High throughput DNA damage quantification of human tissue with home-based collection device

9,931,634

14/634266

February 27, 2015

Kits, methods and systems for providing a service to provide a subject with information regarding the state of a subject's DNA damage. Collection, processing and analysis of samples are also described.

The Regents of The University of California (Oakland, CA, US); Exogen Biotechnology, Inc. (Berkeley, CA, US)

The Regents of the Univeristy of California (Oakland, CA, US), Exogen Biotechnology, Inc. (Berkeley, CA, US)

1. A method for providing a service to provide a subject information regarding the state of a subject's DNA damage, said method comprising: a) receiving from a subject one or more sample tube(s) containing the subject's blood fixed in said tubes, wherein said collection tubes are from a home collection kit, where said kit comprises a package containing: a sterile lancet; a capillary blood collection device coated with an anti-coagulant to avoid coagulation of blood drawn into said capillary; and said one or more sample tubes, where each of said tubes contains a fixative, an anti-coagulant, and a buffer that provide fixation conditions such that blood loaded into said tubes can be shipped and/or stored without substantial changes in DNA break markers; b) isolating T, B and/or NK cells from said blood sample by i. conducting hypotonic lysis of erythrocytes (red blood cells) in fixed blood samples; ii. capturing of T, B, and/or NK cells from said sample using CD-specific antibodies; iii. coupling the antibody captured fixed T, B and/or NK cells to magnetic beads or nanoparticles; iv. exposing the antibody captured fixed T, B and/or NK cells coupled to magnetic beads or nanoparticles tubes to a strong magnetic field to immobilize the cells; and v. washing away unwanted cells and serum components leaving target cells of interest; c) affixing the isolated cells to the surface by an adhesive; d) labeling affixed cells with primary antibodies recognizing DNA damage markers and with secondary antibodies that are covalently conjugated to fluorescent chromophores to provide labeled cells; and e) imaging said labeled cells and scoring for DNA breaks in the cells.

2. A method for providing a service to provide a subject with information regarding the state of a subject's DNA damage comprising: a) receiving from a subject one or more sample tube(s) containing the subject's blood fixed in said tubes, wherein said collection tubes are from a home collection kit, where said kit comprises a package containing: a sterile lancet; a capillary blood collection device coated with an anti-coagulant to avoid coagulation of blood drawn into said capillary; and said one or more sample tubes, where each of said tubes contains a fixative, an anti-coagulant, and a buffer that provide fixation conditions such that blood loaded into said tubes can be shipped and/or stored without substantial changes in DNA break markers; b) isolating and/or identifying specific cell types for DNA damage scoring of cell type-specific DNA damage measurements from said blood sample by: i. conducting a cell type in fixed blood samples; ii. capturing of said cells from said sample using cell type-specific antibodies; iii. coupling the antibody captured cells to magnetic beads or nanoparticles; iv. exposing the antibody captured fixed cells coupled to magnetic beads or nanoparticles tubes to a strong magnetic field to immobilize the cells; and v. washing away unwanted cells and serum components leaving target cells of interest; c) affixing the isolated cells to the surface by an adhesive; d) labeling affixed cells with primary antibodies recognizing DNA damage markers and with secondary antibodies that are covalently conjugated to fluorescent chromophores; and e) imaging said labeled cells and score for DNA breaks in the cells.

3. The method according to any one of claim 1 or 2 , wherein said capillary blood collection device is configured to collect about 10 μl to about 200 μl, or about 10 μl to about 100 μl of blood.

4. The method according to any one of claim 1 or 2 , wherein said anticoagulant in said capillary blood collection device and in said sample tubes comprises EDTA or heparin.

5. The method according to any one of claim 1 or 2 , wherein said fixative is selected from the group consisting of paraformaldehyde, methanol, ethanol, acetone, and urea.

6. The method according to any one of claim 1 or 2 , wherein said fixative comprises paraformaldehyde.

7. The method of claim 6 , wherein said fixative comprises about 0.1 percent up to about 10% paraformaldehyde.

8. The method of claim 7 , wherein said anticoagulant in said sample tubes comprises EDTA and wherein said fixative, when mixed with blood leads to a mix of fixative and blood with a final concentration of 1% paraformaldehyde and 25 mM EDTA.

9. The method of claim 7 , wherein the sample tubes contain about 2% paraformaldehyde and 50 mM EDTA in PBS, and the volume of fixative provided in the sample tube(s) is such that fixative and whole blood are mixed in a 1:1 ratio when the capillary collection device is dispensed into the sample-tube(s).

10. The method of claim 8 , wherein: said anticoagulant in said sample tubes comprises about 10 mM up to about 100 mM EDTA; and said buffer in said sample tubes comprises phosphate buffered saline at about pH 7 to about pH 8.

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