Optical specimen analysis system and method

• US 6,249,593 B1
• Filed: 12/22/1997
• Issued: 06/19/2001
• Est. Priority Date: 02/26/1993
• Status: Expired due to Term

• Current Assignee: E-Y Laboratories, Inc.
• Original Assignee: E-Y Laboratories, Inc.
• Inventors: Tao, Lian, Chu, Albert E.
• Primary Examiner(s): Couso, Jose L.
• Application Number:
  US08/995,590
• Time in Patent Office: 1,275 Days
• Field of Search: 382/100, 382/110, 382/128, 382/129, 382/133, 382/134, 382/162, 382/164, 382/165, 382/173, 382/175, 382/180, 382/181, 382/190, 382/224, 382/274, 382/287, 382/300, 382/321, 356/39, 356/335, 356/246, 435/6, 435/7.23, 436/43, 436/164, 436/513, 436/527, 436/530, 436/532, 436/534, 436/536, 436/538, 436/544, 436/547, 436/805, 436/807, 702/20, 702/21, 702/105, 702/170, 422/67, 422/73
• US Class Current: 382/128
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2021/177 Detector of the video camera type
• CPC Class Codes: G01N 2021/5919 Determining total density of a zone
• CPC Class Codes: G01N 2021/5923 Determining zones of density; quantitating spots
• CPC Class Codes: G01N 2021/593 Correcting from the background density
• CPC Class Codes: G01N 2021/5934 Averaging on a zone
• CPC Class Codes: G01N 2021/8488 the band presenting reference patches
• CPC Class Codes: G01N 21/8483 Investigating reagent band test-element handling not specific to a test method G01N33/4875; analytical elements specific to chemical analysis of biological material G01N33/52; autometer with reagent band G01N35/04
• CPC Class Codes: G06T 2207/10024 Color image
• CPC Class Codes: G06T 2207/30004 Biomedical image processing
• CPC Class Codes: G06T 7/90 Determination of colour characteristics

Abstract

A method and system for automatic analysis of a testing substrate for an analyte in a liquid sample. The sample is applied to a testing substrate having an immobilized thereon, in a limited region of the testing substrate, a receptor capable of binding to the analyte. A labeled reagent, capable of binding to the analyte, is added to the testing substrate. If the analyte is present, a color is generated at the area of the testing substrate where the receptor is immobilized. The testing substrate is then illuminated and, using electronic equipment a digital image of the testing substrate is acquired and automatically scanned to locate an area of the testing substrate having the highest color density and generating a first measurement of color density. Next, an area peripheral to the area of highest color density is located and a second measurement of color density is generated. This peripheral area represents tho background density of the substrate, which can be considered to be the background “noise”. The presence or absence of the analyte in the liquid sample is calculated by adjusting the first measurement with said second measurement in accordance with a predefined mathematical function.

Claims

1. A method for determining the presence of at least one biological analyte in a liquid sample suspected of containing said one analyte comprising:

a) applying said liquid sample to a testing substrate having at least one receptor immobilized thereon in at least one receptor area of said testing substrate, said one receptor being capable of directly or indirectly binding to said analyte, said one receptor area being located in a limited region of said testing substrate;

b) applying a reagent capable of binding directly or indirectly to said one analyte and capable of generating a color at said one receptor area when said one analyte is present;

c) illuminating said testing substrate and acquiring a digital image of said testing substrate;

d) using data processing equipment,

(i) automatically scanning said digital image to locate an optically densest portion of said one receptor area and generating a first measurement of the density of said densest portion;

(ii) locating an area peripheral to said one receptor area and generating a second measurement of density of said area peripheral to said one receptor area; and

(iii) generating an output signal by adjusting said first measurement with said second measurement in accordance with a predefined mathematical function in order to generate an output signal that indicates whether said one analyte is present in said liquid sample.

2. The method of claim 1 wherein said method is also used for the determination of at least a second analyte, said testing substrate having immobilized thereon in a second receptor area wherein said second receptor area is capable of binding directly or indirectly to a second analyte, said step (d) including locating and measuring the density of first and second receptor areas, and generating an output signal for each receptor area.

3. The method of claim 1 wherein the adjusted measurement of step (d)(iii) corresponds to the concentration of analyte present in said sample.

4. The method of claim 1 wherein said testing substrate is a porous membrane.

5. The method of claim 1, wherein said substrate is opaque and includes a marker having a predefined spatial relationship to said receptor area;

said step (d)(i) including locating said marker in said digital image and then locating said receptor area in said digital image based on said markers location.

6. The method of claim 1, further including:

prior to step (d), measuring optical density of a reference substrate and calibrating all subsequent measurements of testing substrates in accordance with said reference substrate'"'"'s measured optical density.

7. The method of claim 1, wherein said digital image comprises an N×

M array of pixels;

said step (d)(i) including;

for each of a selected set of pixel positions spaced apart from each other, measuring the optical density of a region of said digital image associated with said each pixel position; and

selecting a plurality of said measured regions with highest density, interpolating the pixel positions associated with said selected measured regions to generate a final pixel position, and then measuring the optical density of a region of said digital image associated with said final pixel position to generate said first measurement.

8. A method for determining the presence of an analyte in a liquid sample suspected of containing said analyte comprising:

a) applying said liquid sample to a testing substrate having a receptor immobilized thereon at a receptor area of said testing substrate, said receptor capable of directly or indirectly binding to said analyte, said receptor area being located in a limited region of said testing substrate;

b) applying a reagent capable of binding directly or indirectly to said analyte and capable of generating a color at said receptor area when said analyte is present;

c) illuminating said testing substrate and acquiring a digital image of said testing substrate;

d) using data processing equipment;

(i) automatically scanning said digital image to locate a portion of said receptor area having greatest density of a predefined color, and generating a first measurement of the density of said predefined color at said located portion;

(ii) locating an area peripheral to said receptor area and generating a second measurement of the density of said predefined color at said area peripheral to said receptor area; and

(iii) generating an output signal by adjusting said first measurement with said second measurement in accordance with a predefined mathematical function in order to generate an output signal that indicates whether said analyte is present in said liquid sample.

9. The method of claim 8 wherein said method is for the determination of more than one analyte, said testing substrate having immobilized thereon more than one receptor area wherein each receptor area is capable of binding directly or indirectly to a specific analyte, said step (d) including locating and measuring the density of said predefined color at each receptor area, and generating an output signal for each receptor area.

10. The method of claim 8 wherein the adjusted measurement of step (d)(iii) corresponds to the concentration of analyte present in said sample.

11. The method of claim 8 wherein said testing substrate is a porous membrane.

12. A system for analyzing an analyte derived from a biological specimen, wherein said analyte is bound to a receptor area of said substrate wherein a portion of said substrate is not covered by said receptor area, comprising:

means for acquiring a digital image of said substrate; and

data processing means, coupled to said image acquiring means, for automatically scanning said digital image to locate an optically densest portion digital image depicting said analyte and to locate a background portion of said digital image not depicting said analyte;

said data processing means including means for (A) generating a first measurement of the density of said densest portion, (B) generating a second measurement of the density of said background portion of said substrate, and (C) generating an output signal by adjusting said first measurement with said second measurement in accordance with a predefined mathematical function.

13. The system, as defined in claim 12, further including:

means of illuminating said substrate; and

means for sensing the intensity of said means for illumination;

said data processing means including means, coupled to said intensity sensing means, for calibrating said measurements in accordance with said sensed intensity of said means for illumination.

14. The system, as defined in claim 12, wherein:

a multiplicity of distinct analytes are bound to a multiplicity of distinct receptor areas of said substrate;

said data processing means including means for locating an optically densest portion of each said distinct analyte, and for generating a first measurement of the density of said densest portions of each said distinct analyte.

15. The system, as defined in claim 12, wherein said substrate includes a marker having a predefined spatial relationship to said analyte;

and

said data processing means includes means for locating said marker in said digital image and for locating said analyte within said digital image based on said marker'"'"'s location.

16. The system, as defined in claim 12, further including:

a template interposed between said substrate and said means for acquiring a digital image, said template forming a superimposed image on said digitized image of said substrate;

a plurality of guide rails sized to receive said substrate so that said image of said template falls at predefined position with respect to said substrate, thereby indicating where said analyte is deposited on said substrate.