# US 12,006,546 B2 · Method for epigenetic immune cell detection and counting in human blood samples

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- Number: US 12,006,546 B2
- Document ID: US12006546B2
- Status: Granted
- Kind: B2
- Application number: 17/256,390
- Filed: 2019-07-03
- Published: 2022-03-24
- Granted: 2024-06-11
- Earliest claimed: 2018-07-05
- Assignees: Precision for Medicine GmbH
- Applicants: Precision for Medicine GmbH
- Inventors: Sven Olek
- Web page: https://patentgrep.com/patent/US12006546B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/154: Methylation markers
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- 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 invention relates to improved methods for epigenetic blood and immune cell detection and counting, and respective uses and kits.

## Claims

### Claim 1

A method for producing and detecting a panel of amplicons, the method comprising the steps of
a) bisulfite treating isolated genomic DNA from a human blood cell sample containing immune cells to convert unmethylated cytosines into uracil;
b) amplifying the bisulfite treated DNA to produce the panel of amplicons;
wherein said amplifying comprises amplification and/or qPCR using:
a thymidine-cytosine (TpG) template carrying targets of converted genomic regions of CD3gamma/delta, CD4, CD8beta, lipoprotein receptor-related protein 5 (LRP5), mevalonate pyrophosphate decarboxylase (MVD), and lipocalin-2 (LCN2), and
primer pairs and probes for the converted genomic regions of CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2, the primer pairs and probes selected from SEQ ID NOs: 3 to 12 for CD4, SEQ ID NOs: 13 to 20 for CD8beta, SEQ ID NOs: 21 to 28 for LRP5, SEQ ID NOs: 29 to 36 for MVD, SEQ ID NOs: 37 to 44 for LCN2, and SEQ ID NOs: 45 to 56 for CD3gamma/delta, and
c) detecting the panel of amplicons.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising an analysis of the methylation status of an amplicon for CD3 epsilon.

### Claim 3 (depends on 1)

The method according to claim 1, further comprising using a demethylation standard gene selected from a gene expressed in all cells.

### Claim 4 (depends on 3)

The method according to claim 3, further comprising using an in silico bisulfite-converted recombinant nucleic acid comprising a sequence inversing all CpG dinucleotides to GpC of said demethylation standard gene (GAP[GC] construct).

### Claim 5 (depends on 1)

The method according to claim 1, further comprising using a calibrator plasmid comprising one copy of each amplicon sequence in its unconverted genomic state.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said detection of the panel of amplicons further comprises a quantification of CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells, and neutrophils.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said method further comprises an additional flow cytometry (FCM) of said immune cells.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the panel of amplicons comprises amplicons for CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said human blood cell sample is selected from peripheral blood, capillary blood, venous blood, and subfractions thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the human blood cell sample is taken from a newborn.

### Claim 11 (depends on 3)

The method of claim 3, wherein the standard gene is a housekeeping gene.

### Claim 12 (depends on 11)

The method of claim 11, wherein the housekeeping gene comprises glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or beta-actin.

### Claim 13 (depends on 12)

The method of claim 12, wherein the method uses primers and probes selected from SEQ ID NOs: 57 to 61 for GAPDH.

### Claim 14 (depends on 4)

The method of claim 4, wherein the method uses primers and probes selected from SEQ ID NOs: 62 to 64 for the GAP[GC] construct.

### Claim 15 (depends on 5)

The method of claim 5, wherein the unconverted genomic state is in an unmethylated state.

### Claim 16 (depends on 8)

The method of claim 8, further comprising an analysis of the methylation status of the amplicons for CD3gamma/delta, CD4, CD8beta, LRP5, MVD, and LCN2.

### Claim 17 (depends on 9)

The method of claim 9, wherein said human blood cell sample is selected from peripheral blood monocytes, blood clots, and dried blood spots.