PART TWO
Diagnostic Cytology
cytology are an eloquent testimony to this prominence.17,21-36
This chapter explores the contributions made to pulmonary
pathology by morphologic study of sputum, bronchial washings,
bronchial
aspirates,
bronchial
brushings,
bronchoalveolar
lavage specimens (BAL), and transthoracic FNA from the lung.
Sampling and Cytopreparatory Techniques
The purpose of this section is to review the major types of cyto-
logic preparations now used in most laboratories. The specimens
discussed here include sputum, bronchial washings, bronchial
brushings, BALs, and FNAs.
The diagnostic accuracy of cytology begins with a foundation
of excellence in cytopreparation of these specimens. A respira-
tory tract specimen that has been prepared for cytologic examina-
tion should exhibit an abundance of well-preserved and stained
diagnostic cellular material, should have been prepared rapidly
and with relative ease, and should survive permanent slide
storage. Many laboratories have studied techniques for the best
realization of these objectives. Paraffin embedding and section
of sputum is not a productive technical approach to respiratory
cytology, but it is of use in FNA as well as any other specimen
in which visible chunks of tissue are removed along with cells.
Various techniques for freeing tumor cells from mucus by lysis
were interesting in theory but were frequently too laborious
and time consuming to be practical. Several major techniques
have stood the test of time and are the most widely used today.
They are the wet-film preparation and fixation from fresh or pre-
fixed respiratory material, the Saccomanno blender technique,
membrane filtration, cytocentrifugation, and Wright-stained,
air-dried smears. These methods are used for spontaneously
produced sputum, induced sputum, bronchial washings and
brushings, BALs, and FNAs. The Papanicolaou method of stain-
ing is the most generally accepted one, and in the United States
in particular, it has gained widespread popularity. In many Euro-
pean laboratories, air-dried smears stained by a Romanowsky
stain are preferred; however, this technique for FNAs is gaining
acceptance in laboratories in the United States.
The types of cellular specimens and techniques for cellular
preparation discussed next are those currently most commonly
used in the contemporary clinical cytology laboratory. The cyto-
logic pattern may vary depending on the type of preparation,
and knowledge of these variations is essential for accurate cyto-
logic diagnosis.
Sputum
Sputum, a highly specialized product of the respiratory tract, is
the result of the interaction between the mucociliary apparatus
and the immune system of the host and between the animate and
inanimate invaders from the environment. Composed predomi-
nantly of mucus, it also contains a large variety of cellular and
noncellular materials produced by the host and substances that
have been inhaled. It is the most frequently examined specimen
obtained from the respiratory tract. A number of cytoprepara-
tory techniques are used in preparing it for examination.
Fresh and Unfixed Sputum
The simplest sputum specimen is one in which a fresh early
morning specimen, produced by a deep cough, is collected and
brought immediately to the laboratory without any fixation. It
is examined grossly for tissue fragments and blood-tinged areas.
Smears from these areas and other randomly sampled areas are
prepared and fixed immediately in 95% ethyl alcohol. Staining
of these smears is by the traditional Papanicolaou method. Spu-
tum prepared in this manner has the advantage of displaying
cells and other components that exhibit excellent preservation
and staining. It is the method preferred by us and has been in
use in our laboratory for nearly 30 years. Risse and associates, in
a 1987 study, emphasized the importance of blood in sputum.37
They found that in patients with primary lung cancer, the pres-
ence of blood in the sputum was highly significant from the
point of view of its association with a correct positive cytologic
diagnosis in sputum.
Prefixed Sputum
If it is not possible to transmit unfixed material to the labora-
tory, prefixed sputum may be obtained by instructing the patient
to expectorate into a wide-mouthed small jar, half-filled with
70% ethyl alcohol. Preservation of cells trapped in mucus may
be only fair because of failure of the alcohol to penetrate into
the cell. The mucus may also become rubbery, producing greater
difficulties in making a good smear.
Sputum Prefixed with Alcohol and Carbowax
(Saccomanno)
Saccomanno and colleagues described a method of cytoprepara-
tion of sputum that has gained wide popularity. It involves the
collection of sputum in a mixture of 50% ethyl alcohol and 2%
polyethylene glycol (Carbowax). In the laboratory, the specimen
is broken up in a food blender, and smears are prepared from
the centrifuged cell button. This technique has several major
advantages, including concentration of cells and the possibility
of preparing teaching slides from interesting cases.38 It is used in
some institutions for the collection of sputum from outpatients.
In tumor cells, this method produces a number of artifacts dif-
ferent from those found in smears prepared from fresh sputum.
For example, tissue fragments and fungal fragments may be
disrupted. Secretory vacuoles in tumor cells may be exploded,
and cells from small cell undifferentiated carcinoma may be
dispersed.
In a reported study, Perlman and associates reviewed 204
sputum samples that had been made using both slide prepara-
tions directly from fresh, unfixed sputum and the Saccomanno
technique.39 The diagnostic accuracy for 55 squamous carci-
nomas was similar (fresh 95%, Saccomanno 86%) but signifi-
cantly less in the Saccomanno preparations of 22 small-cell
carcinomas (fresh 100%, Saccomanno 24%) and 26 adenocar-
cinomas (fresh 96%, Saccomanno 52%). Four cases negative
on fresh smears were suspicious or diagnostic of cancer on the
Saccomanno slides. In no cases of small-cell undifferentiated
carcinoma did the Saccomanno preparation add information
not available on the fresh smears. In a similar study, Rizzo and
associates concluded that more diagnostic information and
fewer false-negative results were achieved with the Saccomanno
technique.40
Induced Sputum
In those patients who cannot produce sputum spontaneously
by deep coughing, a specimen of sputum may be induced. The
basic principle involves the inhalation of some appropriate solu-
tion that has been aerosolized. The inspired vapor stimulates
mucus production. One popular method uses a heated (37°C)
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