**2.2. White blood cells**

#### *2.2.1. Sample and reagent requirements*

For high-sensitivity WBC analysis using a single tube approach, CD45 is employed for pattern recognition and to exclude unlysed RBCs, other debris not excluded by light scatter thresholding. Thereafter, carefully selected/validated conjugates of CD15 and CD64 are used to accurately delineate/'gate' neutrophils and monocytes, respectively. To detect GPI-deficient CD15-gated neutrophils, FLAER is used in combination with either carefully selected/validated CD24 or CD157 conjugates, and to detect GPI-deficient CD64-gated monocytes, FLAER is used in combination with either carefully selected/validated CD14 or CD157 conjugates (**Table 2** and **3**) [13–16].


staining tube, taking all the precautions noted above for the RBC assay to avoid aerosols and ensure that the sample is not left on the wall of the tube. The reagent set in use should be cocktailed, once optimal volumes of each reagent have been determined by titrations. The appropriate volume of cocktail is added directly into the blood sample at the bottom of the tube and gently admixed by up-and-down pipetting. After gentle swirling to avoid aerosols, the sample is incubated in the dark for 20–30 minutes at room temperature before RBC lysis. There are a variety of commercial lysing agents available such as Versalyse, FACSLyse and Immunoprep and most do a very good job. After lysis following manufacturers' recommendations, the sample is centrifuged and washed with PBS supplemented with 1% serum albu-

Accurate and High Sensitivity Identification of PNH Clones by Flow Cytometry

http://dx.doi.org/10.5772/intechopen.71286

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Optimal instrument setup and standardization is a prerequisite for reproducible results over time and among laboratories. For the analysis of RBCs, the forward scatter (FS) and side scatter (SS) voltages are set in logarithmic mode and voltages adjusted to bring all unstained RBCs into the middle of the plot and above any FS threshold/discriminator. For WBCs, light scatter voltages are set in linear mode at such values that all unstained leukocyte subsets including lymphocytes scatter above the FS threshold and are clearly clustered on scale. Photomultiplier tube (PMT) voltage optimization, standardization and computer-assisted spectral overlap compensation are mandatory steps for instrument standardization of multiparameter assays and can be performed using an instrument platform-based approach (BD

For high-sensitivity RBC analysis, the ICCS Guidelines recommended the use of CD235a (for RBC gating) and CD59 (to detect GPI-deficient cells) [9]. Based on subsequent publications that included rigorous testing and validation of various CD235a and CD59 clones and conjugates, optimal reagent combinations of CD235a-FITC and CD59-PE were identified [13]. Once extensively titrated, these reagents in combination did not cause major aggregation of RBCs while still maintaining a good signal-to-noise ratio and the ability to adequately separate Type II and Type III PNH RBCs from normal (Type I) RBCs [13–15]. Red blood cells are analyzed by a series of gating dot plots beginning with TIME versus SS, FS versus SS with detectors set in logarithmic mode, and CD235a-FITC versus FS to gate singlet RBCs and to quantify and exclude any remaining RBC aggregates (**Figure 1**). TIME is collected as a parameter and monitored during acquisition so that if fluidics problems are encountered, the sample can be reacquired if possible, or if not, data acquired prior to the fluidics hiatus can be 'gated' and only that portion of the data file subsequently analyzed. It is important to adjust the threshold (discriminator) for the FS so that no RBCs are excluded from acquisition. The diagnostic plots include a bivariate CD59 versus CD235a dot plot, a bivariate CD59 versus CD235a density

min. The sample is resuspended in 0.5–1 ml of PBS and acquired.

Biosciences, Beckman Coulter) or interplatform-based approach [17–20].

**3. Analytical phase and data reporting**

**3.1. Instrument setup and standardization**

**3.2. Data acquisition and analysis**

*3.2.1. Red blood cells*

**Table 2.** Recommended clones/conjugates to determine high-sensitivity detection of PNH WBC on Beckman Coulter Cytometers.


**Table 3.** Recommended clones/conjugates for high-sensitivity detection of PNH WBC on Becton Dickinson Cytometers.

#### *2.2.2. Staining procedure*

Undiluted anti-coagulated whole blood is the preferred sample source for the analysis of PNH phenotypes in WBCs. Reverse pipetting is used to dispense 100 μL of sample into the staining tube, taking all the precautions noted above for the RBC assay to avoid aerosols and ensure that the sample is not left on the wall of the tube. The reagent set in use should be cocktailed, once optimal volumes of each reagent have been determined by titrations. The appropriate volume of cocktail is added directly into the blood sample at the bottom of the tube and gently admixed by up-and-down pipetting. After gentle swirling to avoid aerosols, the sample is incubated in the dark for 20–30 minutes at room temperature before RBC lysis. There are a variety of commercial lysing agents available such as Versalyse, FACSLyse and Immunoprep and most do a very good job. After lysis following manufacturers' recommendations, the sample is centrifuged and washed with PBS supplemented with 1% serum albumin. The sample is resuspended in 0.5–1 ml of PBS and acquired.
