Debra A. Ritter, H.T. (ASCP)
Ruth Michels, C.T. (ASCP) CMIAC
Lung cancer is presently the most common cause of fatal
malignancy in both men and women in the United States and
Western Europe and is rapidly becoming pandemic in nature
throughout the world. Based on current smoking patterns,
substantial public health burden will continue during the
next several decades. Despite its high incidence, the
overall curability of lung cancer remains low. Currently,
the five-year survival rates range from ten to thirteen
percent. No other cancer, in men or women, can claim such a
calamitous outcome.
Screening by sputum cytology in high risk asymptomatic
populations is useful for early detection of
roentgenographically occult lesions. The merit lies in the
simplicity and non-invasiveness of the procedure. Patients
who are diagnosed with lung cancer at an early stage and
successfully treated, whether by surgery or radiation
therapy, have shown a longer survival rate than patients
found to have cancer in advanced stages.
Lung carcinogenesis is the result of a series of genetic
mutations that accumulate progressively in the bronchial
epithelium, first manifesting in cytologically identifiable
premalignant lesions and finally resulting in invasive
carcinoma. St. Mary's Hospital & Medical Center in Grand
Junction, Colorado, is undertaking a
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(A)Papanicolaou stained with moderate cell
atypia (B) Cytokeratin 18 stained atypia cell group
(C) Porphyrin stained bright field squamous
carcinoma (D) Fluorescent microscopy of Porphyrin
stained squamous cell carcinoma
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hypothesis that improved methods of detection of
preneoplastic cellular changes in the tracheobronchial
tree will result in earlier clinical interventions and
improved survival in lung cancer.
A methodology to standardize sputum cytology samples
was found to be necessary. The accepted technique for
mechanical homogenization (Saccomanno Technique) was
enhanced using the new Shandon Megafunnel. This
technique resulted in a consistently uniform monolayer
specimen necessary for cytologic and immunocytochemisrty
evaluation. The Saccomanno Method was used for the
collection and processing of the sputum cytology
samples. Once centrifuged, the supernatant was decanted
and the centrifugate vortexed to achieve a homogenized
sample pellet. A 150ul aliquot of the vortexed pellet
was diluted in 18cc of diluent This technique results in
a uniform single cell layer which is contained within
the sample square scribed on the Megafunnel slide.
Photomicrograph A, taken at 40X magnification,
depicts Papanicolaou stained sputum cytology sample
showing a group of cells whose morphology is consistent
with moderate squamous cell atypis (see arrow).
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Photomicrograph B
(see arrow) shows immunocytochemistry stainging of the
same cell group, using a cytokeratin 18 antibody. (50%
Saccomanno Fixative and 50% 0.15M Nalco). One milliliter
of this homogenized sample dilution was then pipetted
into a Megafunnel chamber apparatus for each side. (The
diluent can be adjusted to allow for the desired cell
concentration). Samples were cytocentrifuged at 1250 rpm
for 5 minutes in a Shandon Cytospin 3. Slides were
removed, allowed o air dry and stained using either a
modified Papanicolaou (Pap Stain) or immunocytochemistry
stain.
Note that only one of the cells stains positive using
this antibody. Investigative studies will be done to
determine the difference in staining within this, and
other such groups of cells. Photomicrograpg C
taken at 40X, shows a bright field image of an
immunocytochemistry stained squamous cell carcinoma
using a porphyin stain (see arrow). Photomicrograph D
demonatrates the fluorescence microscopy. |
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