Light Scattering Flow Cytometry for the Minimally Invasive Quantification of Circulating Leukemic Cells.
Greiner, Cherry Anne.
Abstract The detection and quantification of circulating cancer cells is of clinical
importance for the diagnosis and staging of cancer as well as the assessment of a
patient's therapeutic response and risk for relapse. However, the enumeration of these
cells by conventional in-vitro techniques is challenged by the rarity of circulating
cancer cells and extensive blood sample proces... read moresing. To address these limitation some
researchers focused on the development of microfluidic devices to reduce the extensive
isolation and labeling processes while others focused on the development of in-vivo
based detection techniques. However, no one has explored the potential of elastic light
scattering, an intrinsic source of signal contrast known to be sensitive to
morphological differences in cells, for the development of a microfluidic based or
in-vivo based approach to enumerate circulating cancer cells. Using leukemia as a cancer
model, we explored the potential of light scattering to differentiate between Nalm-6
acute lymphoblastic cells and normal red blood cells (RBCs), mononuclear (MNs) and
granulocytic (GRANs) white blood cells. We first performed light scattering spectroscopy
measurements on static pure population cell samples by developing a white light source
flow cytometry system. From these experiments, we noted detectable differences in the
shape of the spectrum and scattering intensities between leukemic cells (Nalm-6) and
normal blood cells. Moreover, we identified wavelengths that best classified the
different populations, which resulted in the development of a laser based in vivo flow
cytometry system with 405, 488 and 633 nm wavelength laser sources. The performance of
the laser flow cytometry system was determined initially by flowing pure population of
each cell types. Results showed that scattering could be detected from a single flowing
cell. Moreover, results from discriminant analysis, showed that leukemic cells can be
identified from normal blood cells based on differences in their light scattering.
Building on these results, we determined the feasibility of light scattering to identify
leukemic cells in mixed population samples. For microfluidic-based applications of light
scattering, Nalm-6 cells in various concentrations from 0.001% to > 20% were mixed
with MNs and GRANs, then flowed in 30 &mum microfluidic channels. From this
experiment, we showed that detection of light scattering from a Nalm-6 cell is possible
in sample proportions as low as 0.01%, i.e. 1 leukemic cell to 10,000 MNs and GRANs.
Classification equations, based on discriminant analysis, were then developed to
automatically classify Nalm-6 cells and normal cells based on their backscattering
intensity properties. Statistical analysis showed that the sensitivity and specificity
was better if samples for light scattering measurements only contained mononuclear
cells, i.e. MNs and leukemic cells. From our statistical analysis we also estimated the
number of cancer cells that need to be detected in order to conclude that leukemic cells
were in the blood; subsequently, the volume of blood to be sampled and light scattering
measurement time were also calculated. Based on our calculations, diagnosis in regards
to the presence leukemic cells in the sample can be made (at a statistical confidence
level of 0.0011 and power of 0.80) in less than 24 hours of blood withdrawal and require
less than 400 microliters of blood sample if at least 1 out of 4,000 cells is leukemic.
For the development of an in-vivo light scattering system results from static sample
measurements were used to determine the number of normal blood cells that would result
in the inability to detect scattering from a single leukemic cell in a 25 &mum blood
vessel. We then determined the performance of the system to detect flowing Nalm-6 cells
in various hematocrit concentrations in a 30 &mum channel, our in-vitro model of a
25 &mum blood vessel. Results showed that light scattering from a single leukemic
cell could be detected from the background scattering of multiple RBCs that surrounded
it. Overall, we found that light scattering based technique can be used to discriminate
between flowing leukemic cells from normal MN, GRANs and RBCs, allowing for the
quantification of these cancer cells in blood. While our study focused on the use
leukemia as a model, overall results from these studies provide the foundations for the
development of minimally and/or non-invasive techniques for quantifying circulating
cancer cells for diagnostic and therapeutic monitoring
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Irene Georgakoudi.
Committee: Sergio Fantini, Charlotte Kuperwasser, and Charles Lin.
Keyword: Biomedical Engineering.read less