Session 1, Saturday, October17th
9:30 - 11:30 Improving the Quality of Diagnosis and Prognosis (I)
Sensitivity of Infrared Spectral Features Toward Differentiation, Maturation, Cell Cycle Dependence and State of Health of Human Cells
Luis Chiriboga1,2, Susie Boydston-White1 and Max Diem
1Department of Chemistry and Biochemistry, City University of New York, Hunter College, 695 Park Avenue, New York, NY 10021, USA
2Molecular Diagnostics Laboratory, Department of Pathology, Bellevue Hospital, New York University, 27th Street and 1st Avenue, New York, NY 10016
The changes in the infrared spectral patterns observed between healthy
and diseased tissues can be classified into two different classes which
we refer to as gross spectral changes, and disease induced changes.
The first class of changes includes, for example, variations of glycogen
content in tissue sections or single cells that are nonspecific for disease.
Similarly, changes in the structural protein content of tissue, increased
vascularity, or the changes accompanying cell maturation and differentiation
can be observed infrared spectroscopically, but these changes are not necessarily
correlated with the occurrence of disease. However, these changes are ideally
suited to create maps of tissues or distributions of cells that can augment
the information obtainable from photomicrographs of stained tissues used
in pathology. In fact, the advantage of infrared (false color) mapping
is that such a map contains more information, per pixel element, than stained
tissue, and can be constructed and interpreted totally objectively by computer
Among the disease induced spectral changes, we and others have found
that certain spectral regions due to nuclear DNA appear to be enhanced
in samples with diagnosed cancerous disease. Since the DNA spectral features
are superimposed on those due to nuclear and cytoplasmic RNA, these changes
are very subtle. In an attempt to quantify and interpret the changes in
the DNA/RNA spectral contributions in healthy and abnormal cells and tissue,
we have found that similar changes in the DNA/RNA spectral features are
observed for cells that are at different stages in the cell division cycle.
This cell cycle dependent spectra were collected for cultured myeloid leukemia
(ML-1) cells that were isolated into pure fractions according to the stage
in the cell cycle. This observation leads to a possible conclusion that
all hitherto observed spectral changes between normal and abnormal cells
and tissues are due to different distributions of cells at given stages
of their development. On the other hand, the possibility exists that a
small number of abnormal cells with large DNA spectral contributions dominates
the observed spectra of abnormal samples.
A recent analysis of spectral data from single cells confirms that spectral
patterns from a sample of exfoliated cells can be interpreted in terms
of the cell maturation, and in terms of distributions of cells according
to their stage in the cell cycle. We, therefore, believe that the understanding
of the infrared spectra of cells in terms of cell differentiation, maturation
and life cycle is crucial for the correct interpretation of infrared spectra
of healthy and abnormal cells and tissues.
IR-Spectroscopy and IR-Microscopy of Breast Tumor Cell Lines and Human Breast Tumor Tissues
Heinz Fabian, Arnfried Schwartz, Ralf Wessel and Iduna Fichtner
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
Peter Lasch and Dieter Naumann
Robert Koch-Institute, Berlin, Germany
Due to recent advances in instrumentation and data processing techniques,
infrared techniques are not only invaluable tools for the structural characterization
of the building blocks of living organisms, but now also allow successful
studies of complex biomedical materials. In order to evaluate the diagnostic
potential of FTIR spectroscopy, we have started to analyse samples that
can be isolated in a highly reproducible manner, and which can be characterized
by other, independent techniques. Such systems are tumor cell lines. Comparative
studies of human breast tumor cell line suspensions revealed that IR methods
have the potential for differentiating between related cell lines and are
sensitive to biochemical changes associated with the cell death. The collection
of IR spectra through microscope optics permits this analysis to be made
from few cells, which raises the possibility of analysing tissues by IR
microscopy and searching for small cluster of abnormal cells. To assess
the potential of the IR technique for the diagnosis of breast tumor tissue,
thin sections of tissue were mapped by IR microspectroscopy. The spectra
of the maps were analysed using functional group mapping or pattern recognition
techniques. The output values of the different approaches were then reassembled
into IR images of the tissues. To allow a definite correlation between
tissue features and spectroscopic properties, alternate sections of tissue
were used for infrared spectroscopic measurements and for light microscopic
staining. Chemical mapping based on single band intensities turned out
to be an easy way to identify major breast tissue constituents, such as
connective tissue, malignant epithelium and fat. Cluster analysis of the
infrared spectra, combined with immunohistochemical staining of the tissue
sections, provided deeper and more detailed insights. Based on these data,
the potential of IR microspectroscopy for the diagnosis of breast cancer
tissue sections is discussed
Examination of Cervical Smears by FTIR Spectroscopy: A Study of the Dilution Effect and the Archiving of Smears
Janie Dubois1, Ashraf A. Ismail1 Jocelyne Arseneau2, and Manon Auger3
1McGill IR Group, Macdonald Campus of McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9.
The potential applicability of FTIR spectroscopy for the examination
of cervical cells has now been under investigation for nearly a decade.
The impetus of these studies is twofold. On a fundamental level, infrared
spectroscopy can provide information about the molecular changes that occur
as cells are transformed from a normal to an abnormal state. A secondary
goal of these studies is the development of FTIR spectroscopy as a diagnostic
tool that can replace or complement the current methods for screening which
predominately rely on visual examination of exfoliated cells to diagnose
cervical pre-cancerous lesions. We have investigated the sample handling
and archiving aspects of cervical smears as part of an ongoing project
aimed at the development of a dedicated FTIR-based cell analyzer. In North
America, Pap smears have to be kept for 5 years, for follow-up purposes,
in cases where no abnormality is dedicated. It is also common practice
to keep all abnormal samples indefinitely. Therefore, it is necessary to
prepare cervical smears for infrared analysis in a form allowing preservation
of the samples. We have established a procedure involving minimal sample
preparation and the use of custom-made polyethylene slides as a disposable
IR support and have evaluated the stability of the samples during 5 years
of storage. Minimal changes in the spectra of the samples have been observed
over this period. The main problems encountered arose from excessive handling
of the samples, resulting in detachment of the cells from the support.
This problem was seen in <10% of the samples examined. Various corrective
measures are under investigation.
2Department of Oncology, McGill University, Montreal, Quebec, Canada.
3Department of Pathology, McGill University, Montreal, Quebec, Canada.
The archived samples were also used to evaluate the feasibility of detecting
abnormal cells in the presence of a large number of visually normal cells
(and in some cases the presence of bacteria resulting from infection).
It was hypothesized that the presence of one or two abnormal cells among
hundreds of normal cells, while sufficient for the classification of a
sample as abnormal by visual microscopy, would not have a significant influence
on the infrared spectrum of the whole sample due to the dilution effect.
FTIR spectra of cervical cell smears recorded from cross-sectional areas
ranging from 5000 ´ 5000 mm
to 20 ´ 20 mm
were compared. Some samples showed definitive differences between the spectra
of the whole sample and those of specific areas. On the other hand, some
samples diagnosed as abnormal by cytopathologists exhibited remarkably
similar spectra from all portions of the sample, whether or not each particular
portion contained cells morphologically classified as abnormal. The explanation
for these findings is not clear yet because exfoliated cervical cells do
not come from one specific area of the cervix and therefore should, in
principle, not be equally affected by pre-cancerous lesions.
Infrared Micro-Spectroscopy of Cervical Smears and Activated Lymphocytes
D. McNaughton, B. Wood and M. Romeo
Chemistry Department, Wellington Rd., Monash University, Clayton, Victoria Australia.
Over the last few years we have built a database of infrared spectra
of cervical smears from patients attending the dysplasia clinic of the
Royal Women�s Hospital (Melbourne). We have also investigated the spectra
of a number of components that may appear as confounding variables in the
spectra of cervical smears. These include bacteria common to the female
genital tract, semen, endocervical mucin and blood components. We find
lymphocytes, particularly activated lymphocytes, which will often be present
due to non specific disease or inflammation, to be of most concern for
analysis and diagnosis. The presence of these confounding variables indicates
that an approach using multivariate statistical techniques or artificial
neural networks is the most appropriate for analysis and subsequent diagnosis.
To date we have subjected some of our data, where biopsy results are available
for comparison, to analysis using the multivariate statistical techniques
of SIMCA (Soft Independent Modelling of Class Analysis) and K-nearest neighbours
(KNN) in addition to developing an analysis based on artificial neural
networks. The methodology we have developed will be described and results
of the preliminary analyses will be discussed and compared.
Lymphocytes activated with the agent phytohaemagluttinate (PHA) show
spectral differences when compared with non activated lymphocytes and we
have carried out a study monitoring lymphocyte activation over time. These
studies show distinct spectral changes over the first few hours after activation
and with the use of principal component analysis (PCA) of derivative spectra
these changes can be attributed to RNA synthesis. These results, which
show that infrared may be useful in the initial screening of donors in
organ transplantation together with the results of a study monitoring the
activation of mixed lymphocytes will be discussed.
How Can Infrared Spectroscopy Contribute to Diagnosis and Prognosis of Chronic Lymphocytic Leukemia?
Christian P. Schultz
Institute for Biodiagnostics, National Research Council of Canada
Chronic lymphocytic leukemia (CLL) is a disorder
of morphologically mature but immunologically less mature lymphocytes and
causes immunosuppression, failure of the bone marrow, and infiltration
of malignant (cancerous) cells into organs. Usually the symptoms and the
course of the disease will develop gradually and occur in the elderly with
90% of the cases in people over 50 years old. Many cases are accidentally
detected by routine blood tests in people with no symptoms. The cause of
CLL is unknown and until today no curative therapy has been established.
Treatment decisions are made by the physician on the basis of a variety
of factors: a) rate of increased lymphocyte count, b) pattern of lymphocyte
spread in the bone marrow, c) size of spleen and lymph nodes and d) rate
development of anemia and low platelet counts.
Infrared spectroscopy can successfully distinguish
normal mature lymphocytes from apparently mature CLL cells, based on IR
band analysis and pattern recognition techniques. The same techniques reveal
that the doubling time of CLL cells is imprinted in the IR spectra and
may therefore be used to predict the course of a patient�s disease, the
treatment and possible outcome. The infrared spectra of CLL cells also
appear to contain many other differentating features, suggesting that the
biochemical composition of these cells varies more than is indicated by
conventional methods. In vitro drug treatment of CLL cells usually provides
information on the cell�s drug sensitivity and may reflect the in vivo
cell response to a particular drug - very important for the selection of
the most effective drug. Infrared spectra of the untreated CLL cells can
be correlated with these values of in vitro drug resistance (e.g. MTT assay)
and then used for the construction of a large training set containing spectral
patterns for sensitive and resistant cells. Applying this model to IR spectra
of undetermined and untreated cell samples then allows the prediction of
drug sensitivity on the original CLL cells without performing the actual
MTT test. Instead of correlating IR spectra with drug resistance values,
the drug testing itself can be performed and monitored by infrared spectroscopy.
This has the benefit of accessing cell changes within cancer cells directly
which may lead to a better understanding of the drug cell interaction and
the actual mechanism. For example, IR spectroscopy was succesfully used
to follow the membrane changing action of Bryostatin 1, a potentially new drug
(in clinical trials) that transforms drug resistant CLL cells back into sensitive ones.
| FT-IR Workshop Series, Berlin, Robert Koch-Institute
|Any opinions, findings and conclusions or recommendations expressed in this publication are those
of the workshop organizers and do not necessarily reflect the views of the Robert Koch-Institute.
© 2021 Peter Lasch