Conventional Testing: Reagents, Instrumentation, and Methods
Flow cytometric enumeration of lymphocyte subsets (CD3, CD4, CD8, CD19, CD20, CD16, and CD56) was performed at Oregon Medical Laboratories (OML, Eugene, OR), a clinical reference laboratory, using an EPICS-XL flow cytometer. All instrumentation, software, reagents, and monoclonal antibodies used by OML were obtained from Beckman Coulter (Miami, FL). Conventional four-color analysis using four fluorophores, fluorescein isothiocyanate (FITC), phycoerythrin (R-PE), phycoerythrin-Texas Red®-X dye (ECD) and phycoerythrin-cyanin 5.1 (PC5) was performed in a four-tube panel using CYTO-STAT/COULTER CLONE directly labeled monoclonal antibodies in the following antibody combinations:
Tube 1: CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5
Tube 2: CD45-FITC/CD56-PE/CD19-ECD/CD3-PC5
Tube 3: CD19-FITC/CD20-PE/CD45-PC5
Tube 4: CD16-FITC/CD56-PE/CD45-PC5
For each tube, monoclonal antibodies were added to 100 μl of whole blood. These tubes were vortexed, incubated for 10 min at room temperature, then lysed with the TQ Prep Workstation and ImmunoPrep Reagent System. Data were acquired on the EPICS-XL, equipped with a 488-nm argon-ion laser with 525-, 575-, 620-, and 675-nm band-pass filters. At least 5,000 lymphocyte events per tube were accumulated. Tubes 1 and 2 were evaluated using the tetraONE software system, which employs automated CD45/Side Scatter (SS) lymphocyte gating to derive percentages for lymphocytes expressing CD3, CD4, CD8, CD19, and CD56. Tubes 3 and 4 were evaluated with CD45/SS gating on lymphocytes to determine the percentage of lymphocytes positive for the FITC- and PE-conjugated markers (9). Instrument alignment was checked daily using Flow-Check Fluorospheres, instrument compensation was performed weekly using CYTO-COMP Reagent and Cell Kits, and normal control cells were run daily using Immuno-Trol Control Cells. OML has demonstrated good agreement with other laboratories for parameters in this study in proficiency testing via both the College of American Pathology and Center for Disease Control.
Fluorescence-Intensity Multiplexed Zenon Reagent Labeling Testing: Reagents, Instrumentation and Methods
Whole blood lysis using ammonium chloride was performed and cell concentrations were adjusted to 1 × 107 cells/ml in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (8, 10). Mouse anti-human monoclonal antibodies (BD Pharmingen, San Diego, CA) used: IgG1 isotype (clone MOPC-21), IgG2b isotype (clone 2–35), CD3 (clone UCHT1, IgG1), CD4 (clone RPA-T4, IgG1), CD8 (clone HIT8a, IgG1), CD16 (clone 3G8, IgG1), CD19 (clone HIB19, IgG1), CD20 (clone 2H7, IgG2b), CD56 (clone B159, IgG1), and CD45 (clone HI30, IgG1). Zenon Alexa Fluor 488 Mouse IgG1 Labeling Reagent, Zenon R-PE Mouse IgG1 Labeling Reagent, Zenon R-PE Mouse IgG2b Labeling Reagent, and Zenon Alexa Fluor 647-R-PE Mouse IgG1 Labeling Reagent (Molecular Probes, Inc.) were used. Testing was performed using an Elite Flow Cytometer (Beckman Coulter) equipped with a 488 nm argon-ion laser and 525-, 575-, and 675-nm bandpass emission filters. Acquisition was performed using Expo32 version 1.2 software (Beckman Coulter), collecting both compensated data and uncompensated data. Analysis of compensated data was performed using Expo32 version 1.2 software. Uncompensated data analysis was performed using Win List 5.0 3D software (Verity Software House, Topsham, ME).
In forming a complex between Zenon labeling reagents and antibodies (Fig. 1), the molar ratio (MR) of the Zenon labeling reagent to the primary antibody must be selected. A MR of 3, i.e., 3 Fab fragments (∼50,000 MW each) to 1 primary antibody (∼150,000 MW), is equivalent to a mass ratio of 1:1 (Fab fragment to IgG target). Each complex is formed by adding 1 μg primary antibody to the Zenon labeling reagent of the appropriate IgG type (i.e., 5 μl (1 μg) of the Fab fragment at 200 μg/ml for MR3, or 8.5 μl for MR5).
To determine the optimal MR for combined antibody testing, assays were performed with Zenon Alexa Fluor 488 Mouse IgG1 Labeling Reagent complexed with 1 μg CD4 and CD8 antibodies at molar ratios of 2 through 10. Each complex was tested individually, and the resulting histograms were overlaid.
Initial testing for differentiating targets based on fluorescence intensity combining two colors was performed using the following complexes and molar ratios: CD4 at MR2 and CD8 at MR8 complexed with the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Reagent, and CD3 at MR3 complexed with the Zenon R-PE Mouse IgG1 Labeling Reagent. Complexes were tested in one, two, and three antibody combinations per tube.
For fluorescence-intensity multiplex testing, complexes were made using 1 μg monoclonal antibody combined with Zenon labeling reagents at various molar ratios, staining cells in one tube for simultaneous testing: Zenon Alexa Fluor 488 Mouse IgG1 Labeling Reagent with CD19 at MR3, CD4 at MR4, CD8 at MR4, and CD16 at MR14; Zenon R-PE Mouse IgG1 Labeling Reagent with CD56 at MR6 and CD3 at MR3; Zenon R-PE Mouse IgG2b Labeling Reagent with CD20 at MR14; Zenon Alexa Fluor 647-R-PE Labeling Reagent with CD45 at MR3. CD45/SS gating on lymphocytes was used.
The Zenon labeling reagent and 1 μg of the primary antibody were combined at the desired MR. Complexes were incubated for 5 min at RT. Zenon blocking reagent (mouse IgG) was then added to each complex in the same volume as Zenon mouse IgG labeling reagent used, and incubated 5 min at RT. All Zenon reagent-labeled complexes were added to 100 μl of cell suspension, incubated 30 min at RT, washed once in PBS, and resuspended in 1% formaldehyde/PBS (8). A lymphocyte gate was made on CD45/SS and 50,000 events were collected (10–13). The IgG1 isotype complexed with Zenon Alexa Fluor 488 Mouse IgG1 Labeling Reagent at MR14 and the IgG2b isotype complexed with Zenon R-PE Mouse IgG2b Labeling Reagent at MR14 served as negative controls (8, 10, 11). Single-color compensation controls were generated using the primary antibody complexed with the Zenon labeling reagent at the highest MR for each fluorophore used; standard compensation procedures were followed. (8, 10, 11). Data analysis using Expo32 used simple regions around distinct multivariate cell populations. Win List data analysis used regions around distinct multivariate cell populations to define subsets, with individual marker values then determined using Boolean Gate Analysis. In this analysis, multivariate populations were gated and Boolean expressions were applied to define populations of interest (14). Subsequent color gating was used to assign an individual color to each discrete population. Statistical interpretation was performed using Microsoft Excel 2000 and the Analysis Toolpack Add-in.