cent, 2.5 per cent
and 2.0 per cent, the smears were surveyed
for the sex chromatin of the above morphology and the incidence was 11.5 per cent,
10.6 per cent and 15.8 per cent of all the
polymorphonuclear leukocytes, respectively.
Although no attempt was made to determine
the incidence of this structure in all the
smears of blood from the women subjects,
the above figures amply prove that their
recognition is of great help in sexing the
unknown smear of blood. It is interesting to
point out that no drumstick or any other nuclear appendage resembling the drumstick
was encountered in the few segmented polymorphonuclear leukocytes that contained
the plano-convex or elliptical mass of sex
chromatin.
The heavy chromatin structure of the
nuclei of lymphocytes precluded the recognition of the mass of sex chromatin, although
the presence of such a structure is suggested
to an experienced eye. The young lymphocyte occasionally contained an unmistakable
clump of sex chromatin in its usual location
(Fig. 3). Such a structure was seen only in
the women, although in the men, smaller
clumps of chromatin could be seen frequently. The relative paucity of young
lymphocytes in the peripheral blood made us
abandon early in the studies the counting
of sex chromatin in lymphocytes.
Monocytes proved much better subjects
for study. The lacy pattern of their chromatin permitted the detection of the planoconvex or elliptical mass of sex chromatin.
I t was located at random in the nucleus but
almost always hugging the inner aspect of
the nuclear membrane. Frequently it was
close to the hof or bay of the nucleus, some-
Vol. SO
times atop the crest of the billows of the
nucleus or just adjacent to this (Fig. 4).
Care had to be exercised so that nuclear
folds or other chromatin clumps were not
mistaken for the sex chromatin. The smooth
outlines of the sex chromatin, its location
on the nuclear membrane, and its well
defined margins served to distinguish this
structure from the other nonspecific chromatin clumps mentioned previously. Graph
2 presents its incidence in 23 of our women
subjects, which varied from 7 to 33 per cent
with an average of 18.3 per cent of all the
monocytes in the women subjects. Such
structure was never encountered in the
monocytes of the men.
Table 2 presents the distribution of the
plano-convex or elliptical sex chromatin
among the different types of white blood
cells in 12 apparently normal women. In
each case, 100 neutrophilic leukocytes were
counted. The distribution as computed for
all white blood cells varied between 2.0 and
7.0 per cent, which is lower than the percentage distribution of typical drumsticks
among segmented polymorphonuclear leukocytes (Table 1). However, the observation
of this type of sex chromatin should be
helpful in those cases where the drumsticks
are scarce.
DISCUSSION
Our findings not only confirm but also
greatly supplement the observations of
Davidson and Smith.3 The random distribution of drumsticks is such that they might
not be encountered in a differential count of
200 leukocytes. The technical quality of the
smear of blood is of great importance. We
feel, therefore, that several fields of a
suitably stained smear should be examined
F[C. 1 (upper left). A polymorphonuclear neutrophilic leukocyte with a typical "drumstick" projecting from the tip of one of the lobules of the nucleus. The contrast with the small club projecting
from the lobule above is striking. Wright's stain. X 1800.
FIG. 2 (upper right). An elliptical dense mass of chromatin interpreted as sex chromatin closely
applied to the inner aspect of the nuclear membrane in a segmented leukocyte. Wright's stain.
X 1200.
FIG. 3 (lower left). "Sex chromatin" in a lymphocyte in the peripheral blood of a woman. The
larger size, the well-defined margins, and the greater intensity of staining distinguish this structure
from other clumps of chromatin in the nucleus. Wright's stain. X 1800.
Fio. 4 (lower right). "Sex chromatin" in a monocyte in the peripheral blood of a woman. The
elliptical shape, the location on the nuclear membrane, and .the well-defined outlines are helpful
in distinguishing this structure from other clumps of chromatin and nuclear folds. Wright's stain.
X 1800.
Sept. 1958
SEX CHROMATIN IN LEUKOCYTES
FIGS. 1 to 4
219
220
Vol. SO
MURTHY AND VON HAAM
TABLE 1
INCIDENCE OP SEX CHROMATIN IN THE LEUKOCYTES OF WOMEN
Total Leukocytes Countec
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
IS
19f
20f
21
22
23
24
25
l- 100
with
No. of Cells
chroPMN* sex
matin
51
48
53
52
71
52
67
43
62
57
51
41
66
56
46
62
36
57
39
56
54
59
39
56
37
5
10
8
2
8
3
1
4
S
5
0
3
2
3
4
2
5
5
1
2
3
5
2
4
1
101-200
with
No. of Cells
sex chroPMN
matin
201-300
with
No. of Cells
sex chro-J
PMN
matin .
53
59
46
48
65
61
59
49
47
53
42
46
61
44
36
56
46
51
49
58
59
63
45
61
46
45
54
51
50
54
57
61
36
59
56
53
57
65
50
46
51
50
53
50
36
67
56
34
55
59
5
11
6
0
6
0
2
5
7
3
4
3
1
2
1
3
2
6
1
3
4
6
2
4
0
8
11
6
1
8
0
0
4
S
3
2
1
4
6
1
4
3
4
0
5
6
1
1
3
1
Percentage
301-400
Distribution of
Chromatin
Sex
with
in PMN
No. of Cells
sex chromatin
PMN
58
65
43
39
68
63
55
57
64
58
44
53
67
49
39
63
47
70
39
0
58
67
28
63
56
6
S
3
3
11
3
3
5
6
0
3
2
3
5
2
1
6
5
1
0
6
4
0
4
2
11.6
17.8
11.8
3.1
12.S
2.6
2.5
9.7
12.5
4.9
5.2
4.5
3.9
8.0
4.8
5.2
8.9
8.7
1.7
6.6
7.9
6.5
3.4
6.4
2.0
* PMN = polymorphonuclear neutrophils.
f Not included in the computation of averages.
before the absence of drumsticks can be
regarded as absolute and a diagnosis of
"male" made on an unknown smear.
Thorough familiarity with the morphology
of drumsticks is essential in order to distinguish them from small clubs and other
nuclear appendages. Davidson and Smith
originally suggested that at least 500 polymorphonuclear leukocytes should be counted;
other authors suggest 1000. In no case in
our series did we fail to find a drumstick
in 4.00 polymorphonuclear leukocytes in women. However, in endocrinopathic cases
and those of indeterminate sex, where the
incidence of drumsticks is reported to be
very low,7 counting of 500 to 1000 neutrophils may be necessary.
The figures reported for the frequency of
the drumsticks vary depending on whether
only the typical drumsticks as described by
Davidson and Smith were counted, or
whether all possible forms of the drumsticks were enumerated. The average number of drumsticks per 500 polymorphonuclear leukocytes according to the first
criterion is 13.2,3 11.4,8 and 17.5.4 Using
the latter method, we found an average value
of 35.2, which compares favorably with 41.1
per 500 polymorphonuclear leukocytes as
given by Kosenow and Scupin.4
Davidson and Smith3 and Briggs and
Kupperman 2 reported a greater frequency of
the chromatin-rich sessile nodules and tags
in women. The latter workers stated that
it is probably characteristic of the female,
and considered it as 1 of the features of the
Sept. 1958
221
SEX CHROMATIN IN LEUKOCYTES
polymorphonuclear leukocytes in the female
and hence to be utilized as a clue in "sexing"
the unknown blood smear. Kosenow and
Scupin4 added the sessile nodules and the
drumsticks, and divided the total by the
number of small clubs in 500 polymorphonuclear leukocytes. If this quotient was
greater than 0.4, the diagnosis was female,
if below 0.4 the diagnosis was male. We have
found the incidence of the chromatin-rich
sessile nodules to be about equal or even
slightly higher in men. Sun and Rakoff5 also
found the incidence of these structures to be
approximately the same in both sexes. The
discrepancies in these figures might well be
caused by the different criteria used by each
author to identify a nuclear appendage as a
chromatin-rich sessile nodule. We included
all nuclear appendages other than small
clubs in the group of sessile nodules.
The occurrence of the sex chromatin in
its recognized form in the nuclei of white
blood cells other than segmented polymorphonuclear leukocytes confirms the unique
morphology of this structure in women. Its
practical application is even more important
in cases with a shift to the left of leukocytes
or a low incidence of drumsticks. In such
cases a clue to the correct sex can be obtained
if planoconvex or elliptical dark-staining
clumps of chromatin are found in the white
blood cells. We agree that at the present
time the drumstick should be the sine qua non
for the diagnosis of female sex in the examination of smears of blood. However, we feel
that the finding of the planoconvex sex
chromatin contributes at least a strong clue
to the sex. One may indeed speak of patterns
of male and female in smears of blood. The
pattern of the male is characterized by the
absence of the typical drumsticks, the
greater frequency of the small clubs, especially those that project from the tip of one of
the lobules of the polymorphonuclear leukocyte, and the absence of the elliptical or
planoconvex mass of sex chromatin in nonsegmented neutrophilic leukocytes or in
other white blood cells. The female pattern,
on the other hand, is characterized by the
occurrence of the typical drumstick in segmented leukocytes and of the planoconvex
mass of sex chromatin in monocytes, lym-
10
NUMBER OF CASES
20
GRAPH 1. The incidence of typical "drumsticks," expressed as percentages of the total number of polymorphonuclear neutrophilic leukocytes.
0
10
NUMBER OF CASES
20
GRAPH 2. The incidence of the elliptical or
plano-convex mass of sex chromatin in monocytes,
expressed as percentages of the total number of
monocytes in the peripheral blood of 23 women.
phocytes, nonsegmented polymorphonuclear
leukocytes and also in those segmented polymorphonuclear leukocytes that do not contain the typical drumstick. I t is our belief
that the recognition of such a male and female pattern offers considerable help in arriving at the correct diagnosis of sex in cases
where the paucity of drumsticks renders the
diagnosis difficult. Using the above criteria,
we examined 147 unknown smears of blood
from the clinical laboratories at the University Hospital. A correct diagnosis of the sex
was made in 12 cases where no typical drumstick was encountered in the first 100 polymorphonuclear neutrophils counted. In all
these cases, the diagnosis of female sex was
made mainly on the basis of the occurrence
of the planoconvex or ellipital mass of sex
chromatin in leukocytes. The diagnosis was
subsequently confirmed by finding at least 1
222
Vol. SO
MURTHY AND VON HAAM
TABLE 2
INCIDENCE OP THE PLANOCONVEX OR ELLIPTICAL MASS OF SEX CHROMATIN IN LEUKOCYTES OP WOMEN
Case
No.
Segmented Polymor- Nonsegmented Polyphonuclear Leuko- morphonuclear Leucytes
kocytes
No. of
cells
1
2
3
4
5
6
7
8
9
10
11
12
100
100
100
100
100
100
100
100
100
100
100
100
Monocytes
Lymphocytes
Total
No. Percentage
No. of Total
of Sex
of Sex
Leuko- Chromatin
Chromatin
Sex chro- No. of Sex chro- No. of Sex chro- No. of Sex chro- cytes
matin
cells
matin
matin cells
matin
cells
2
5
3
1
6
2
3
4
2
6
3
4
2
13
63
4
32
55
17
9
47
15
9
12
2
2
1
0
2
3
0
0
4
3
0
1
7
9
9
7
20
S
5
8
6
9
15
12
typical drumstick in a survey of the entire
blood smear.
SUMMARY AND CONCLUSIONS
1. The distribution of drumsticks in a
differential count of 4.00 polymorphonuclear
leukocytes in 25 apparently normal, healthy
women has been studied. Polymorphonuclear
leukocytes bearing the sex chromatin were
so distributed that at least 1 such cell was
encountered in 100; however, in a few cases
200 or more polymorphonuclear leukocytes
had to be counted before 1 typical drumstick was encountered.
2. A planoconvex or elliptical mass of
dark-staining chromatin closely applied to
the inner aspect of the nuclear membrane
has been identified as "sex chromatin" in
monocytes, lymphocytes, the nonsegmented
leukocytes and a few segmented polymorphonuclear leukocytes that do not contain
the drumstick.
3. The male pattern of the peripheral
blood is characterized by the absence of
typical drumsticks, the greater frequency of
the small clubs and the absence or paucity of
sex chromatin of the classical morphology.
4. The female pattern of the peripheral
blood is characterized by the finding of
typical drumsticks in segmented polymorphonuclear leukocytes and by the presence
0
i
3
2
0
1
0
1
3
0
1
2
12
11
IS
37
20
31
10
22
5
18
18
18
0
0
I
0
0
0
0
2
0
1
0
0
121
133
190
14S
172
194
132
139
15S
142
142
142
4
8
S
3
S
6
3
7
9
10
4
7
3.3
6.0
4.2
2.0
4.6
3.0
2.2
5.0
5.7
7.0
2.S
4.9
of a dark-staining planoconvex or elliptical mass of sex chromatin closely applied
to the inner aspect of the nuclear membrane
in monocytes, lymphocytes and nonsegmented polymorphonuclear leukocytes.
SUMMARIO IN 1NTERLINGUA
1. Esseva studiate le distribution de
"bachettas de tambur" in un numeration
differential de 400 leucocytes polymorphonuclear in 25 apparentemente normal feminas in bon stato de sanitate. Leucocytes
polymorphonuclear que portava le chromatina de sexo esseva distribute de maniera
que al minus 1 tal cellula esseva incontrate
in 100. Tamen, in alicun casos, 200 leucocytes polymorphonuclear o plus debeva
esser contate ante que 1 typic "bachetta de
tambur" esseva trovate.
2. Un massa plano-convexe o elliptic de
chromatina a colorabilitate obscur, applicate
intimemente al aspecto interior del membrana nuclear ha essite identificate como
"chromatina de sexo" in monocytes, lymphocytes, non-segmentate leucocytos, e un
micre numero de segmentate leucocytos
polymorphonuclear non continente le "bachetta de tambur."
3. Le configuration mascule del sanguine
peripheric es characterisate per le absentia
del typic "bachettas de tambur," le plus
Sept. 1958
SEX CHROMATIN IN LEUKOCYTES
alte frequentia de micre clavas, e le absentia
o paucitate de chromatina de sexo del
morphologia classic.
4. Le configuration feminin del sanguine
peripheric es characterisate per le presentia
de typic "bachettas de tambur" in segmentate leucocytos polymorphonuclear e de un
massa plano-convexe o elliptic de chromatina de sexo a colorabilitate obscur, intimemente applicate al aspecto interior del membrana nuclear in monocytos, lymphocytos, e
non-segmentate leucocytos polymorphonuclear.
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2S3-297, 1950.
2. BUIOGS, D. K., AND KUI'I'ERMAN, H. S.: Sex
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differentiation by leukocyte morphology. J.
Clin. Endocrinol., 16: 1163-1179, 1956.
3. DAVIDSON, W. M., AND SMITH, D. R.: A mor-
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4. KOSENOW, W., AND SCUPIN, R.: Die Bestim-
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