Method and Apparatus for Time-Resolved Measurements
of Lymphocyte and Aggregate Structure Using
Computer-Automated Microscopy
Kyriacos Zygourakis 1
John L. Bednarczyk 2
Bradley W. McIntyre 2
Michael W. Glacken 1,3
1 Department of Chemical Engineering, Rice University, Houston,
Texas
2 Department of Immunology, M.D. Anderson Cancer Center, University
of Texas, Houston, Texas
3 Currently with Bristol Myers
ABSTRACT
The invention is a method and apparatus for direct testing and quantification
of the physiological function of cells of the immune system. This assay
can become a clinical tool to identify and differentiate between normal
and diseased immune cells from the blood of patients with cancer, AIDS
or other diseases of the immune system.
SIGNIFICANCE
All current assays used for clinical diagnosis simply test for the presence
or amount of easily measurable physical or chemical parameters. For instance,
blood from patients who have recently had tumor surgery may be tested
for the presence of tumor-associated proteins to determine whether or
not the surgery was successful. Or, suspected leukocyte adhesion deficiency
patients (LAD, an immunodeficiency disease) may have their lymphocytes
(cells of the immune system) tested for the absence of the protein LFA-1,
a critical adhesion molecule. The point to be made from these examples
is that present diagnostic assays test for specific molecular properties,
but not for physiological function. Function can usually only be inferred
indirectly, if at all, from a battery of tests and the analysis of an
experienced expert.
Our group is developing a diagnostic tool that will be able to directly
assay the functional properties of cell populations taken from patients.
Presently, we are using this assay to investigate a critical function
of lymphocytes: cell-cell adhesion. Lymphocytes are the primary line of
defense against tumors and viruses, and the adhesion of lymphocytes to
tumors and virally infected cells is required before any killing action
by the lymphocyte against the target cell can occur. Our goal is to develop
a clinical assay that will delineate between cell populations based on
molecular content, morphology, adhesiveness, cell surface phenotype, and
the extent and the ability to which various adhesion events may be stimulated.
We envision the implementation of a clinical study in the near future
in which cancerous cells and/or cells from a patient with multiple sclerosis
or other autoimmune diseases may be distinguished from normal cells based
on the values of the functional and structural indices measured using
this assay. Our goal is that this assay becomes a standard clinical tool
for evaluating patient health, formulating treatment strategies and analyzing
their effectiveness.
BRIEF DESCRIPTION OF ASSAY AND SOME CURRENT RESULTS
The assay, as currently developed involves:
- inducing aggregation of lymphocytes using monoclonal antibodies (or
other protocols) in a custom-built incubator placed on the motorized
stage of a microscope equipped with a video camera;
- using a digital time-lapse technique to acquire and store on disk
a sequence of high-resolution digital images showing the aggregation
of cells at specified times; and
- processing the sequence of digitized images with in-house developed
software to obtain morphological indices that quantitatively characterize
the kinetics of the aggregation event.
We have so far identified several indices that describe the morphological
structure of lymphocytes induced to aggregate by chemical or biological
means. The most useful indices are (a) the moments of the size distribution
of the aggregates; (b) the average aggregate size at specified time points;
and (c) the fractional decrease of the viewing area covered with aggregates.
The last index accurately characterizes the three-dimensional structure
of the formed aggregates. Another useful morphological index is the shape
factor of the cell aggregates.
By following the temporal evolution of these morphological indices, we
have shown that the assay can accurately quantify the rates of aggregation
events and allow comparisons of mechanical binding strengths and cytoskeletal
activity (Munn et. al., 1993; Neelamegham
and Zygourakis, 1996). Our results also show that the assay has small
repeatability errors and is sensitive enough to compare aggregation events
induced through distinct molecular epitopes (Munn
et. al., 1993; Neelamegham and
Zygourakis, 1996). Used in conjunction with current biochemical detection
assays and adhesion pathway modulation experiments, the developed assay
will facilitate the study of cellular adhesion and aggregation mechanisms.
Using the temporal evolution patterns of the morphological indices, we
have also shown that spontaneous adhesion of a lymphocyte cell line was
morphologically similar to chemically induced adhesion and different from
the adhesion induced by antibodies to adhesion molecules. This is intriguing,
since we know that both the spontaneous chemically induced adhesion are
mediated by the same adhesion molecule, LFA-1, while antibody-induced
aggregation is not. Clearly, this is an indication that this assay has
the ability to identify and distinguish between various adhesion mechanisms
and functions.
OTHER APPLICATIONS OF THE IMAGE ANALYSIS ASSAY
The developed assay is not limited to the study of homotypic lymphocyte
aggregation. Using a variation of the approach outlined above, we were
successful in quantifying the kinetics of lymphocyte activation and proliferation
(Teague et. al, 1993). Our approach
has several advantages over the other currently used assays (like MTT
or 3H-thymidine incorporation): it is non-invasive, allows for continuous
monitoring of cells, does not require sample harvesting, does not produce
radioactive waste, allows us to observe single cells or detect subpopulations,
and scores directly cell activation, cell division and DNA synthesis.
The use of this assay could aid the clinician in making diagnostic and
prognostic assessments of leukemia patients by providing for quick detection
and enumeration of blasting and proliferating lymphocytes.
REFERENCES
- L.L. Munn, M.W. Glacken, B.W. McIntyre and K. Zygourakis, "Analysis
of Lymphocyte Aggregation Using Digital Image Analysis, " Journal
of Immunological Methods, 166, 11-25 (1993).
[ABSTRACT]
- T. K. Teague, L.L. Munn, K. Zygourakis and B.W. McIntyre, "Analysis
of Lymphocyte Activation and Proliferation by Video Microscopy and Digital
Imaging," Cytometry, 14, 772-782 (1993).
[ABSTRACT]
- S. Neelamegham and K, Zygourakis, "A Quantitative Assay for Intercellelular
Aggregation," Annals Biomed. Engrg., 25, in press (1996).
[ABSTRACT]
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