TECHNICAL REPORT
Clinical application of the liquid-based cytological test in
cytological screening of sputum for the diagnosis of lung cancer
GUANG-PING WU, EN-HUA WANG, JIAN-HUA LI, ZHI-MIN FU AND SHUO HAN
Department of Pathology, College of Basic Medical Sciences and Department of Pathology,
The First Affiliated Hospital, China Medical University, Shenyang, China
ABSTRACT
Background and objective: The liquid-based cytological test (LCT) is successfully and widely used to
assess cervical cytology. This study aimed to compare
the cytological findings and diagnostic sensitivity of
LCT with those of the pick-and-smear (PS) method for
diagnosing lung cancer.
Methods: Sputum specimens from 101 patients diagnosed with lung cancer were studied.
Results: LCT slides showed decreased areas of cell
monolayers, a clearer background and distinct, stereoscopic cytological features. The LCT had a significantly
higher diagnostic sensitivity for lung cancer (80.2%)
than the PS method (63.4%, P < 0.05), particularly for
small cell lung carcinoma (P < 0.05). Combination of
the LCT with the conventional PS method showed significantly higher diagnostic sensitivity for the detection of adenocarcinoma (80.6%) compared with the PS
method alone (55.6%, P < 0.05).
Conclusions: LCT is a useful and easily performed
technique that can be widely applied, and is suitable
for mass screening for the early diagnosis of lung
cancer.
or no value in the identification of peripheral cancers.6 The major limitation of the sputum smear
method has been variable quality due to contamination with blood, saliva or inflammatory cells.7
The liquid-based cytological test (LCT) is a useful
cytological technique and its clinical application was
approved by the Food and Drug Administration in
1999. It is successfully used in cervical cytology for
the preparation of clear cell monolayers through the
removal of mucus, necrotic material and inflammatory cells.8,9 This study aimed to directly compare
the LCT with the conventional pick-and-smear (PS)
method, with respect to the quality of the slides
and diagnostic sensitivity. The use of the LCT for the
diagnosis of lung cancer was also evaluated.
METHODS
Correspondence: En-Hua Wang, Department of Pathology,
College of Basic Medical Sciences and Department of Pathology,
The First Affiliated Hospital, China Medical University, Shenyang
110001, China. Email: [email protected]
Received 24 July 2007; invited to revise 31 December 2007, 1
April 2008, 27 April 2008; revised 20 February 2008, 12 April 2008,
28 April 2008; accepted 18 June 2008 (Associate Editor: Jeffrey
Swigris).
The study was conducted in accordance with the
regulations of the institutional review boards at
China Medical University. From January to October
2004, 708 outpatients and inpatients with histories of
respiratory symptoms were recruited at The First
Affiliated Hospital, China Medical University. The
patients were instructed to rinse their mouths and
brush their teeth before coughing sputum, to reduce
contamination with food dregs and bacteria. They
were also instructed to increase their intake of liquids
and to expectorate into a plastic cup following a deep
cough upon awakening. This procedure was to be
repeated on at least three consecutive days. Of these
708 patients, 101 were diagnosed with lung cancer on
the basis of histopathology and/or cytological findings at bronchoscopy. These 101 patients, as well as
40 randomly selected patients without lung cancer,
were included as study subjects and control subjects,
respectively. In the study group there were 68 men
and 33 women, ranging in age from 30 to 93 years.
Histological examination confirmed that there were
48 cases of squamous cell carcinoma (SCC), 36 cases
of adenocarcinoma (ADC) and 17 cases of small cell
lung carcinoma (SCLC).
After macroscopic examination, four smears
were prepared from fresh, unfixed sputum and
immediately fixed in 95% ethanol and stained with
© 2008 The Authors
Journal compilation © 2008 Asian Pacific Society of Respirology
Respirology (2009) 14, 124–128
doi: 10.1111/j.1440-1843.2008.01399.x
Key words: cytopathology, liquid-based cytological
test, lung neoplasm, sputum.
INTRODUCTION
Sputum cytology is regarded by many clinicians as a
non-invasive, cheap and simple test for the diagnosis
of lung cancer.1 It complements radiology in the diagnostic workup of lung cancer patients and can be
useful for identifying lung carcinoma,2 especially at
the early and occult stages.3–5 However, it has shown a
low yield in prospective screening trials and is of little
125
LCT in cytological screening of sputum
Papanicolaou. The residual sputum was transferred to
a small bottle with the same volume of CytoRich
liquid (Tripath Imaging Inc., Burlington, NC, USA).
One millilitre of mucolytic agent (Tripath Imaging)
was added for every 10 mL of sputum, incubated at
room temperature for 30 min and vortexed for 10 s.
Additional mucolytic agent was added to the mixture
until the mucus was completely lysed. The mixed
liquid was then transferred to a 50-mL tube and centrifuged at 2000 g for 10 min. The supernatant was
removed and the pellet was resuspended in 7.5 mL of
distilled water. This suspension was vortexed again
and centrifuged at 2000 g for 5 min. The supernatant
was removed and the pellet was vortexed and transferred to the AutoCyte PREP system (Tripath
Imaging), in which slides were automatically prepared and stained. Two slides were prepared from
each tube and were stained with Papanicolaou.
For all sputum samples, slides prepared by both the
LCT and conventional PS methods were screened and
assessed independently by two cytologists.
The sensitivities of the two methods were compared by the c2-test using the SPSS 10.0 software
package. Statistical significance was defined as
P < 0.05.
RESULTS
In comparison with the conventional PS method, LCT
demonstrated a number of advantages. First, screening time was reduced and screening efficiency was
increased due to the decreased areas of cell monolayers. Second, the slides had a clearer background due
to the dissolution of mucous material, the destruction
of most of the red blood cells and significantly
reduced numbers of inflammatory cells. Thus, abnormal and tumour cells were more readily discernible.
Regardless of cell type, the cells were clearly stained
and their morphology was well preserved. The microscopic fine structure of the nuclear envelope, nucleoli
and chromatin was also clearly discernible and
stereoscopic.
In the LCT slides, SCC cells were evenly distributed
in a perfect monolayer pattern, without mucus
(Fig. 1). Single ADC cells showed globular and stereoscopic morphology in the LCT slides and small aggregates of ADC cells were arranged in an acinic pattern
(Fig. 2). SCLC cells showed mulberry structures
(Fig. 3). A comparison of slide quality between the PS
method and the LCT is shown in Table 1.
Table 2 shows comparisons of the LCT, PS and
combination of the LCT and PS methods for detecting lung cancer. Of the 101 sputum samples from
patients with lung cancer examined by the conventional PS method, only 64 were found to have cancer
cells, due to obscuring by mucous material, inflammatory cells and necrotic debris. The diagnostic
sensitivity was only 63.4% (64/101). However, the
LCT showed 80.2% (81/101) diagnostic sensitivity
due to a clearer background and well-preserved cell
morphology. This difference in diagnostic sensitivity
between the two methods was significant (P < 0.05).
When the PS method and the LCT were evaluated in
(a)
(b)
(a)
(b)
Figure 1 Squamous cell carcinoma. (a) A slide prepared by
the conventional pick-and-smear
method; (b) A slide from the same
patient prepared with the liquidbased cytological test, showing
homogeneous cell distribution in
a perfect monolayer and absence
of mucus (Papanicolaou stain,
¥ 400).
Figure 2 Adenocarcinoma. (a) A
slide prepared by the conventional
pick-and-smear method; (b) A
slide from the same patient prepared with the liquid-based cytological test, showing a clearer
background. The small aggregates
of cancer cells are arranged in an
acinic pattern (Papanicolaou stain,
¥ 400).
© 2008 The Authors
Journal compilation © 2008 Asian Pacific Society of Respirology
Respirology (2009) 14, 124–128
126
G-P Wu et al.
(a)
(b)
Figure 3 Small cell lung carcinoma. (a) A slide prepared by
the conventional pick-and-smear
method; (b) A slide from the same
patient prepared with the liquidbased cytological test, showing a
clearer background, crowded cell
clusters, small uniform nuclei and
scanty cytoplasm (Papanicolaou
stain, ¥ 400).
Table 1 Comparison of the differences in quality between slides generated by the conventional pick-and-smear (PS)
method and the liquid-based cytological test (LCT) for the identification of cancer cells
Method
SCC
ADC
SCLC
PS
LCT
Large amounts of mucus, necrotic material, inflammatory
cells and red blood cells were observed among the SCC
cells
Mucous and inflammatory cells were observed around
the ADC cells, which showed silkworm-like, spindle
structures
The dispersed cells were arranged in strip or band
structures
The original cellular structures were well preserved,
without mucus or white blood cells among the
SCC cells
The small aggregates of ADC cells were arranged
in an adenoid pattern and had stereoscopic
morphology
The crowded cell clusters showed mulberry or
inlay-like structures
ADC, adenocarcinoma; SCLC, small cell lung carcinoma; SCC, squamous cell carcinoma.
Table 2 Comparison of the liquid-based cytological test (LCT), the pick-and-smear (PS) method, and a combination of
LCT and the PS method for detection of lung cancer in 101 patient samples
Pathology
SCC
ADC
SCLC
Total
n
LCT
positive (percentage)
PS
positive (percentage)
LCT + PS
positive (percentage)
48
36
17
101
38 (79.2%)
28 (77.8%)
15 (88.2%)*
81 (80.2%)*
37 (77.1%)
20 (55.6%)
7 (41.2%)
64 (63.4%)
42 (87.5%)
29 (80.6%)*
15 (88.2%)
86 (85.1%)**
*P < 0.05, **P < 0.01, when compared with the PS method.
ADC, adenocarcinoma; SCLC, small cell lung carcinoma; SCC, squamous cell carcinoma.
combination, the diagnostic sensitivity for detecting
lung cancer was 85.1%, significantly higher than the
PS method alone (63.4%, P < 0.01). No cancer cells
were found in the 40 samples from patients without
lung cancer.
Of the 38 positive SCC cases as determined by LCT,
five were negative by the PS method, and 33 were
positive by either the LCT or the PS method. On the
other hand, of the 37 positive SCC cases as determined by the PS method, four were positive by the PS
method but negative by LCT, and 33 were positive by
either LCT or the PS method. The diagnostic sensitivity for the detection of SCC was 87.5% when the LCT
and PS methods were combined. There was no significant difference when compared with either method
alone (P > 0.05).
Of the 28 positive ADC cases as determined by LCT,
nine were negative by the PS method and 19 were
positive by either LCT or the PS method. Of the 20
positive ADC cases as determined by the PS method,
one was positive by the PS method but negative by
LCT, and 19 were positive by either LCT or the PS
method. Although there was no significant difference
in diagnostic sensitivity between the two methods
(P > 0.05), when LCT and the PS method were combined, the diagnostic sensitivity for ADC was 80.6%,
significantly higher than that of the PS method alone
(55.6%, P < 0.05).
Of the 15 positive SCLC cases as determined by the
LCT, seven were positive by the PS method and the
remaining eight were negative. The diagnostic sensitivity of the LCT for the detection of SCLC was 88.2%,
Respirology (2009) 14, 124–128
© 2008 The Authors
Journal compilation © 2008 Asian Pacific Society of Respirology
127
LCT in cytological screening of sputum
significantly higher than that of the PS method alone
(41.2%, P < 0.05).
DISCUSSION
The detection of cancer cells by sputum smear has
been used to diagnose lung cancer for over 100 years.
Diagnostic sensitivity is reportedly quite variable due
to different preparation methods in different institutions. Recently, the diagnostic sensitivity of sputum
cytology was reported to be 84.4%2 and 31%,7 whereas
the sensitivity of LCT was 80.2% in this study. There
was a significant difference in diagnostic sensitivity
between the LCT and the PS method, and when
the two methods were combined, the diagnostic
sensitivity for the detection of lung cancer was 85.1%,
significantly higher than that of the PS method alone
(63.4%, P < 0.01).
The well-known Saccomanno method was used to
collect specimens in a fixative, blend the material to
liquefy the mucus and disperse the cells in an evenly
distributed monolayer on slides.10 This method
provided more information for the diagnosis of
lung cancer and decreased false negative results.10
However, Perlman et al. reported that this method
was not better than the fresh smear method, particularly for the diagnosis of SCLC.11 Recently, Tang et al.
used dithiothreitol as a mucolytic agent and found
this approach to be more sensitive than the conventional PS method for the diagnosis of lung cancer.12
However, this method did not remove inflammatory
cells and red blood cells from the slides.
The area of a conventional smear is generally
1375 mm2 and the time needed for screening is 7 min,
whereas the area of the LCT monolayer is only
134 mm2, and the time needed for screening is
reduced to 2.5 min. Forty-eight samples could be processed simultaneously and the cells stained automatically using standard procedures in one hour. The
cell monolayers showed a clearer background and
stronger contrast qualities due to the programming of
specimen processing, slide preparation and staining.
Therefore, the cytologists were more easily able to
concentrate on screening every field of vision with a
notable increase in screening efficiency. This is in
agreement with a previous report by Rana et al.13
With the conventional sputum smear method,
SCLC slides contained necrotic material, and showed
small cells in loose arrangement. The malignant cells
were ‘flooded’ with mucus, epithelial cells and other
impurities in the sputum, and as a result the diagnostic sensitivity was lower. Wang et al. reported that the
diagnostic sensitivity for SCLC was 50% when using
the sputum processing method.7 However, the
present study showed that with the LCT the diagnostic sensitivity for SCLC increased to 88.2% from 41.2%
observed with the conventional PS method. The LCT
therefore represents a useful method of sputum processing not previously used in diagnostic cytology,
and could be a potentially powerful tool for the diagnosis of SCLC.
In this study, the epithelial cells were deposited
naturally on glass slides coated with poly-L-lysine in
© 2008 The Authors
Journal compilation © 2008 Asian Pacific Society of Respirology
an automatic procedure. There was no smearing or
distortion, the cells were distinctly stereoscopic and
cellular structures were well preserved, especially in
the ADC slides. Twenty specimens were positive for
ADC when examined using the conventional PS
method, whereas 28 specimens were positive with the
LCT. Although there was no significant difference in
diagnostic sensitivity between the two methods, the
diagnostic sensitivity for ADC was 80.6% when the PS
method and LCT were combined, which was significantly higher than that for the PS method alone
(55.6%, P < 0.05).
On the other hand, the diagnostic sensitivity of the
LCT for SCC was not as good. The number of cases
positively diagnosed using the PS method was 37,
whereas with the LCT only 38 cases were positively
diagnosed. This may have been due to the large
number of squamous cells obtained with the PS
method,14 and the fact that the number of monolayer
slides prepared for each specimen may not have been
sufficient. In the case of the seven SCC and one ADC
specimen examined using the LCT, the first slide
showed no cancer cells, although carcinoma cells
were found on the second. This is likely to be due to
the non-globular morphology of SCC cells, which are
not readily sedimented and therefore lost during
centrifugation.
Motherby et al. reported a diagnostic sensitivity of
82.6% for the diagnosis of lung tumours if one slide was
prepared from the bronchial secretions, 88.8% with
two slides and 94.0% with seven to eight slides.15 This
indicates that a single slide may not adequately represent all the cellular elements from a sputum specimen.
We suggest that at least four slides be prepared for each
sample. Further studies are needed to use all residual
material and investigate the diagnostic accuracy of
LCT in detecting cancer cells in sputum from patients
with lung cancer.
The LCT provides automatic preparation of cell
monolayers from sputum that are highly representative of the cellular content of sputum samples and are
superior to those obtained by the PS method. LCT
results in a clearer background, smaller areas to be
screened and well-preserved, distinctly stereoscopic
original cellular structures. The method is therefore
suitable for mass screening for the early diagnosis of
lung cancer.
ACKNOWLEDGEMENTS
This work was supported by grants from the Science
Foundation of Liaoning Province (Wang En-Hua,
Grant No. 20033002) and the Shenyang Science
and Technology Bureau (Wu Guang-Ping, Grant No.
1053125-1-45).
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© 2008 The Authors
Journal compilation © 2008 Asian Pacific Society of Respirology