# US 5,702,884 A · Whole blood sample preparation for polymerase chain reaction using ammonium chloride and a carboxylic acid or metal carboxylate for selective red blood cell lysis

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- Number: US 5,702,884 A
- Document ID: US5702884A
- Status: Granted
- Kind: A
- Application number: 08/615,848
- Filed: 1996-03-12
- Granted: 1997-12-30
- Earliest claimed: 1996-03-12
- Assignees: Johnson & Johnson Clinical Diagnostics, Inc.
- Inventors: Tobias E. Ekeze; JoAnne Hansen Kerschner
- Web page: https://patentgrep.com/patent/US5702884A

## CPC

- G01N 33/5094: {for blood cell populations (red blood cells G01N33/80)}
- C12N 1/06: Lysis of microorganisms
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

Leucocytes can be quickly and selectively separated from erythrocytes by subjecting a whole blood sample to a series of steps including lysing the erythrocytes and washing the remaining leucocytes with a solution containing ammonium chloride and a carboxylic acid or a metal carboxylate. The resulting white blood cells can be readily lysed and subjected to polymerase chain reaction to amplify and detect a target nucleic acid. The test kit useful in practicing the amplification method includes a labeled primer, a PCR reagent and a reagent mixture containing ammonium chloride and a carboxylic acid or a metal carboxylate for sample preparation.

## Claims

### Claim 1

A method for the selective preparation of leucocytes comprising:
A) mixing a whole blood sample with an erythrocyte lysing solution comprising between 50 mM and 100 mM of ammonium chloride and between about 0.001 weight percent to about 0.1 weight percent of a monocarboxylic acid or salt thereof selected from the group consisting of acetic acid, monosubstituted acetic acid, disubstituted acetic acid, and trisubstituted acetic acid, and wherein said lysing solution has a pH of between 6 and 8,
B) centrifuging the resulting mixture to form a pellet of leucocytes from said whole blood sample,
C) after removing the supernatant, washing said leucocyte pellet in a fresh sample of said lysing solution, and
D) centrifuging and isolating said leucocyte pellet, provided that steps A) through D) are carried out within about 20 minutes.

### Claim 2 (depends on 1)

The method of claim 1 wherein said monocarboxylic acid or salt thereof is between about 0.005 and 0.05 weight percent.

### Claim 3 (depends on 1)

The method of claim 1 wherein said monocarboxylic acid or salt thereof is selected from the group consisting of monohalo acetic acid, dihalo acetic acid, trihalo acetic acid and acetic acid.

### Claim 4 (depends on 3)

The method of claim 3 wherein said monocarboxylic acid is acetic acid.

### Claim 5 (depends on 1)

The method of claim 1 wherein the pH is between 6.5 and 7.5.

### Claim 6 (depends on 1)

The method of claim 1 wherein steps A) through D) are carried out within from about 10 to about 16 minutes.

### Claim 7 (depends on 1)

The method of claim 1 wherein said whole blood sample has a volume of from about 0,01 to about 10 mL.

### Claim 8 (depends on 1)

The method of claim 1 wherein each centrifugation step is carried out within less than about 10 minutes.

### Claim 9

A method for the amplification and detection of a target nucleic acid comprising:
I) selectively preparing leucocytes suspected of containing a target nucleic acid in a whole blood sample by:
A) mixing a whole blood sample with an erythrocyte lysing solution comprising between 50 mM and 100 mM of ammonium chloride and between about 0.001 weight percent to about 0.1 weight percent of a monocarboxylic acid or salt thereof selected from the group consisting of acetic acid, monosubstituted acetic acid, disubstituted acetic acid and trisubstituted acetic acid, and wherein said lysing solution has a pH of between 6 and 8,
B) centrifuging the resulting mixture to form a pellet of leucocytes from said whole blood sample,
C) after removing the supernatant, washing said leucocyte pellet in a fresh sample of said lysing solution, and
D) centrifuging and isolating said leucocyte pellet, provided that steps A) through D) are carried out within about 20 minutes,
II) lysing the leucocytes in said washed pellet to release said target nucleic acid,
III) amplifying said released target nucleic acid using polymerase chain reaction and a set of primers that are specific to and hybridizable with the opposing strands of said target nucleic acid, and
IV) detecting said amplified target nucleic acid.

### Claim 10 (depends on 9)

The method of claim 9 wherein steps A) through D) are carried out within from about 10 to about 16 minutes.

### Claim 11 (depends on 9)

The method of claim 9 wherein said whole blood sample has a volume of from about 0.01 to about 10 mL.

### Claim 12 (depends on 9)

The method of claim 9 wherein each centrifugation step is carried out within less than about 10 minutes.

### Claim 13 (depends on 9)

The method of claim 9 wherein the polymerase chain reaction is carried out in the presence of a thermostable DNA polymerase and wherein one or both of said primers are labeled for detection.

### Claim 14 (depends on 13)

The method of claim 13 wherein said labeled primers are labeled with biotin, and detection is achieved using a conjugate of avidin and an enzyme which can provide a detectable signal in the presence of its substrate.

### Claim 15 (depends on 9)

The method of claim 9 for the detection of a viral, bacterial, fungal or protozoan RNA or DNA.

### Claim 16 (depends on 15)

The method of claim 15 for the detection of RNA or DNA from any of a Streptococcus species, Mycobacterium species, Pneumocystis carinii, herpes simplex viruses, Epstein Barr virus, cytomegalovirus, hepatitis viruses, retroviruses, Candida species, or Aspergillus species.

### Claim 17 (depends on 16)

The method of claim 16 for the detection of HIV-I, HIV-2, Mycobacterium tuberculosis, Mycobacterium avium complex or cytomegalovirus RNA or DNA.

### Claim 18 (depends on 9)

The method of claim 9 wherein said amplified target nucleic acid is detected using, a reagent which provides a colorimetric or chemiluminescent signal in response to said label on said labeled primer.

### Claim 19

A kit for polymerase chain reaction comprising:
a) a set of two primers specific to and hybridizable with opposing strands of a target nucleic acid, one or both of said primers being labeled with a detection moiety, and in the same or different package, at least one additional PCR reagent, and
b) in a separate package, a solution or a dry composition containing, when said dry composition is reconstituted with water, between 50 mM and 100 mM of ammonium chloride and between about 0.001 weight percent to about 0.1 weight percent of a monocarboxylic acid or salt thereof selected from the group consisting of acetic acid, monosubstituted acetic acid, disubstituted acetic acid, and trisubstituted acetic acid, and wherein said solution has a pH of between 6 and 8.

### Claim 20 (depends on 19)

The kit of claim 19, wherein one or both of said primers are labeled with biotin, and said kit comprises additionally a thermostable DNA polymerase cofactor which is a magnesium or manganese salt, and dATP, dCTP, dGTP, and dTTP.

### Claim 21 (depends on 19)

The kit of claim 19, wherein said set of primers comprises two primers specific to and hybridizable with the opposing strands of HIV-I, HIV-2, Mycobacterium tuberculosis, Mycobacterium avium complex or cytomegalovirus DNA or RNA.