Preserved cell preparations for flow cytometry and immunology

• US 5,478,722 A
• Filed: 02/17/1991
• Issued: 12/26/1995
• Est. Priority Date: 02/17/1991
• Status: Expired due to Fees

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: Caldwell, Charles W.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): LANKFORD JR, LEON B
• Application Number:
  US07/652,095
• Time in Patent Office: 1,773 Days
• Field of Search: 435/7.4, 435/1, 435/2, 435/240.1, 435/240.2, 435/240.3, 435/219, 435/7.1, 435/7.2
• US Class Current: 435/1.1
• CPC Class Codes: C12N 1/04 Preserving or maintaining viable microorganisms immobilised microorganisms C12N11/00
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]
• CPC Class Codes: Y10T 436/106664 Blood serum or blood plasma standard or control

Abstract

Cells are treated to preserve them in non-frozen hydrated form with minimal alterations to their antigens or nucleic acids. This makes the cells useful as quality control (QC) reagents for processes such as calibrating and standardizing flow cytometry equipment, for use as "unknown" test samples for QC testing programs, and to prepare patient specimens for archival storage. This method includes: (1) treating the cells to inhibit proteolysis, preferably using several protease inhibitors; (2) fixing the cells, either by contacting them with a crosslinking agent such as formalin or paraformaldehyde, or by exposure to microwave radiation, while the cells are being agitated to promote the formation of crosslinking bonds within a single cell while inhibiting the formation of cell clumps; and (3) if the cells are treated with a chemical crosslinking agent, removing the crosslinking agent and contacting the cells with a quenching agent. The cells should be stored in a solution containing protease inhibitors. This method causes little or no detectable alteration of antigenic or DNA histograms. It has been used to preserve cells that have been stored for months at room temperature, or for more than a year at refrigerated temperatures, with no substantial deterioration of surface or cytoplasmic antigens or DNA content.

Claims

1. A method of preserving cells to render them useful for subsequent immunologic analysis, wherein the cells express cellular proteins which function as antigenic determinants that can be used to characterize the cells by binding to complementary monoclonal antibodies, comprising the following treatment steps:

a. treating a population of cells with at least one protease inhibitor under conditions which allow said protease inhibitor to chemically react with cellular enzymes to an extent which inhibits cell-related proteolytic activity;

b. contacting the cells which have been treated with a chemical crosslinking agent while the cells are being agitated under conditions which promote the formation of molecular bonds within a single cell while inhibiting the formation of crosslinking bonds between different cells; and

,

c. contacting the cells with a quenching compound that will inhibit subsequent crosslinking,

wherein said treatment steps do not substantially alter the ability of the cellular proteins to bind to complementary monoclonal antibodies.

2. The method of claim 1 wherein at least two different protease inhibitors are user to treat the cells.

3. The method of claim 1 wherein at least one protease inhibitor is selected from the group consisting of sodium azide, phenylmethylsulfonyl fluoride, and ethylenediaminetetraacetate.

4. The method of claim 1 wherein the first protease inhibition treatment is carried out in an aqueous solution that also contains a stabilizing agent comprising a compound which contains sulfhydryl groups.

5. The method of claim 4 wherein the stabilizing agent is selected from the group consisting of dithiothreitol, cysteine, and 2-mercaptoethanol.

6. The method of claim 1 wherein the crosslinking agent comprises an aldehyde compound.

7. The method of claim 6 wherein the crosslinking agent comprises formaldehyde.

8. The method of claim 1 wherein the cells are stored, after the crosslinking agent is removed, in a solution containing at least one protease inhibitor which remains in contact with the cells throughout a storage period until the cells are needed for analysis.

9. The method of claim 1 wherein the cells are contacted, after the crosslinking agent is removed, with a membrane-modifying agent that renders membranes of the cells permeable.

10. The method of claim 9 wherein the membrane-modifying agent comprises a detergent.

11. A population of cells which has been treated by the method of claim 1.

12. A population of cells which has been treated by the method of claim 4.

13. A population of cells which has been treated by the method of claim 8.