# US 2019/0376033 A1 · PREPARATION OF FETAL NUCLEATED RED BLOOD CELLS (NRBCs) FOR DIAGNOSTIC TESTING

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0376033 A1
- Document ID: US20190376033A1
- Status: Published application
- Kind: A1
- Application number: 16/237,179
- Filed: 2018-12-31
- Published: 2019-12-12
- Earliest claimed: 2014-05-15
- Assignees: Kellbenx Incorporated
- Applicants: Kellbenx Incorporated
- Inventors: Hassan BENNANI; Leonard H. KELLNER
- Web page: https://patentgrep.com/patent/US20190376033A1

## CPC

- G01N 33/80: involving blood groups or blood types {or red blood cells (white blood cells G01N33/56972)}
- C07K 16/2881: {against CD71}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G01N 33/54326: {Magnetic particles}
- G01N 33/56966: {Animal cells}
- G01N 33/582: {with fluorescent label}
- G01N 33/6878: {in epitope analysis}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- C12N 5/0641: {Erythrocytes}
- C12Q 2531/113: PCR
- C12Q 2565/501: being an array of oligonucleotides
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- G01N 2800/385: Congenital anomalies
- G01N 2800/387: Down syndrome; Trisomy 18; Trisomy 13
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The disclosure relates to methods of preparation of fetal nucleated red blood cells (NRBCs) from biological samples for diagnostic testing.

## Claims

### Claim 1

A method of enriching for fetal nucleated red blood cells (fNRBCs) from a biological sample, comprising:
(a) subjecting the biological sample to density separation to obtain a fNRBC-containing cell fraction;
(b) subjecting the fNRBC-containing cell fraction obtained in step (a) to magnetic activated cell sorting (MACS) using at least one fNRBC positive selection reagent to obtain a MACS-sorted cell population;
(c) fluorescently labeling cells in the MACS-sorted cell population obtained in step (b) with at least one fNRBC positive selection reagent to obtain a fluorescently labeled cell population; and
(d) sorting the fluorescently labeled cell population obtained in step (c) by flow cytometry to select for fNRBCs, thereby obtaining a population of cells enriched for fNRBCs.

### Claim at position 2

2-3. (canceled)

### Claim 4 (depends on 1)

The method of claim 1, wherein at least one fNRBC positive selection reagent of step (b) comprises monoclonal antibody 4B9, an anti-CD235a antibody, or a nuclear stain.

### Claim at position 4

5-9. (canceled)

### Claim 10 (depends on 1)

The method of claim 1, which does not comprise a negative selection step.

### Claim 11 (depends on 1)

The method of claim 1, wherein the fNRBC-containing cell fraction obtained in step (a) is subject to negative selection prior to step (b).

### Claim 12 (depends on 1)

The method of claim 1, wherein the MACS-sorted cell population obtained in step (b) is subject to negative selection prior to step (c).

### Claim 13 (depends on 11)

The method of claim 11, wherein the negative selection is negative immunoselection.

### Claim 14

(canceled)

### Claim 15

A method of enriching for fNRBCs from a biological sample, comprising:
(a) subjecting the biological sample to density separation to obtain a fNRBC-containing cell fraction;
(b) subjecting the fNRBC-containing cell fraction obtained in step (a) to MACS using at least two fNRBC positive selection reagents to obtain a MACS-sorted cell population;
(c) performing micromanipulation on the MACS-sorted cell population obtained in step (b) to isolate individual fNRBCs or groups of fNRBCs.

### Claim 16 (depends on 15)

The method of claim 15, wherein at least one fNRBC positive selection reagent of step (b) comprises monoclonal antibody 4B9 or an anti-CD235a antibody.

### Claim 17

(canceled)

### Claim 18 (depends on 15)

The method of claim 15, which does not comprise a negative selection step.

### Claim 19 (depends on 15)

The method of claim 15, wherein the fNRBC-containing cell fraction obtained in step (a) is subject to negative selection prior to step (b).

### Claim 20 (depends on 15)

The method of claim 15, wherein the MACS-sorted cell population obtained in step (b) is subject to negative selection prior to step (c).

### Claim 21 (depends on 19)

The method of claim 19, wherein the negative selection is negative immunoselection.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

The method of claim 1, wherein the biological sample is maternal blood.

### Claim at position 19

24-25. (canceled)

### Claim 26 (depends on 1)

The method of claim 1, which further comprises validating the identity of at least one fNRBC as a fetal cell.

### Claim 27 (depends on 1)

A cell population enriched in fNRBCs obtained or obtainable by the method of claim 1.

### Claim 28

A FACS-sorted cell population containing (a) at least 2, at least 5 or at least 10 and/or (b) up to 15, up to 25, or up to 35 fNRBCs enriched from maternal blood.

### Claim at position 23

29-30. (canceled)

### Claim 31 (depends on 27)

A method of detecting a fetal abnormality, comprising analyzing at least one fNRBC from the cell population of claim 27 for a fetal abnormality.

### Claim 32

(canceled)

### Claim 33 (depends on 31)

The method of claim 31, which comprises analyzing a single fNRBC for the fetal abnormality.

### Claim 34 (depends on 31)

The method of claim 31, which comprises analyzing a group of fNRBCs for the fetal abnormality.

### Claim at position 28

35-38. (canceled)

### Claim 39 (depends on 31)

The method of claim 31, which further comprises validating the fNRBC or fNRBCs as fetal cells.

### Claim at position 30

40-42. (canceled)