# US 12,768,833 B2 · Process for multi-analyses of rare cells extracted or isolated from biological samples through filtration

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- Number: US 12,768,833 B2
- Document ID: US12768833B2
- Status: Granted
- Kind: B2
- Application number: 19/010,663
- Filed: 2025-01-06
- Published: 2025-11-13
- Granted: 2026-10-20
- Earliest claimed: 2012-05-24
- Assignees: Narada SAS; Université CôTe D'Azur; Centre Hospitalier Universitaire De Nice; Université Paris Cité; Assistance Publique-Hopitaux De Paris (Ap-HP); Institut National De La Sante Et De La Recherche Medicale (Inserm)
- Applicants: Narada SAS; Université CôTe D'Azur; Centre Hospitalier Universitaire De Nice; Université Paris Cité; Assistance Publique-Hopitaux De Paris (Ap-HP); Institut National De La Sante Et De La Recherche Medicale (Inserm)
- Inventors: Patrizia Paterlini-Brechot; Paul Hofman; Sophie Marie Genevieve Laget; Thierry Capiod
- Web page: https://patentgrep.com/patent/US12768833B2

## CPC

- G01N 33/5091: {for testing the pathological state of an organism}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G01N 33/554: the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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

A process for isolating or extracting rare cells from a biological sample including filtering a biological sample, which may be treated or diluted, through a filter that has a pore size, pore density or other physical properties that retain rare cells, but which permits other kinds of cells to pass through the filter.

## Claims

### Claim 1

A process for analyzing DNA molecules in rare cells isolated from blood obtained from a subject, the method comprising:
a) treating a diluted blood sample with an anticoagulant, selectively only lysing red blood cells in the diluted blood sample that is treated with the anticoagulant, and producing a fixed blood sample by fixing the diluted blood sample that is treated with the anticoagulant and has lysed red blood cells;
b) filtering the fixed diluted blood sample from step a) by vertical filtration with a negative pressure through a polycarbonate filter, wherein the filter has a pore size from 3 μm to 30 μm, has a pore density from 3×103 pores/cm2 to 5×106 pores/cm2, and is suitable for retention of the rare cells but permits passage of cells smaller than the rare cells, wherein the rare cells are nucleated cells larger than neutrophils and mature lymphocytes, and wherein the filter comprises elementary filtration areas, each of the elementary filtration areas having a surface which equals to the area of a disk with a diameter between 0.6 cm and 3 cm, wherein the surface of each of the elementary filtration areas and the number of elementary filtration areas of the filter are chosen according to the nature of a fluid to be filtered, the nature of biological particles to be separated when the filter is used, and the volume of the fluid to be filtered, and wherein the ratio of a total volume of fluid filtered through the surface of each of the elementary filtration areas of the filter to the area of each of the elementary filtration areas of the filter is less than 40 ml/cm2;
c) recovering the rare cells on the filter or the rare cells detached from the filter cells; and
e) analyzing the DNA molecules from the recovered rare cells, thereby analyzing the DNA molecules in the rare cells isolated from the blood obtained from the subject.

### Claim 2 (depends on 1)

The process of claim 1, wherein the subject has a cancer or a tumor; or is suspected of having a cancer or a tumor; or is at risk of having a cancer or a tumor.

### Claim 3 (depends on 2)

The process of claim 2, wherein the recovered rare cells comprise circulating tumor cells (CTCs).

### Claim 4 (depends on 3)

The process of claim 3, wherein DNA molecules from the CTCs are different from DNA molecules from leukocytes.

### Claim 5 (depends on 4)

The process of claim 4, wherein the DNA molecules analyzed in step e) comprise mutated DNA molecules and the analyzing step comprises identifying the mutated DNA molecules.

### Claim 6 (depends on 1)

The process of claim 1, wherein, in step e), said analyzing DNA molecules from the recovered rare cells comprise one or more of PCR analyses, high throughput sequencing, and methylation analyses.

### Claim 7 (depends on 1)

The process of claim 1, wherein the DNA molecules analyzed in step e) comprise mutated DNA molecules and the analyzing step comprises identifying the mutated DNA molecules.

### Claim 8 (depends on 1)

The process of claim 1, wherein said analyzing the DNA molecules from the recovered rare cells comprises detecting whether DNA mutations are present in the recovered rare cells.

### Claim 9 (depends on 1)

The process of claim 1, wherein the blood sample is, further treated with a proteolytic agent prior to the filtering step.

### Claim 10 (depends on 1)

The process of claim 1, wherein the pore size from 3 μm to 30 μm is a pore size from 5 μm to 25 μm.