Patent No. 5,487,112

Dated: January 23, 1996

Entitled: "Method and Apparatus for Time-Resolved Measurements of Lymphocyte Function and Aggregate Structure Using Computer-Automated Microscopy"

Inventor(s): Kyriacos Zygourakis et al.

 

 

BACKGROUND OF THE INVENTION

 

A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

The invention relates to a method and apparatus for time-resolved measurements of lymphocyte function and cell aggregate structure using computer-automated micros copy.

Presently accepted methods for quantifying adhesion mechanics among cell aggregates are insensitive to the fine details of molecular pathways and structures which govern the cell aggregation process. Current methods of data presentation loose much information, and many physical characteristics of the aggregated cells which are important to the understanding of cell adhesion mechanics are never considered.

Known methods for quantifying cell aggregation often blur the distinction between aggregate shapes by use of rather simplistic, subjective scoring methods. For example, one known method for quantifying aggregate structure is through the use of a relative number scale presented below.

 

l+=<10% of cells in small aggregates (5-10 cells/clump)

2+=10-40% of cells aggregated (size varies)

3+=40-90% of cells aggregated (size varies)

4+=>90% of cells aggregated, usually in medium to large clumps

5+=approximately 100% of cells aggregated in large clusters

 

In order to eliminate the effects of personal bias, assays using this number scale are usually scored double-blind. While this has proved somewhat helpful in eliminating errors resulting from the subjectivity of the observer, the scoring method is still subject to human error and to variability between the perception of different observers. More importantly, this number scale does not reflect the potential differences in the physical dynamics or morphology that may exist between various adhesion events.

For example, using this number scoring method, adhesion induced in a human lymphoblastoid T-cell line, JURKAT, by anti-VLA-4 antibody was 5+, adhesion induced by the lectin PHA (phytohemagglutinin) was 5+, while adhesion induced by the phorbol ester PMA (phorbol 12-myristate 13-acetate) was scored a 4+. These similar number scores occur despite the fact anti-VLA-4 induced aggregates are large round and compact, with very few free non-aggregated cells, aggregates induced by PHA are long-chained structures, and those induced by PMA are smaller, more loosely structured and contain far more non-aggregated cells. Stated otherwise, application of the known number scoring method to these three aggregate structures results in very similar numerical scores despite a markedly different appearance of the aggregates.

Another limitation of the simplistic numerical scoring method is the inability to establish rates of aggregate formation. Again, this results in a substantial loss of important information. For example, two antibodies that induce lymphocyte adhesion have recently been discovered; L25 (anti VLA-4) and IC9 (ligand presently unknown). Both antibodies induce 5+ adhesion, but it has been observed that the rate of IC9-induced aggregation is considerably faster than that of L25-induced adhesion. A complete understanding of the different adhesion mechanisms induced by these two anti bodies requires assessment of the relative rates of aggregation, an assessment which is not possible using presently available methodologies.

 

 

SUMMARY OF THE INVENTION

 

The present invention avoids the above-noted drawbacks of the prior art by quantifying morphological features of cell aggregates with several new structural measurements obtained by digital image analysis. The invention involves obtaining magnified images of cell aggregates, digitizing and filtering the images, segmentation of the processed images to identify the aggregates, and analysis of the segmented images to determine size, perimeter, shape and texture of each aggregate within the images.

The new structural measurements of the present invention include aggregate size distribution, aggregate size frequency distribution, aggregate shape factor distribution, the coverage of the image area by cell aggregates, and the integral optical density of the digitized image. These new structural measurements offer information regarding the size distribution, texture, shape and optical density of the cell aggregates, and also provide an indication of the percentage of the total image area that is covered by aggregates. These new parameters provide additional information which is useful in classifying subtle differences in the character of aggregation in various cell and induction systems, and may provide new insight regarding the physical and molecular bases of each.

The apparatus of the present invention includes a television camera for producing a video image of a magnified number of cell aggregates, a digitizer for digitizing the video image, and a computer for analyzing the digitized images, and for calculating the above structural parameters.

In addition, in order to assess the time rate of cell aggregation, a number of video images can be taken at spaced apart time intervals, and the structural parameters can be calculated for each image in the sequence. This will provide cell aggregation rate information, also valuable in morphological studies.

 

 

BRIEF DESCRIPTION OF THE DRAWINGS

 

FIG. 1 is the apparatus of the present invention used to practice the method of the present invention.

FIG. 2 illustrates the image acquisition of the present invention.

FIG. 3 is a flow chart of the operation of the apparatus of FIG. 1.

FIG. 4 is a flow chart of the calculation of the morphological indices of the present invention.

FIGS. 5A-D are computer-generated images of different aggregated cell structures used to illustrate the present invention.

FIGS. 6A-D are graphs of the aggregate frequency distribution parameter of the present invention calculated for the cell aggregations of FIGS. 5A-D.

FIGS. 7A-D are graphs of the aggregate size distribution parameter of the present invention calculated for the cell aggregate structures of FIGS. 5A-D.

 

 

CLAIMS:

 

What is claimed is:

1. A method of characterizing the structure of cells and aggregates of cells in a cell suspension, including the steps of:

  • incubating a cell suspension and reagent to produce non-aggregated cells and aggregates of cells;
  • producing at least one image of said non-aggregate cells and aggregates of cells;
  • characterizing an extent of aggregation of said cell suspension according to a ratio of a total area of said at least one image covered by sells and cell aggregates to total area of said at least one image covered by non-aggregated cells before aggregation begins.

2. The method of claim 1, wherein said producing step comprises producing a time sequence of multiple images of said aggregates of cells, and wherein said characterizing step comprises characterizing a structure of said aggregates of cells according to a time rate of change of the said shape factor distribution.

3. The method of claim 1, wherein said incubating step includes creating a uniform cell distribution on a bottom of an inverted frusto-conical tapered well.

4. The method of claim 3, wherein said producing step comprises producing quadrant images of said aggregates of cells in four quadrants of said well; and combining said quadrant images to form a single image of said aggregates of cells.

5. A method of characterizing lymphocyte function in a cell suspension, including the steps of:

  • incubating a cell suspension to produce aggregates of cells;
  • producing at least one image of said aggregates of cells;
  • adding to said cell suspension monoclonal antibodies to cell surface molecules;
  • producing at least one image of said aggregates of cells after adding said monoclonal antibodies; and
  • characterizing lymphocyte function in said cell suspension according to the variations resulting from adding said monoclonal antibodies in a size distribution, s(A)dA, of said aggregates of cells, wherein s(A)dA is a fraction of total area of each said at least one image covered by aggregates of cells attributable to aggregates having an area between A and A+dA.

6. A method of characterizing lymphocyte function in a cell suspension, including the steps of:

  • incubating a cell suspension to produce aggregates of cells;
  • producing at least one image of said aggregates of cells;
  • changing at least one chemical, physical, or biological condition of said aggregates of cells;
  • producing at least one image of said aggregates of cells after said condition has been changed; and
  • characterizing lymphocyte function in said cell suspension according to the variations resulting from said changed condition in a shape factor distribution, f(T)dT, wherein f(T)dT is a fraction of a total area of said at least one image covered by aggregates attributable to aggregates having a shape factor between T and T+dT, and wherein:

    T = P2 / 4A

  • P being a perimeter of an aggregate of cells, and A being an area of said aggregate of cells.

7. The method of claim 6, wherein said changing step includes adding to said cell suspension monoclonal antibodies to cell surface molecules.

8. The method of claim 6, wherein said incubating step comprises incubating a cell suspension and a reagent, and wherein said changing step includes adding at least one additional reagent.

9. A method of characterizing the structure of cells and aggregates of cells in a cell suspension, comprising the steps of:

  • incubating a cell suspension and reagent to produce a mixture of non-aggregated cells and aggregates of cells;
  • producing a time sequence of images of said mixture of non-aggregated cells and aggregates of cells; and
  • characterizing a structure of said non-aggregated cells and aggregates of cells according to a time rate of change of a size distribution, s(A)dA, of said non-aggregated cells and aggregates of cells, wherein s(A)dA is a fraction of total area of each image of said time sequence of images covered by non-aggregated cells and aggregates of cells attributable to non-aggregated cells and aggregates of cells having an area between A and A+dA.

10. A method of characterizing the structure of cells and aggregates of cells in a cell suspension, comprising the steps of:

  • incubating a cell suspension and reagent to produce a mixture of non-aggregated cells and aggregates of cells;
  • producing quadrant images of said non-aggregated cells and aggregates of cells in four quadrants, and combining said quadrant images to form a single image of said non-aggregated cells and aggregates of cells; and
  • characterizing a structure of said non-aggregated cells and aggregates of cells according to a size distribution, s(A)dA, of said non-aggregated cells and aggregates of cells, wherein s(A)dA is a fraction of total area of said single image covered by non-aggregated cells and aggregates of cells attributable to non-aggregated cells and aggregates of cells having an area between A and A+dA.

11. A method of characterizing a structure of cells and aggregates of cells in a cell suspension, comprising the steps of:

  • incubating a cell suspension and reagent to produce a mixture of non-aggregated cells and aggregates of cells;
  • producing a time sequence of images of said non-aggregated cells and aggregates of cells; and
  • characterizing an extent of aggregation of said cell suspension according to a time rate of change of a ratio of total area of each image of said sequence of images covered by non-aggregated cells and aggregates of cells to total area of each image.

12. A method of characterizing lymphocyte function in a cell suspension, including the steps of:

  • incubating a cell suspension to produce aggregates of cells; producing at least one image of said aggregates of cells;
  • adding to said cell suspension monoclonal antibodies to cell surface molecules;
  • producing at least one image of said aggregates of cells after adding of said monoclonal antibodies; and
  • characterizing lymphocyte function in said cell suspension according to variations resulting from adding of said monoclonal antibodies in a ratio of a total area of said at least one image covered by cell aggregates to total area of said at least one image.

[Return to List of Publications]