# US 10,870,891 B2 · Diagnostic test system for specific, sensitive and reproducible detection of circulating nucleic acids in whole blood

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- Number: US 10,870,891 B2
- Document ID: US10870891B2
- Status: Granted
- Kind: B2
- Application number: 15/862,896
- Filed: 2018-01-05
- Published: 2018-07-19
- Granted: 2020-12-22
- Earliest claimed: 2017-01-05
- Assignees: Biodesix, Inc.
- Applicants: Biodesix, Inc.
- Inventors: Hestia Mellert; Leisa Jackson; Gary A. Pestano
- Web page: https://patentgrep.com/patent/US10870891B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6851: Quantitative amplification
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present disclosure relates to a rapid diagnostic test system that includes the prospective collection of whole blood, preservation of circulating nucleic acids at ambient temperature, and the reproducible detection of nucleic acids including DNA and mRNA (including fusion transcripts and differentially expressed transcripts) by different genomic methodologies.

## Claims

### Claim 1

A method of detecting a fragmented RNA in a freshly-collected whole blood sample from a mammalian subject comprising:
a) recovering blood components from a whole blood sample from a mammalian subject, said blood components including plasma and buffy coat from the whole blood sample;
b) isolating free RNA, RNA associated with, exosomes and/or platelets in platelet enriched plasma (PEP), RNA associated with PBMCs, or a combination thereof from said blood components;
c) purifying and concentrating the isolated RNA of step (b);
d) amplifying said RNA of step (c) or a complimentary DNA generated from said RNA of step (c) wherein generating complimentary DNA comprises performing reverse transcription of said isolated RNA using SuperScript IV reverse transcriptase;
e) purifying and concentrating the amplified RNA; and
f) identifying and/or quantifying amplified said RNA or complimentary DNA from step (e).

### Claim 2 (depends on 1)

The method of claim 1, wherein the RNA is of sizes ranging between 50 and 250 nucleotides.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (b) comprises isolating said RNA from a whole blood sample that has been collected from mammalian donors into formaldehyde or formaldehyde-free preservatives and/or inhibitors of enzymes known to degrade RNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein steps (a)-(f) are performed in less than three days.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian subject is a human.

### Claim 6 (depends on 1)

The method of claim 1, wherein step (b) comprises isolating free RNA and/or RNA associated with exosomes and/or platelets in said PEP.

### Claim 7 (depends on 1)

The method of claim 1, further comprising generating an integrated test result report (iTRR) integrating physician information, patient information, reimbursement information, and/or treatment recommendation or impact, with the results from step (e), such as a serum protein test result.

### Claim 8 (depends on 1)

The method of claim 1, further comprising one or more control reactions.

### Claim 9 (depends on 1)

The method of claim 1, further comprising quantifying said RNA or complimentary DNA from step (d) using dPCR, qPCR, or NGS.

### Claim 10 (depends on 1)

The method of claim 1, wherein 2 or more copies of variant target are detected by dPCR and are validated against mammalian donor samples as the cut-off for a positive DNA or RNA-fusion mutation.

### Claim 11 (depends on 1)

The method of claim 1, wherein elevated levels of differentially-expressed target are detected by dPCR when validated compared to normal healthy mammalian donor samples as the cut-off for a positive result.

### Claim 12 (depends on 1)

The method of claim 1, where the RNA is obtained from components of the buffy-coat fraction of processed whole-blood from cancer patients, including but not limited to malignant tumor cells, lymphocytes, granulocytes, neutrophils, dendritic cells.

### Claim 13 (depends on 1)

The method of claim 1, further comprising obtaining said whole blood sample from the subject.

### Claim 14 (depends on 13)

The method of claim 13, wherein said whole blood sample is obtained using a specimen collection kit configured for collection and ambient temperature shipment of fractionated or whole blood, wherein the collection kit is configured to allow downstream molecular proteomic and/or genomic analysis of the blood components.

### Claim 15

A method of detecting a cytokeratin nucleic acid in a sample comprising:
a) obtaining a cytokeratin RNA containing sample;
b) annealing forward and reverse primers that hybridize to said cytokeratin nucleic acid with said sample;
c) amplifying said cytokeratin RNA; and
d) detecting amplified cytokeratin RNA.