Products and methods for single parameter and multiparameter phenotyping of cells

• US 20010041343A1
• Filed: 07/20/2001
• Published: 11/15/2001
• Est. Priority Date: 05/04/1999
• Status: Active Grant

• Current Assignee: Hematotypes, Inc.
• Original Assignee: Dan A. Pankowsky
• Inventors: Pankowsky, Dan A.
• Granted Patent: US 6,682,940 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/2525 Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

A method of single parameter and multiparameter characterizing of cells, particularly immunophenotyping of cells, is provided. The method preferably uses antibody-coated microspheres which are adapted to bind to specific types of cells. One or more sets of coated microspheres are contacted simultaneously or sequentially with a suspension of cells and bind the cells they are adapted to bind to form bead-cell complexes. Cells may bind to one or more microspheres. The bead-cell complexes are then separated from the suspension The complexes are preferably stained and then examined to characterize the cells, preferably the cells bound to the microspheres. A method of quantitating a specific cell type is provided. A kit and apparatus for performing the method are also provided.

Claims

1. A method of characterizing cells comprising the steps of:

a) providing a suspension of cells in a liquid medium, said cells including first cells,

b) contacting a group of first beads with said suspension, each of said first beads being coated with a binding substance or being magnetic such that each first bead is adapted to bind to at least one of said first cells,

c) incubating said first beads with said suspension for a period of time effective to permit said first cells to bind to said first beads to form first bead-first cell complexes, each first bead-first cell complex comprising a first bead and a first cell,

d) separating said first bead-first cell complexes from said suspension, and

e) examining said separated first bead-first cell complexes and characterizing said first cells.

2. A method according to

claim 1

, wherein said suspension contains an effective amount of an enhancement reagent.

3. A method according to

claim 2

, wherein said enhancement reagent is albumin.

4. A method according to

claim 1

, further comprising the step of adding to said suspension an effective amount of an additive to inhibit phagocytosis of beads.

5. A method according to

claim 1

, further comprising the step of adding to a suspension of said cells a fixative in an amount effective to inhibit phagocytosis of beads and subsequently washing out said fixative prior to said contacting step.

6. A method according to

claim 1

, further comprising the step of adding to a suspension of said cells an effective amount of a fixative to preserve said cells for later analysis and subsequently washing out said fixative prior to said contacting step.

7. A method according to

claim 1

, further comprising the step of providing a substrate, said group of first beads being affixed to said substrate during said contacting step.

8. A method of quantitating a specific cell type in a cell suspension comprising the following steps:

a) providing a suspension of cells in a liquid medium, said cells including first cells, said first cells comprising second cells and optionally third cells,

b) counting the number of first cells per unit volume of said suspension,

c) performing one of steps (c1) and (c2)

(c1) incubating in said suspension first beads and second beads, each of said first beads being adapted to bind to at least one of said first cells, each of said second beads being adapted to bind to at least one of said second cells, said incubation being for a period of time effective to permit said first beads to bind to said first cells to form first bead-cell complexes, said incubation being for a period of time effective to permit said second beads to bind to said second cells to form second bead-cell complexes,

(c2) incubating in said suspension first beads and second beads, each of said first beads being adapted to bind to at least one of said second cells, each of said second beads being adapted to bind to at least one of said third cells, said incubation being for a period of time effective to permit said first beads to bind to said second cells to form first bead-cell complexes, said incubation being for a period of time effective to permit said second beads to bind to said third cells to form second bead-cell complexes,

d) separating said first bead-cell complexes and said second bead-cell complexes from said suspension,

e) estimating the relative percentages of

     1) the cells bound to the first beads and

     2) the cells bound to the second beads, and

f) calculating the number of second cells or the number of third cells per unit volume of said suspension.

9. A method for preparing an enriched population of a target cell comprising the following steps:

a) providing a suspension of cells in a liquid medium, said cells including target cells,

b) contacting a group of first beads with said suspension, each of said first beads being coated with a binding substance or being magnetic such that each first bead is adapted to bind to at least one of said target cells,

c) incubating said first beads with said suspension for a period of time effective to permit said target cells to bind to said first beads to form first bead-target cell complexes, each first bead-target cell complex comprising a first bead and target cell,

d) separating said first bead-target cell complexes from said suspension, and

e) resuspending said target cells in another liquid medium.

10. A method according to

claim 9

, wherein said resuspended target cells are still attached in said first bead-target cell complexes.

11. A method according to

claim 9

, wherein said resuspended target cells have been released from said first bead-target cell complexes.