Cellular Composition of the Nipple Aspirate
Specimen of Breast Fluid
I. The Benign Cells
EILEEN
B.
KING,
M.D.,
DIANA B A R R E T T ,
C T (ASCP),
AND N I C H O L A S L. PETRAKIS,
MARY-CLAIRE KING, PH.D.,
M.D.
TheG. W. Hooper Foundation and the Department ofPathology, University of California, San Francisco, California 94143
ABSTRACT
King, Eileen B., Barrett, Diana, King Mary-Claire, and Petrakis, Nicholas L.:
Cellular composition of the nipple aspirate specimen of breast fluid. I. T h e
benign cells. Am J Clin Pathol 64: 728-738, 1975. A nipple aspirator
device was used to obtain breast secretions for cytologic examination,
as well as for viral and biochemical analysis. Examination of the first
1,456 specimens from 796 women revealed ductal epithelium in 54%.
Ductal epithelial cells were often absent in specimens from normal women;
however, 78.5% of women with benign breast disease on tissue biopsy
had specimens containing ductal epithelium. Apocrine metaplastic cells were
a further indication of the presence of breast disease, and were rarely
found in specimens from asymptomatic women. Foam cells were often
abundant in specimens from normal breasts, but were found in decreased
numbers in specimens from women with benign breast disease. Differences in the occurrence of ductal epithelial cells, apocrine metaplastic
cells, and foam cells suggest an alteration in the rate of maturation of
ductal epithelium in women with both benign and malignant breast disease.
The finding of a relative abundance of cells in nipple aspirate specimens
from women wiui breast disease and few or no cells in specimens from
women with normal breasts is believed to be of great importance in the
cytologic evaluation of nipple aspirate specimens. (Key words: Cytology;
Breast secretion; Nipple aspirate; Benign breast cells.)
IN T H E PAST, cytologic methods were not
successfully applied to the early diagnosis of breast cancer, due to problems in
obtaining representative cellular specimens
Received March 20, 1975; accepted for publication March 20, 1975.
Supported by Public Health Service grant CA
13556 and contract N01 CB 33882 from the National
Cancer Institute, and by a gift from Mrs. Viola
K. Schroeder.
Reprints of this entire Research Symposium are
available from the ASCP Meeting Services Department, 2100 West Harrison Street, Chicago, Illinois
60612, for $5.00 per copy.
728
from early and pre-clinical lesions. Material
previously available for cytologic examination consisted of spontaneous nipple discharges, breast secretions obtained with a
conventional breast pump, and needle aspirates from cystic and solid masses. Now,
with a modified breast p u m p method of
specimen collection that utilizes a simple
nipple aspirator device, 9 workers can obtain fluid from a much larger number of
women, and can often predict successful
aspiration on the basis of race, age, menopausal status, and cerumen type. 8 Study-
December 1975
729
NIPPLE ASPIRATE BENIGN CELLS
ing breast fluid specimens is, of course,
potentially valuable not only for diagnosing
breast cancer but also for understanding
the normal physiology and the pathophysiology of the human breast, especially
as related to neoplasms. Our investigations
included biochemical, viral, and cytologic
analysis of breast fluid specimens obtained
by the nipple aspiration technic. 7 In this
paper, we describe the cellular composition and the range of normal cellular
findings detected in nipple aspirate
specimens.
Materials and Methods
Subjects for our study were women from
a variety of racial and ethnic backgrounds.
The majority of women were asymptomatic. A number of women had symptoms
of benign breast disease, but only a few
of them required breast biopsy. Specimens
were obtained from the remaining breasts
of women who had had a mastectomy
for carcinoma of the breast. (These women
are referred to as post-mastectomy patients.) This report includes a review of
normal findings in 1,456 specimens from
796 women, and a more detailed analysis
of 136 of these specimens from 82 women.
Each breast fluid specimen obtained with
the aspirator device consists of 5 - 1 0 0
A of fluid from the ducts, so it must be
carefully conserved. A variety of cell concentration technics were considered for this
purpose. T h e efficiency of cell recovery
utilizing either membrane filters (Nuclepore, Gelman, and Millipore) or the Cytocentrifuge (Shandon) was evaluated with
cell suspensions of known concentrations.
Millipore filters consistently provided the
best results in terms of both cell recovery
(81 ± 3%) and preservation of cell morphology. 1 T h e Cytocentrifuge continues to
be of great interest to us because of its
speed and simplicity of operation, which
give it a practical advantage over the
filter method for mass screening. Although
satisfactory cell detail has been achieved
using the Cytocentrifuge, cell recovery
remains poor (11 ± 1%). We used the
Millipore filter method for all specimens
and processed some of them using additional technics. Direct smearing onto a
glass slide is the simplest preparatory
method but has proven unsatisfactory due
to scant cellularity of the specimens and
difficulty in controlling both distribution
of the cells and thickness of the smear.
Examination of each specimen consisted
of first screening every microscopic field
for the presence of abnormal or malignant cells. Then a more thorough evaluation of cellular content was made in cases
that were selected because they fell within
the range of clinical categories in this
study. Total cell determinations and differential counts were made in 136 specimens.
Cell counts were based upon determinations using both the Coulter Counter and
the hemocytometer. 1 Differential counts
were based on established cytomorphologic
criteria for ductal epithelial cells, apocrine
metaplastic cells, and foam cells. 2-4_6 T o
confirm the accuracy and usefulness of
these morphologic features, the same three
types of cells were obtained in suspension by scraping them directly from breast
tissue and collecting them onto Millipore
filters for comparison with cells from
nipple aspirate specimens.
Results
Fluid Yield
Petrakis and associates8 studied many
of the same women that we studied, and
obtained fluid from one or both breasts
of 60.7% of women in whom collection
was attempted. However, Petrakis and
associates found that the fluid yield from
Caucasians was much higher than this
average (70.2%); in contrast, the fluid
yield from Orientals was very low (24.1%).
Total Cellularity
Total cell counts were made in 37 specimens from 21 women. Tables 1 and 2
730
KING ET
Table 1. Average Cellularity and
Clinical Status
Clinical Status
Normal
Benign breast symptoms
Post-mastectomy
Pregnant
Average No. of Cells
per Specimen
x 103
16.2
24.9
6.3
221.5
±
6.5
± 13.1
±
3.2
± 149.7
Table 2. Average Cellularity and Age
in Nonpregnant Women
Age (by Decade)
Average No. of Cells per
Specimen
x 103
Third
Fourth
Fifth
Sixth*
13.0 ± 7.0
30.6 ± 12.2
1.8 ±
.8
7.6
* Only one specimen.
show the clinical status and ages of these
patients related to average total numbers
of cells in nipple aspirates. Specimens
from women in any trimester of pregnancy were characteristically so highly cellular that pregnancy was frequently suspected on the basis of the cellular specimen. The statistical evaluation of this
finding by analysis of variance revealed
that the correlation was statistically significant (F test, Sum of Squares Simultaneous
Test Procedure, p < .05). Total cellularity
AL.
A.J.C.P. —Vol. 64
in nonpregnant women did not vary
significantly with age. Although average
cellularity was greater in specimens from
women with symptoms of benign breast
disease than in normal women, the difference was not statistically significant.
Fewer cells were found in specimens from
post-mastectomy patients than in those
from women with no clinical evidence of
breast abnormality, but again the difference was not statistically significant.
Distribution of Cell Types
Epithelial cell types were classified as
ductal and apocrine. Foam cells, although
possibly of ductal epithelial origin, have
histiocytic characteristics and were considered as a separate category. 3 T h e morphologic features used to identify ductal
and apocrine cell types were studied in
cells obtained directly from breast tissue
and are illustrated in Figures 1-13. The
following morphologic descriptions are
based upon membrane filter preparations
with Papanicolaou staining.
Ductal epithelial cells are typically 10-15
/j, in diameter, but may be as large as
30 (i. Cytoplasm is cyanophilic; either it is
homogeneous or it contains vacuoles,
which are variable in size, single and discrete, or small and multiple. The nucleus
is large for the size of the cell, occupying from half to two thirds of the diameter
FlG. 1 (upper left). Cells of the normal nipple aspirate specimen include anucleate keratotic plaques from ductal
opening and foam cells from deeper ductal lumen. x250.
FlG. 2 (upper right). Cells from superficial part of the ductal system originate in the zone of transition
from squamous to columnar epithelium. This sheet of cells is typical. At one end are cells of squamoid
appearance and at the other end are typical ductal cells. x250.
FlG. 3 (lower left). A longitudinal section through the nipple shows duct opening plugged with keratotic material,
which must be removed before ductal secretions can be obtained with the nipple aspirator device.
FlG. 4 (lower right). Longitudinal section of same duct atjunction of squamous and columnar epithelium. x250.
December 1975
NIPPLE ASPIRATE BENIGN CELLS
I
• *
/
731
732
KING ET
of the cytoplasm. It is most often oval,
but may be round or indented. T h e chromatin is fine, uniform, compact, and dark,
and is arranged in a thin, uniform, chromatinic membrane at the nuclear border.
There is usually a distinct nucleolus or
chromocenter. Single ductal epithelial cells
are often similar in size to histiocytes and
lymphocytes. They are differentiated by
paying attention to differences in chro-
A.J.C.P.—Vol.
AL.
64
matin pattern, shape of nucleus, and less
well-defined cytoplasm: the nucleus of the
histiocyte is characterized by a delicate
chromatin mesh, absence of nucleoli or
chromocenters, and much variability in
shape from one cell to another; the
lymphocyte nucleus is usually spherical,
may vary in size, is usually about two
thirds the diameter of the ductal cell
nucleus, and has blocky, dense, hyper-
W^fflP
**f
t
FIG. 5 (left). Intralobular duct with uniform low columnar lining epithelium and myoepithelial cell layer. X250.
FIG. 6 (upper right). Individual ductal epithelial cells scraped directly from tissue on Millipore filter. Small,
uniformly granular, dark-staining nuclei with smooth borders and narrow cytoplasmic margins. x250.
Fie. 7 (middle left). Single ductal epithelial cell (middle center), histiocytes (middle right), lymphocyte
(lower right), file-like grouping of ductal cells. x250.
December 1975
NIPPLE ASPIRATE BENIGN CELLS
^^^r
^!MF
0%
JU
ft
J
1
733
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w
FIG. 8 (/e/i)- Breast tissue section with duct lined by apocrine metaplastic epithelium
showing papillary infolding. X250.
FIG. 9 (upper right). Cells of apocrine metaplastic type obtained directly from breast tissue scraping.
Cytoplasm is dense and contains fine granules and vacuoles. Nuclei are round with uniform granular
chromatin, accentuated chromatinic membrane, and prominent nucleolus. x250.
FIG. 10 (lower right). Cells of apocrine metaplastic type from nipple aspirate specimen show both cytoplasmic
density and vacuolization. Nuclei vary in both size and staining reaction. x250.
chromatic chromatin. Ductal cells are more
easily recognized in groups than as single
cells. Cell groups are usually tightly arranged, show knobby borders where individual cells are protruding beyond the
main mass, and have overlapping and
multilayering centrally. They are interpreted as surface epithelium splitting from
underlying cells and rolling up in the ductal
secretions to form discrete groupings. Cells
may be arranged in a monolayer, sheet,
or strand, especially when cell groups
are small (Figs. 5-7).'
In the ampullary portion of the nipple
duct, epithelial cells undergo a transition
from columnar to squamous-type cells.
The epithelial cells in transition are very
close to the opening of the duct and may
easily become part of the cellular content
of nipple aspirates. These transitional
cells vary from squamous-like spherical
and oval cells to flat, intermediate, or
superficial forms, sometimes containing
keratohyaline granules in their cytoplasm
(Figs. 2 and 4).
Apocrine metaplastic cells are usually
two to three times the diameter of ductal
epithelial cells, and have very dense, sometimes distinctly granular, basophilic or
amphophilic cytoplasm and central or
734
KING ET AL.
AJ.C.P.—Vol.
64
FIG. II (left). Ductal lining with foam cells located in the epithelial layer and appearing in juxtaposition to
well-preserved ductal epithelial cells. x250.
FIG. 12 (upper right). Foam cell scraped from breast tissue and collected on Millipore filter. Nucleus has smooth
borders, uniform chromatin, and prominent nucleolus, similar to apocrine metaplastic cells. x250.
FIG. 13 (lower right). Foam cells from nipple aspirate specimen. Note similarity of nuclei to those of ductal
epithelial cells and apocrine metaplastic cells. x250.
eccentric nuclei. T h e nuclear diameter is
approximately one fifth to one third that
of the cell cytoplasm; the nucleus is usually
round, but may be oval or bean-shaped,
It is distinguished by a prominent, uniform, chromatinic membrane at the nuclear border, fine granular chromatin,
and a prominent nucleolus. Apocrine
metaplastic cells appear singly or in clusters (Figs. 8-10). It is not always possible
to classify all cells with these characteristics as apocrine, because ductal cells may
be quite large in size and similar in
cytoplasmic and nuclear structure and
foam cells may sometimes have similar,
very dense cytoplasm.
December 1975
NIPPLE ASPIRATE BENIGN CELLS
Foam cells tend to be larger than
epithelial or apocrine cells, ranging from
15 to 50 jn or larger. Cytoplasm is abundant,
finely vacuolated, and cyanophilic. The
nucleus is usually small and round with
distinct fine chromatin and parachromatin
and a prominent nucleolus. The nuclear
borders are smooth and accentuated by
a distinct chromatinic membrane. Hyperchromasia and pyknoses are not uncommon. Foam cells comprise a large proportion of the benign cells found in nipple
aspirates. T h e more numerous they are
in a given specimen, the more likely that
they will be found in clumps as well as
singly (Figs. 11-13).
Squamous epithelial cells and anucleate
keratotic plaques originating from the
ductal opening were noted but were not
included in the differential counts. They
were considered significant as evidence of
clearing the ductal opening before obtaining an adequate specimen (Figs. 1
and 3).
A more detailed analysis of the occurrences of different cell types was made
in 136 specimens, 68 from 48 nonpregnant women and 68 from 34 pregnant
women. Total cells counted and classified
per specimen ranged from 100 to 500,
with the exception of 12 specimens in
which fewer than 100 cells were available and all cells were counted. Of particular interest is the number of ductal
epithelial cells: 2% of the total 14,607
cells counted were single ductal epithelial
cells. Although only single cells were in-
735
cluded in the differential count, the total
number of groups of ductal cells was
also tabulated for each specimen. Whereas
single ductal cells were found in only
34 of the 136 specimens, groups of ductal
cells were found in 82 specimens, and a
total of 92 specimens (67.6%) contained
single and/or grouped ductal epithelial
cells. Forty-nine of 68 specimens (72.1%)
from pregnant women contained ductal
cells, compared with 43 of 68 (63.2%)
from nonpregnant women (Table 3).
(Among the remainder of the 796 women
were 28 who had had breast tissue removed surgically either by biopsy alone
or by biopsy and mastectomy: 22 [78.5%]
had satisfactory specimens containing
ductal epithelial cells, and only 6 [21.5%]
had unsatisfactory specimens lacking ductal epithelial cells.) From 1 to 80 groups
of cells were found in individual specimens. T h e total number of cells making up the individual tissue fragments
tended to be much greater in pregnant
and premenopausal women than in postmenopausal women. However, comparison
(using the Kolmogorov-Smirnov test) of
data from the three groups revealed the
only significant difference to be the presence of more cells per group in pregnant than in postmenopausal women
(.001 < p < .005). T h e number of single
ductal cells was significantly higher in
specimens from premenopausal women
with benign breast disease symptoms than
in specimens from the other women shown
in Table 4 (F test, SS-STP, p < .05).
Table 3. Occurrence of Ductal Cells
Ductal Cells
Clinical
Status
of Patients
No.
Specimens
None
Single
Cells
Pregnant
Nonpregnant
TOTAL
68
68
136
19
25
44
1
9
10
Groups of
Cells
Single Cells
and Groups
of Cells
Single Cells
and/or
Groups of
Cells
41
17
58
7
17
24
49
43
92
736
A.J.CP.—Vol.
KING ET AL.
64
Table 4. Distribution of Cell Types
Clinical Status
Single Ductal
Epithelial
Cells
Foam Cells
Histiocytes
Lymphocytes
Neutrophils
Necrotic
Cells
Postmenopausal,
asymptomatic
.5 ± .3
50.3 ± 12.6
10.3 ± 4.6
27.0 ± 6.5
7.3 ± 2.9
7.0 ± 2.7
Postmenopausal,
symptomatic
1.3 ± .9
39.3 ± 8.4
11.3 ± 5.0
20.3 ± 6.2
13.3 ± 5.2
15.3 ± 2.6
Premenopausal,
symptomatic
3.1 ± 1.7
48.8 ± 5.5
18.2 + 4.6
16.3 ±4.1
8.7 ± 1.5
7.1 ± 1.3
Premenopausal,
symptomatic
10.5 ±7.5
21.0 ± 9.2
21.3 ± 3.1
33.5 ± 6.0
5.5 ± 1.7
8.5 ± 1.0
.5±
29.2 ± 2.2
19.1 ± 1.1
13.7 ± 1.6
30.6 ± 2.9
6.6 ± .6
Pregnant
.2
Note: In this table, "symptomatic" means symptomatic for benign breast disease. No specimens from women with malignant breast disease
are included in this tabulation.
Similarly, when the number of ductal epithelial cell groups present was analyzed
according to clinical status, a higher incidence was found in women with symptoms of benign breast disease than in either
normal women or in those who had treated
or untreated malignant breast disease
(F test, SS-STP, p < .05) (Table 5).
Apocrine metaplastic cells comprised an
insignificant number of the total cells present, and were observed only occasionally,
in the 136 specimens. However, in all
1,456 specimens, apocrine metaplastic cells
were rarely present in specimens from
normal nonpregnant women, were frequent in specimens from women who had
either benign or malignant breast disease,
and were very common in specimens from
pregnant women.
Foam cells were an important component of the 136 specimens in terms of
percentage of total cells. They were most
numerous in asymptomatic women, either
pre- or postmenopausal (48.8 ± 5.5% and
50.3 ± 12.6%, respectively); in women with
symptoms of breast disease and in pregnant women, single foam cells formed a
smaller percentage of cells counted (Table 4). Histiocytes were more numerous
in premenopausal than in postmenopausal
women. Lymphocytes were more numer-
ous in symptomatic premenopausal women
than in asymptomatic premenopausal
women (F test, SS-STP, p < .05). Neutrophils were significantly more numerous
in pregnant women than in all others
(F test, SS-STP, p < .05) (Table 4).
T h e comparison of total and differential
counts between right and left breasts (using
the paired T test) for the 136 specimens
revealed no significant difference.
Discussion
From the data collected, we determined
the cellular composition of nipple aspirate
specimens from women in various clinical
states, hoping to establish a baseline for
normal cytology that might define an adequate normal cell specimen. In planning
this study, we recognized two major problems: (1) the need to determine the adequacy of specimen collection with the
nipple aspirator device, and (2) the need
to select an appropriate preparatory
method to conserve and preserve diagnostic features in a specimen of small
volume with unknown cellular content.
A fluid specimen was obtained from one
or both breasts of 60.7% of women in
whom nipple aspiration was attempted. 8
T h e low fluid yield in the series being
December 1975
737
NIPPLE ASPIRATE BENIGN CELLS
reviewed is related to striking differences
in fluid availability correlated with race.
Among the Chinese in our series, 24.1%
yielded nipple secretions with the aspirator,
in sharp contrast to the 70.2% successful
collection rate among Caucasians. Of critical importance to the success of this method
in producing a fluid specimen is the care
and time taken to prepare the nipple
by loosening and removing plugs of keratotic debris that block the duct openings
(Fig. 1). Allowing adequate time for the
action of softening agents to take effect
is absolutely essential. Afterward, plugs
and debris must be removed mechanically.
The problem of specimen preparation
was considered with reference to the efficiency of different technics in recovering total cell content as well as in preserving details of cell morphology. O u r
studies conducted with body cavity fluids
have shown reproducible results: the
highest percentage of recovery and the
best preservation of cell morphology occur
using the Millipore filter.1 T h e expected
error in our determinations of total cellularity of the nipple specimens was based
upon the determination made with Millipore filter recovery rates.
We found a disappointingly small number of cells per specimen for the vast
majority of women aged 40 years or more,
who are in the age group most likely
to have clinical breast cancer. However,
it was encouraging that among women
in the fourth decade of life, there was
an average of 30.6 ± 12.2 cells per specimen. Women in this younger age group
might be expected to harbor the precursors of clinical breast cancer; therefore,
knowledge of the group is especially significant if a cytologic approach to diagnosis
of early lesions is to be effective.
Cell types are believed to be even more
important than cell numbers in establishing specimen adequacy. If epithelial cells
of ductal lining are identified, the specimen is considered satisfactory; foam cells
Table 5. Occurrence of Ductal Epithelial
Cell Groups
Clinical Status
Normal
Benign breast disease symptoms
Post-mastectomy
Carcinoma
Average No. of
Ductal Epithelial
Cell Groups
per Specimen
4.2
10.8
1.3
3.5
± 1.5
± 3.2
± .5
± 1.3
alone are viewed as representative of ductal
content but the specimen containing just
foam cells may be of equivocal value
even though large numbers of such cells
are often present. By this interpretation,
54.1% of 1,456 consecutively collected
specimens were satisfactory. However, only
a few ductal cells were found in many
cases, and we questioned the adequacy
of those specimens. When specimens with
fewer than ten ductal cells were excluded
from the acceptable category, only about
15% of the specimens appeared satisfactory; however, 67.6% of the 136 specimens
in which detailed differential cell counts
were made contained ductal cells, either
single or grouped (Table 3). This marked
contrast appears in part related to pregnancy status: half of the specimens in the
group of 136 were from pregnant women,
whereas only a small fraction of the
1,456 specimens were from pregnant
women. Furthermore, both single cells and
groups of cells of ductal epithelial origin
were better represented in specimens from
symptomatic premenopausal women than
in those from others; fewer foam cells
in these same specimens may relate to an
alteration in the normal turnover rate for
ductal epithelium (accepting the foam cell
as representative of a degenerate form
of ductal epithelium). This concept of an
altered turnover rate is also supported by
knowledge of the anatomic changes in the
breasts of pregnant women and by the
observation of fewer foam cells in specimens from pregnant women.
738
KING ET
Our observations thus far have led us to
believe that the normal, nonpregnant
woman who has no symptoms of breast
disease can be expected to have a nipple
aspirate specimen containing very few
cells. Foam cells will comprise a significant percentage of the normal cells, while
well-preserved ductal epithelial cells will
often be absent or very few in number.
If symptoms of benign breast disease are
present, the sample can be expected to have
an average of 25,000 cells, with ductal
epithelial cells present in 78.5% of cases.
Apocrine metaplastic cells, a further indicator of breast disease, will rarely be found
in specimens from asymptomatic women.
We believe that our finding of relatively
cellular specimens associated with benign
breast disease and comparatively scantly
cellular specimens from normal breasts is
very important in the routine evaluation of
nipple aspirate specimens.
AL.
2.
3.
4.
5.
6.
7.
8.
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9.
AJ.CP.—Vol.
64
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