Method for discriminating between intact and damaged cells in a sample

• US 5,057,413 A
• Filed: 06/13/1988
• Issued: 10/15/1991
• Est. Priority Date: 06/13/1988
• Status: Expired due to Term

• Current Assignee: Becton
• Original Assignee: Becton, Dickinson & Co
• Inventors: Terstappen, Leon W. M. M., Loken, Michael R., Shah, Virendra O.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): SCHEINER, TONI R
• Application Number:
  US07/206,454
• Time in Patent Office: 1,219 Days
• Field of Search: 435/9, 435/6, 435/7, 435/29, 435/257, 435/810, 436/63, 436/94, 436/172, 436/43, 436/548, 436/800, 530/380, 250/459.1, 250/461.2
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1402 Data analysis by thresholding or gating operations performed on the acquired signals or stored data
• CPC Class Codes: G01N 2015/1477 Multiparameters
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

A method is disclosed for the discrimination between intact and damaged cells in a sample, and more particularly, the method uses a vital nucleic acid dye to selectively stain intact versus damaged cells which then may be counted and sorted by flow cytometry means. The method also may be used in conjunction with fluorescently labelled monoclonal antibodies to simultaneously identify cellular antigens.

Claims

1. A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of:

a) taking a sample of peripheral blood from an individual;

b) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye;

c) adding a fixative to the sample;

d) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR2## wherein n is 0;

m is 1;

R is a lower alkyl;

R₁ and R₂ are lower alkyls;

R₃ is hydrogen;

Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and

X represents an anion;

e) analyzing the sample in an automated instrument capable of passing the cells in the sample through a sensing region substantially one at a time and capable of detecting and recording both fluorescence and light scattered in more than one direction by each cell passing through said sensing region; and

f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.

2. The method of claim 1 wherein erythrocytes in the sample are lysed.

3. The method of claim 1 wherein the automated instrument is a flow cytometer.

4. The method of claim 1 wherein the fixative is paraformaldehyde.

5. The method of claim 1 wherein the fluorescent label is fluorescein isothiocyanate.

6. The method of claim 1 wherein the fluorescent label is phycoerythrin.

7. The method of claim 1 wherein the fluorescent labels are fluorescein isothiocyanate and phycoerythrin.

8. The method of claim 1 wherein the intact and damaged cells are further discriminated based upon relative fluorescence intensity of fluorescent labels and light scattered.

9. A method for discriminating between a heterogeneous population of intact and damaged cells in a peripheral blood sample comprising the steps of:

a) taking a sample of peripheral blood from an individual;

b) lysing erythrocytes in the sample;

c) adding to the sample one or more monoclonal antibodies to cell surface antigens, wherein the monoclonal antibodies are labeled with fluorescent labels and wherein the fluorescent labels have peak emission spectra that are distinguishable from each other and from a nucleic acid dye;

d) adding paraformaldehyde to the sample;

e) adding to the sample a nucleic acid dye which preferentially binds DNA, wherein the nucleic acid dye comprises the formula ##STR3## wherein n is 0;

m is 1;

R is a lower alkyl;

R₁ and R₂ are lower alkyls;

R₃ is hydrogen;

Z is a quinoline nucleus which may be substituted with lower alkyl, halo, nitro, amino and dialkylamino; and

X represents an anion;

e) analyzing said sample in a flow cytometer equipped with a single laser tuned to 488 nm; and

f) discriminating between damaged and intact cells in the sample based upon relative fluorescence intensity of the nucleic acid dye in each cell and light scattered.

10. The method of claim 9 wherein the intact and damaged cells are further discriminated based upon relative fluorescence intensity of fluorescent labels and light scattered.