System and method for analyzing samples labeled with 5, 10, 15, 20 tetrakis (4-carboxyphenyl) porphine (TCPP)

• US 8,877,509 B2
• Filed: 07/19/2010
• Issued: 11/04/2014
• Est. Priority Date: 07/17/2009
• Status: Active Grant

• Current Assignee: BioAffinity Technologies, Inc.
• Original Assignee: BioAffinity Technologies, Inc.
• Inventors: Dorian, Constance, Cousins, John, Bennett, Gordon
• Primary Examiner(s): Sisson, Bradley L
• Application Number:
  US12/839,283
• Publication Number:
  US 20110014647A1
• Time in Patent Office: 1,569 Days
• Field of Search: None
• US Class Current: 436/64
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 2800/12 Pulmonary diseases
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/57423 of lung
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

One embodiment of the present invention provides for a method of determining if a sputum sample contains dysplastic or carcinomic cells by obtaining a sputum sample containing cells. The sputum sample is labeled with TCPP to stain cells suspected to be dysplastic or carcinomic. The labeled sputum sample is excited with an excitation wavelength of light of about 475 nm+/−30 nm and emission at about 560 nm+/−30 nm is detected from cells identified to be macrophages. An imager focuses on the plasma membrane of one or more cells suspected to be dysplastic or carcinomic and emission at about 655 nm+/−30 nm, if present, is detected for TCPP labeled cells of the sputum sample after focusing on the plasma membrane of the cells of the sputum sample. Photon flux for each pixel of a sensor is measured to obtain a value for the imaged cell. The measured value is scored to determine if a cell is cancerous or dysplastic.

Claims

1. A method of determining if a biological sample of cells contains dysplastic or carcinomic cells the method comprising:

obtaining a biological sample suspected of containing dysplastic or carcinomic cells from human sputum from a patient suspected of having lung cancer;

labeling the biological sample with between about 0.05-1.0 μ

g/ml of Meso Tetra (4-Carboxyphenyl) Porphine (TCPP) in an aqueous alcohol of between 50% and 90% isopropanol alcohol solution at a pH of between 6.0 and 10.5 to stain cells suspected to be dysplastic or carcinomic;

exciting the sample with an excitation wavelength from a light source having a controlled emission rate for a wavelength of light limited to 475 nm+/−

30 nm;

focusing an imager on the plasma membrane of i) one or more cells suspected of containing dysplastic or carcinomic cells and ii) normal cells to capture an image of i) and ii) at a sensor having pixels capable of quantifying the photon flux at any given pixel on the sensor wherein the sensor comprises a CCD and wherein the imager comprises a fluorescent microscope;

detecting at the sensor emission limited to 655 nm+/−

30 nm if present from TCPP-labeled cells after focusing on the plasma membrane of i) one or more cells suspected of containing dysplastic or carcinomic cells and ii) normal cell one or more cells of the biological sample suspected of containing dysplastic or carcinomic cells;

measuring photon flux at each pixel of the sensor to obtain a quantitative value for the imaged cells; and

automatically scoring with an appropriately-programmed processor whereby the measured quantitative value to determine if a cell is cancerous or dysplastic based upon a preferential binding of TCPP to the plasma membrane of a cancerous or dysplastic cell as compared to the plasma membrane of a non-cancerous cell.

2. The method of claim 1 further comprising:

detecting emission from cells identified to be cells of interest at 560 nm+/−

30 nm.

3. The method of claim 1 wherein the excitation wavelength is 475 nm+/−

5 nm.

4. The method of claim 1 wherein emission of macrophages is 560 nm+/−

5 nm.

5. The method of claim 1 wherein the emission of TCPP labeled cells is 655 nm+/−

5 nm.

6. The method of claim 1 wherein the scoring further comprises comparing via the programmed processor the measured value to a database of stored values for cancerous, dysplastic and non-cancerous cells to assigning a score based upon the results of the comparison.