System for microvolume laser scanning cytometry

• US 7,336,812 B2
• Filed: 02/03/2004
• Issued: 02/26/2008
• Est. Priority Date: 07/21/1999
• Status: Expired due to Fees

• Current Assignee: PPD Biomarker Discovery Sciences LLC (CellCarta Biosciences, Inc.)
• Original Assignee: PPD Biomarker Discovery Sciences LLC (CellCarta Biosciences, Inc.)
• Inventors: Walton, Ian, Dietz, Louis J, Chung, Chih-Hua, Winkler, James L, Kantor, Aaron B, Tsur, Shalom, Lee, Byron, Norton, Scott
• Primary Examiner(s): Bella; Matthew C.
• Assistant Examiner(s): TUCKER, WESLEY J
• Application Number:
  US10/771,585
• Publication Number:
  US 20040161143A1
• Time in Patent Office: 1,484 Days
• Field of Search: 382/133, 382/134, 382/165, 382190-194, 382270-273
• US Class Current: 382/134
• CPC Class Codes: G01N 15/1433 using image recognition

Abstract

The present invention provides an improved integrated system for biological marker identification. The system uses Microvolume Laser Scanning Microscopy (MLSC) in order to measure patterns of expression of biological markers in biological fluids. The system includes improved instrumentation for performing MLSC, and also includes improved particle detection and analysis methods. The system further comprises an informatics architecture for the analysis of data obtained from the MLSC in tandem with other medical information.

Claims

1. A method for analyzing a sample containing particles to detect and characterize target particles having a plurality of detectable characteristics in a fixed volume capillary that contains a fluorescent background and which exhibits background characteristics, the method comprising:

(a) scanning the fixed volume capillary containing the sample to generate a plurality of channels of data, wherein each channel of data comprises a distinct detectable characteristic and a distinct background characteristic;

(b) sampling each of the channels of data to produce corresponding sets of pixel values;

(c) generating sets of enhanced pixel values by independently modifying each set of pixel values to selectively enhance spatial features that are indicative of a target particle;

(d) removing from one or more sets of enhanced pixel values the distinct background characteristic for the corresponding channel;

(e) independently establishing threshold values for the detection of said particles for each set of enhanced pixel values;

(f) independently identifying, in each set of enhanced pixel values, groups of above-threshold pixels located in patterns that are diagnostic of said particles;

(g) independently identifying, for each group of above-threshold pixels located in a diagnostic pattern in a particular set of enhanced pixel values, the corresponding below-threshold or at-threshold pixels in the remaining sets of enhanced pixels in the remaining spectral channels;

(h) characterizing the target particles in the sample by analyzing the pixels independently identified in steps (f) and (g); and

(i) calculating at least one of;

(I) a volume of the sample scanned; and

(II) an absolute particle count;

wherein particles are initially identified and analyzed in channels with above-threshold pixels located in patterns diagnostic of said particles, and said particles are then independently analyzed in all remaining channels by locating pixels in the same positions as the above-threshold pixels initially identified.

2. A method for analyzing a sample containing particles to detect and characterize target particles having a plurality of detectable characteristics in a fixed volume capillary that contains a fluorescent background and which exhibits background characteristics, the method comprising:

(a) scanning the fixed volume capillary containing the sample to generate a plurality of channels of data, wherein each channel of data comprises a distinct detectable characteristic and a distinct background characteristic;

(b) sampling each of the channels of data to produce corresponding sets of source pixel values;

(c) suimning the sets of source pixel values to generate a composite image;

(d) calculating a threshold for particle detection in said composite image independently in each set of composite pixel values;

(e) performing particle detection in said composite image using said threshold independently in each set of composite pixel values using the corresponding threshold;

(f) identifying, for each particle identified in a particular set of composite pixel values in step (e), the corresponding pixels in the remaining sets of source pixel values;

(g) analyzing the pixels identified in step (f); and

(h) calculating at least one of;

(I) a volume of the sample scanned; and

(II) an absolute particle count.

3. A method for analyzing a sample containing particles to detect target particles having a plurality of detectable characteristics in a fixed volume capillary that contains a fluorescent background and which exhibits backgronnd characteristics, the method comprising:

(a) scanning the fixed volume capillary containing the sample to generate a plurality of channels of data, wherein each channel of data comprises a distinct detectable characteristic and a distinct background characteristic;

(b) sampling each of the channels of data to produce corresponding sets of source pixel values;

(c) calculating a threshold for particle detection independently in each set of source pixel values without first summing the source images;

(d) performing particle detection independently in each set of source pixel values using the corresponding threshold; and

(e) calculating at least one of;

(I) a volume of the sample scanned; and

(II) an absolute particle count.