Methods, reagents and kits for flow cytometric immunophenotyping

Patent metadata

Field	Value
Publication number	US-9880158-B2
Application number	US-201013376103-A
Country	US
Kind code	B2
Filing date	Jun 2, 2010
Priority date	Jun 3, 2009
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The invention relates to the field of flow cytometry and more particularly to a panel of antibody reagents conjugated to fluorescent compounds. Provided are reagent compositions, comprising at least eight distinct fluorochrome-conjugated antibodies comprising a set of at least three identification antibodies for the identification of a leukocyte population of interest and at least four characterization antibodies for further characterization and/or classification of said leukocyte population. Also provided are kits and methods related to the reagent compositions.

First claim

The invention claimed is:

1. A set of a first reagent composition and a second reagent composition for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, wherein the second reagent composition comprises fluorochrome-conjugated antibodies, and the first reagent composition is selected from the group consisting of:
(a) fluorochrome-conjugated antibodies against the following combinations of markers: CD20, CD4, CD45, CD19, Igγ, CD8, Igκ, CD56, CD5, T-cell receptor (TCR) γδ, CD3, and CD38, wherein:
(i) the antibody against CD20 and the antibody against CD4 are both conjugated to the same fluorochrome;
(ii) the antibody against Igγ and the antibody against CD8 are both conjugated to the same fluorochrome; and
(iii) the antibody against CD19 and the antibody against TCRγδ are both conjugated to the same fluorochrome,
wherein the fluorochrome in (i) has a different fluorescence emission from the fluorochrome in (ii) and (iii), and the fluorochrome in (ii) has a different fluorescence emission from the fluorochrome in (iii);

(b) fluorochrome-conjugated antibodies against the following combinations of markers: CD20, CD45, CD8, Igλ, CD56, Igκ, CD4, CD19, CD3, CD14, and CD38, wherein:
(i) the antibody against CD8 and the antibody against Igλ are both conjugated to the same fluorochrome;
(ii) the antibody against CD56 and the antibody against Igκ are both conjugated to the same fluorochrome; and
(iii) the antibody against CD3 and the antibody against CD14 are both conjugated to the same fluorochrome,
wherein the fluorochrome in (i) has a different fluorescence emission from the fluorochrome in (ii) and (iii), and the fluorochrome in (ii) has a different fluorescence emission from the fluorochrome in (iii);

(c) fluorochrome-conjugated antibodies against the following combinations of markers: CD45, CD138, CD38, CD56, β2 microglobulin (β2micro), CD19, CyIgκ, and CyIgλ, wherein the antibody against CD45, the antibody against CD138, the antibody against CD38, the antibody against β2micro, the antibody against CD19, the antibody against CyIgκ, and the antibody against CyIgλ are each conjugated to a fluorochrome that has a different fluorescence emission from the other fluorochromes in (c); and
(d) fluorochrome-conjugated antibodies against the following combinations of markers: cyCD3, CD45, cytoplasmic myeloperoxidase (cyMPO), cyCD79a, CD34, CD19, CD7, and CD3, wherein the antibody against cyCD3, the antibody against CD45, the antibody against cyMPO, the antibody against cyCD79a, the antibody against CD34, the antibody against CD19, the antibody against CD7, and the antibody against CD3 are each conjugated to a fluorochrome that has a different fluorescence emission from the other fluorochromes in (d).

2. The set of claim 1 , wherein the first reagent composition is any one of (a), (b), or (c).

3. The set of claim 2 , wherein the second reagent composition comprises fluorochrome-conjugated antibodies against one of the following combinations of markers:
(1) CD20, CD45, CD23, CD10, CD79b, CD19, CD200, and CD43;
(2) CD20, CD45, CD31, LAIR1, CD11c, CD19, IgM, and CD81;
(3) CD20, CD45, CD103, CD95, CD22, CD19, CXCR5, and CD49d;
(4) CD20, CD45, CD62L, CD39, HLADR, CD19, CD27, and CD31;
(5) CD4, CD45, CD7, CD26, CD3, CD2, CD28, and CD8;
(6) CD4, CD45, CD27, CCR7, CD3, CD45RO, CD45RA, and CD8;
(7) CD4, CD45, CD5, CD25, CD3, HLADR, cyTCL1, and CD8;
(8) CD4, CD45, CD57, CD30, CD3, CD11c, and CD8;
(9) CD4, CD45, cyPerforin, cyGranzyme, CD3, CD16, CD94, and CD8;
(10) CD4, CD45, CD279, CD3, and CD8;
(11) CD2, CD45, CD7, CD26, CD3, CD56, CD5, and CD19;
(12) CD16, CD45, CD57, CD25, CD3, CD56, CD11c, and CD19;
(13) HLADR, CD45, cyPerforin, cyGranzyme, CD3, CD56, CD94, and CD19; and
(14) CD45, CD138, CD38, CD28, CD27, CD19, CD117, and CD81.

4. The set of claim 3 , wherein the first reagent composition is (a) or (b), and the second reagent composition is (1), (2), (3), or (4).

5. The set of claim 3 , wherein the first reagent composition is (a) or (b), and the second reagent composition is (5), (6), (7), (8), (9), or (10).

6. The set of claim 3 , wherein the first reagent composition is (a) or (b), and the second reagent composition is (11), (12), or (13).

7. The set of claim 3 , wherein the first reagent is (c) and the second composition is (14).

8. The set of claim 3 , wherein the second reagent composition is selected from the group consisting of:
(1) an antibody against CD20 that is conjugated to PacB or Horizon V450; an antibody against CD45 that is conjugated to PacO or AMCA; an antibody against CD23 that is conjugated to FITC or Alexa488; an antibody against CD10 that is conjugated to PE; an antibody against CD79b that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD19that is conjugated to PE-Cy7; an antibody against CD200 that is conjugated to Alexa647; and an antibody against CD43 that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(2) an antibody against CD20 that is conjugated to PacB or Horizon V450; an antibody against CD45 ; that is conjugated to PacO or AMCA; an antibody against CD31 that is conjugated to FITC or Alexa488; an antibody against LAIR1 that is conjugated to PE; an antibody against CD11c that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD19that is conjugated to PE-Cy7; an antibody against IgM that is conjugated to Alexa647; and an antibody against CD81 that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(3) an antibody against CD20 that is conjugated to PacB or Horizon V450; an antibody against CD45 that is conjugated to PacO or AMCA; an antibody against CD103 that is conjugated to FITC or Alexa488; an antibody against CD95 that is conjugated to PE; an antibody against CD22 that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD19 that is conjugated to PE-Cy7; an antibody against CXCRS that is conjugated to Alexa647; and an antibody against CD49d that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(4) an antibody against CD20 that is conjugated to PacB or Horizon V450; an antibody against CD45 ; that is conjugated to PacO or AMCA; an antibody against CD62L that is conjugated to FITC or Alexa488; an antibody against CD39 that is conjugated to PE; an antibody against HLADR that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD19 that is conjugated to PE-Cy7; an antibody against CD27 that is conjugated to Alexa647; and an antibody against CD31 that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(5) an antibody against CD4 that is conjugated to PacB or Horizon V450; an antibody against CD45 that is conjugated to PacO or AMCA; an antibody against CD7 that is conjugated to FITC or Alexa488; an antibody against CD26 that is conjugated to PE; an antibody against CD3 that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD2 that is conjugated to PE-Cy7; an antibody against CD28 that is conjugated to Alexa647; and an antibody against CD8 that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(6) an antibody against CD4 that is conjugated to PacB or Horizon V450; an antibody against CD45 that is conjugated to PacO or AMCA; an antibody against CD27 that is conjugated to FITC or Alexa488; an antibody against CCR7 that is conjugated to PE; an antibody against CD3 that is conjugated to PerCP-Cy5.5, PerCP, or PE-Texas Red; an antibody against CD45RO that is conjugated to PE-Cy7; an antibody against CD45RA that is conjugated to Alexa647; and an antibody against CD8 that is conjugated to APC-H7, APC-Cy7, Alexa680, or Alexa700;
(7) an antibody against CD4 that is conjugated to PacB or Horizon V450; an anti

Assignees

• Van Dongen Jacobus Johannes Maria
• Orfao De Matos Correia E Vale José Alberto
• Flores-Montero Juan Alejandro
• Almeida Parra Julia Maria
• Van Der Velden Vincent Henricus Johannes
• Böttcher Sebastian
• Rawstron Andrew Craig
• De Tute Ruth Mary
• Lhermitte Ludovic Bernard Simon
• Asnafi Vahid
• Mejstríková Ester
• Szczepanski Tomasz
• Monteiro Da Silva Lucio Paulo Jorge
• Ayuso Marta Marín
• Pedreira Carlos Eduardo
• Univ Erasmus Med Ct Rotterdam

Inventors

• Van Dongen Jacobus Johannes Maria
• Orfao De Matos Correia E Vale José Alberto
• Flores-Montero Juan Alejandro
• Almeida Parra Julia Maria
• Van Der Velden Vincent Henricus Johannes
• Böttcher Sebastian
• Rawstron Andrew Craig
• De Tute Ruth Mary
• Lhermitte Ludovic Bernard Simon
• Asnafi Vahid
• Mejstríková Ester
• Szczepanski Tomasz
• Monteiro Da Silva Lucio Paulo Jorge
• Ayuso Marta Marín
• Pedreira Carlos Eduardo

Classifications

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title

• G01N33/5094 Primary

  for blood cell populations (red blood cells G01N33/80) · CPC title

• G01N2015/1402

  Data analysis by thresholding or gating operations performed on the acquired signals or stored data · CPC title

• G01N33/533

  with fluorescent label · CPC title