Mobile Site ›
Print Friendly View

Test ID: CMVC8    
Cytomegalovirus (CMV) CD8 T-Cell Immune Competence, Quantitative Assessment by Flow Cytometry

Method Description Describes how the test is performed and provides a method-specific reference

Assessment of cytomegalovirus (CMV)-immune competence measures 3 different components of the immune response: 1) numerical analysis of the CMV-specific CD8 T-cell population (enumeration), which provides absolute counts of the antigen-specific CD8 T cells, 2) functional and 3) cytotoxic analysis of the CMV-specific CD8 T cells (functionality and cytotoxic activity).

 

Enumeration:

Enumeration of CD8 T cells is performed on whole blood using Beckman Coulter MHC class I tetramers, specific for 5 major histocompatibility complex (MHC) class I alleles-A1, A2, B7, B8, and B35.

 

Panel I consists of whole blood stained with CD3 PE-Cy5 (PhycoErythrin-Cy5), CD8 fluorescein isothiocyanate (FITC), and CD4 PhycoErythrin (PE) followed by a 20-minute incubation at ambient temperature in the dark. The sample is then treated with the tetramer lyse reagent for 10 minutes at ambient temperature in the dark. The flow count bead solution is then thoroughly mixed and added at a volume exactly equal to the volume of whole blood added. After the addition of beads, the sample is analyzed on a BD FACS Canto flow cytometer within 1 hour and exactly 3,000 bead events are counted.(Package insert: Beckman Coulter, 2002, Beckman Coulter, Inc., Fullerton, CA)

 

The number of CD3 and CD8 cells per mcL of whole blood is counted using the following formula:

 

No. of CD3+cells/uL = No. of events in CD3+ gate X concentration of beads/mcL*

                                                No. of events in the bead capture gate (3,000)

Multiply by 1,000 to obtain results as cells x 10(3)/mL

  

No. of CD8+cells/mcL = No. of events in CD8+ gate X concentration of beads/mcL*

                                                No. of events in the bead capture gate (3,000)

Multiply by 1,000 to obtain results as cells x 10(3)/mL

 

*This number is provided by the manufacturer for each lot.

 

In Panel II, whole blood is stained with CD3 (PE-Cy5), CD8 (FITC) and incubated with the relevant MHC class I tetramer-PE for 20 minutes at ambient temperature in the dark. The whole blood is then lysed using the tetramer lyse reagent and washed with the wash buffer. The sample is then run on the flow cytometer and the CMV-specific CD8 T cells are analyzed as a percentage of the total CD8+ T cells. The absolute count of the CMV-specific CD8 T cells is obtained using the following formula:

 

Absolute count of CMV-specific CD8 T cells =

 % of CMV specific CD8 T cells X absolute count of CD8 T cells/mcL**

                                                            100

Multiply by 1,000 to obtain results as cells x 10(3)/mL

 

 **This number will be obtained from Panel I.

 

The final absolute counts for CD3 T cells, CD8 T cells, and CMV-specific CD8 T cells are expressed as cells/mL of whole blood.

 

Functionality (Intracellular Interferon-gamma: IFN-gamma) and Cytotoxic Activity (Surface CD107a/b):

Peripheral blood mononuclear cells (PBMC) are isolated from the same sample of whole blood used for enumeration. PBMCs are stimulated with HLA allele-specific CMV peptides and costimulatory molecules (CD28/CD49d) in a polypropylene tube. Antihuman CD107a/b conjugated with FITC is added along with the peptides to capture the transient expression of the CD107a and CD107b, which are markers for cytotoxic activity. A mixture of Brefeldin A (BFA) and monensin is also added during the stimulation to facilitate the intracellular accumulation of IFN-gamma, which is detected using the antihuman IFN-gamma antibody conjugated with allophycocyanin (APC). A similar tube is prepared for the patient sample with the absence of exogenous CMV peptide and this tube serves as the unstimulated control (back-ground stimulation). Thus, there are 2 tubes for every HLA allele. After peptide stimulation for 5 hours, EDTA is added to the sample to arrest activation and to remove adherent cells from the activation tube. Antihuman CD8 antibody and the relevant MCH-class I tetramer are added and incubated at ambient temperature for 20 minutes in the dark. This step is followed by a simultaneous lysis and fixation of the cells to prepare them for permeabilization. Cells are then washed and permeabilized with BD FACS Perm II solution. The antihuman IFN-gamma antibody is added and the sample is incubated for 30 minutes at ambient temperature in the dark. Finally, the cells are washed and analyzed by flow cytometry. The delta percent (stimulation in the presence of CMV-specific peptide stimulation in the absence of specific peptide) CMV-specific CD8 T cells expressing IFN-gamma and CD107a/b is used to calculate the absolute count of activated and functionally cytotoxic CMV-specific CD8 T cells.(Functionality: IFN-gamma assay, unpublished Mayo method; Cytotoxic Activity: Betts MR, Brenchley JM, Price DA, et al: Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 2003;281:65-78; Betts MR, Price DA, Brenchley JM, et al: The functional profile of primary human antiviral CD8 T cell effector activity is dictated by cognate peptide concentration. J Immunol 2004;172[10]:6407-6417)

 

Global CD8 T-Cell Immune Competence:

Peripheral blood mononuclear cells (PBMC), which contain CD8 T cells, are stimulated with a mixture of phorbol myristate acetate (PMA) and ionomycin, and with stimulatory signals derived using antibodies against the costimulatory molecules CD28/CD49d. The cells are simultaneously treated with a mixture of BFA and monensin, which blocks extracellular secretion of IFN-gamma, enabling intracellular retention and detection of the protein. PBMCs that have not been stimulated are used as a control to determine the background levels of IFN-gamma and CD107a and CD107b. The cells are analyzed on the BD FACS Canto flow cytometer and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma and CD107a and CD107b signals are determined within the "gated" CD8 T cell population. Global CD8 T cell immune competence is measured by the amount of IFN-gamma produced (CD8 T-cell functional activity) and surface expression of CD107a/b (cytotoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)

 

The method for T- and B-cell natural killer cell quantitation is described under TBBS / T- and B-Cell Quantitation by Flow Cytometry.

Supplemental Report Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.

Monday through Friday

Specimens are required to be received in the laboratory on weekdays and by 4 p.m. on Friday. No weekend processing

Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.

3 days

Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result

4 days

Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

PBMC's are stored for 7 days at -70 degrees C

Performing Laboratory Location The location of the laboratory that performs the test

Rochester