**3.2. Data acquisition and analysis**

#### *3.2.1. Red blood cells*

*2.2.2. Staining procedure*

CD24-PE CD24-APC

CD24-PE CD24-APC

CD14-PE CD14-APC700

CD64-PC5 CD64-ECD CD64-PC7

CD15-PC5 CD15-PerCP-eF710 CD15-PerCPCy5.5

CD45-PC7 CD45-KO CD45-eF450

Cytometers.

CD14-PE CD14-APC

CD64-APC CD64-PECy7

CD15-APC CD15-PerCP-eF710 CD15-PerCPCy5.5

CD45-eF450 CD45-PerCP CD45-APC-H7

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

Debris/unlysed RBC exclusion + pattern

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

recognition

**Target Antibody Conjugates Purpose Clone (Vendor)** WBC FLAER-Alexa488 GPI-linked (Neuts + Monos) NA (Cedarlane)

recognition

**Target Antibody Conjugates Purpose Clone (Vendor)** WBC FLAER-Alexa488 GPI-linked (Neuts + Monos) NA (Cedarlane)

4 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

GPI-linked (Neuts) SN3 (eBio), ML5 (BD)

GPI-linked (Neuts) SN3 (eBio), ALB9 (BC)

GPI-linked (Monos) 61D3 (eBio), RMO52 (BC)

CD157-PE GPI-linked (Neuts + Monos) SY11B5 (eBio, EXBIO, BD, BC,

Gating on Monocytes 22 (BC)

Gating on Neutrophils 80H5 (BC)

Debris/unlysed RBC exclusion + pattern

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

GPI-linked (Monos) 61D3 (eBio),

Gating on Monocytes 10.1 (BD, eBio)

Gating on Neutrophils HI98 (BD)

CD157-PE GPI-linked (Neuts + Monos) SY11B5 (eBio, EXBIO, BD, BC,

SN3 (eBio, EXBIO)

SN3 (eBio, EXBIO)

Tuk4 (Invitrogen) RMO52 (BC)

22 (BC), 10.1 (EXBIO)

Sysmex)

22 (BC)

MMA (eBio) MEM158 (EXBIO)

J33 (BC) J33 (BC) 2D1 (eBio)

> Tuk4 (Invitrogen) MoP9 (BD)

10.1 (EXBIO), 22 (BC)

Sysmex)

MMA (eBio) MEM 158 (EXBIO)

2D1 (eBio) 2D1 (BD) 2D1 (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

*3.2.2.1. FLAER/CD24/CD14-based assay*

neutrophils and monocytes.

For laboratories equipped with modern cytometers with 6-, 8- or 10 PMTs (Canto, Canto II and Navios), it is possible to configure 6-color cocktails based on FLAER, CD24 and CD14 (**Tables 2** and **3**). **Figure 2** shows the sequence of bivariate gating and diagnostic dot-plots from a Naviosspecific reagent set comprising FLAER-Alexa488, CD24-PE (clone ALB9), CD15-PC5 (clone 80H5), CD64-PC7 (clone 22), CD14-APCA700 (clone RMO52) and CD45-KO (clone J33). The FS versus SS plot is gated from TIME versus SS plot (not shown) and light scatter voltages are set so that all WBC subsets are clearly visible and optimally separated; the threshold/discriminator is set to ensure that no lymphocytes are excluded. A debris exclusion gate (or WBC inclusion gate) can then be established to exclude any debris above the threshold/discriminator but below the

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**Figure 2.** Sequence of bivariate gating and diagnostic dot-plots for FLAER/CD24/CD14- based analysis of PNH

**Figure 1.** Sequence of bivariate gating and diagnostic dot-plots for analysis of PNH RBCs.

plot and a single parameter histogram of CD59 staining (**Figure 1**). Bivariate dot plots and/ or density plots are recommended over single-parameter histograms, especially for samples containing small numbers of PNH phenotypes, for identifying poorly stained samples that need to be re-stained and for detecting media contamination and troubleshooting instrumentation issues [13]. However, while data regarding clone sizes come predominantly from the two-dimensional plots, in which the gating regions are linked across the dot plot and density plots, the single parameter histogram can also be useful in some situations. All three plots work in concert for optimal adjustment of the regions for Type III PNH cells and Type II PNH cells. An additional utility of the single parameter histogram is in comparing old versus new plots of CD235a-FITC/CD59-PE cocktails when tested on non-PNH samples.

#### *3.2.2. White blood cells*

High-sensitivity methodologies to detect PNH phenotypes in neutrophils and monocytes have been published previously. These methods were initially based on two separate 4-color neutrophil (FLAER, CD24, CD15 and CD45) and monocyte (FLAER, CD14, CD64 and CD45) tubes. In this earlier setting, samples were stained first with the RBC and neutrophil cocktails and if PNH phenotypes were detected, the 'reflex' monocyte tube was thereafter set up. The current document uses the same gating strategy used in earlier assays but discuss the more modern single tube assays on newer flow cytometers with 5, 6, or more PMTs that allow the simultaneous detection and quantification of both neutrophils and monocytes.

#### *3.2.2.1. FLAER/CD24/CD14-based assay*

plot and a single parameter histogram of CD59 staining (**Figure 1**). Bivariate dot plots and/ or density plots are recommended over single-parameter histograms, especially for samples containing small numbers of PNH phenotypes, for identifying poorly stained samples that need to be re-stained and for detecting media contamination and troubleshooting instrumentation issues [13]. However, while data regarding clone sizes come predominantly from the two-dimensional plots, in which the gating regions are linked across the dot plot and density plots, the single parameter histogram can also be useful in some situations. All three plots work in concert for optimal adjustment of the regions for Type III PNH cells and Type II PNH cells. An additional utility of the single parameter histogram is in comparing old versus new

High-sensitivity methodologies to detect PNH phenotypes in neutrophils and monocytes have been published previously. These methods were initially based on two separate 4-color neutrophil (FLAER, CD24, CD15 and CD45) and monocyte (FLAER, CD14, CD64 and CD45) tubes. In this earlier setting, samples were stained first with the RBC and neutrophil cocktails and if PNH phenotypes were detected, the 'reflex' monocyte tube was thereafter set up. The current document uses the same gating strategy used in earlier assays but discuss the more modern single tube assays on newer flow cytometers with 5, 6, or more PMTs that allow the

plots of CD235a-FITC/CD59-PE cocktails when tested on non-PNH samples.

**Figure 1.** Sequence of bivariate gating and diagnostic dot-plots for analysis of PNH RBCs.

6 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

simultaneous detection and quantification of both neutrophils and monocytes.

*3.2.2. White blood cells*

For laboratories equipped with modern cytometers with 6-, 8- or 10 PMTs (Canto, Canto II and Navios), it is possible to configure 6-color cocktails based on FLAER, CD24 and CD14 (**Tables 2** and **3**). **Figure 2** shows the sequence of bivariate gating and diagnostic dot-plots from a Naviosspecific reagent set comprising FLAER-Alexa488, CD24-PE (clone ALB9), CD15-PC5 (clone 80H5), CD64-PC7 (clone 22), CD14-APCA700 (clone RMO52) and CD45-KO (clone J33). The FS versus SS plot is gated from TIME versus SS plot (not shown) and light scatter voltages are set so that all WBC subsets are clearly visible and optimally separated; the threshold/discriminator is set to ensure that no lymphocytes are excluded. A debris exclusion gate (or WBC inclusion gate) can then be established to exclude any debris above the threshold/discriminator but below the

**Figure 2.** Sequence of bivariate gating and diagnostic dot-plots for FLAER/CD24/CD14- based analysis of PNH neutrophils and monocytes.

smallest lymphocytes. The CD45 versus SS plot is then gated through Boolean gating on Time and "not debris" with a gate drawn around the CD45+ cells. The CD45 versus SS plot is useful not only for pattern recognition but also for excluding any unlysed RBCs and other debris not removed by the debris exclusion gate. The CD15 versus SS plot is gated on the CD45+ populations and includes a gate drawn around the CD15++ neutrophils excluding as well as possible the CD15 dim + eosinophils visible to the left of the neutrophil/granulocyte population. The diagnostic FLAER/CD24 plot is gated on the CD15++ neutrophils and a region is drawn to encompass the FLAER-negative/CD24-negative cells, which represent the PNH neutrophils. The CD64 versus SS plot is also gated on the CD45+ cells and a region is drawn around the CD64++ monocytes. The FLAER versus CD14 dot plot is gated on the CD64++ monocytes and a region is drawn to delineate the FLAER-negative/CD14-negative cells, which represent the PNH monocytes. The lymphocytes gated on the CD64-negative/low SS plot are not a suitable target population for the PNH clone quantification due to their long lifespan. However, they serve as internal controls for verification of antigen expression and compensation settings. Plotting FLAER versus CD24, CD14, CD15 and CD64 verifies the instrument voltage and compensation settings as visible and clustered populations in the "correct" location. Plot FLAER versus CD24 shows B-cells (FLAER+/CD24+) verifying that both reagents were added, FLAER+/CD24 negative NK and T-cells and no dual-negative cells as this is a PNH-negative sample.

#### *3.2.2.2. FLAER/CD157-based assay*

ADP-ribosyl cyclase 2 (CD157) is a GPI-anchored cell surface enzyme encoded by the bone marrow stromal cell antigen-1 gene, which plays a role in pre-B cell growth [21]. Within the hematopoietic system, CD157 is highly expressed on both mature neutrophils and monocytes [22] leading to the possibility that CD157 could replace both CD24 and CD14, allowing the development of a single tube, high sensitivity 5C assay to identify and quantify both PNH neutrophils and PNH monocytes on cytometers with five or more PMTs [14–16]. The ability to perform simultaneous evaluation of both PNH neutrophils and PNH monocytes is particularly attractive to laboratories equipped with 5-C instruments such as the FC500 due to the major cost and time savings involved over running two separate 4-color assays for neutrophils and monocytes [14–16]. The gating and analysis strategies are similar to the ones used for the above described single-tube 6-color assay, except for the diagnostic FLAER/CD157 dot plots gated on CD15++ neutrophils and CD64++ monocytes (**Figure 3**). Three control lymphocyte plots (FLAER/CD15, FLAER/CD64 and FLAER/CD157) are also shown to monitor instrument setup and compensation.

of FLAER, CD157, CD24, CD14, CD15, CD64 and CD45. While the CD157 failed to stain normal neutrophils in this sample, FLAER and CD24 stained the neutrophils in the expected manner. Similarly, while CD157 failed to stain normal monocytes in this sample, FLAER and

**Figure 3.** Sequence of bivariate gating and diagnostic dot-plots for FLAER/CD157- based analysis of PNH neutrophils

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**1.** The presence or absence of PNH clones. It is important to be clear and to avoid potentially misleading ambiguous terminology. A report stating that a CD59 test is negative may imply to some providers that the target population is negative for the GPI marker CD59 (thus indicating a PNH clone) or that absence of CD59 is not seen (thus indicating the absence

**2.** PNH clone size in the RBCs (total PNH clone size as well as the percentages for Type II and Type III PNH populations). There is a clinical significance associated for Type II and

CD14 stained monocytes in the expected manner.

The following components are recommended for a PNH report:

**3.3. Reporting**

and monocytes.

of a PNH clone).

It is important to note that several CD157-negative, non-PNH cases have been observed in the authors' laboratories (unpublished data). For these rare cases, the inclusion of the second GPI reagent (FLAER) as part of the built-in robustness of the assay prevents the misinterpretation of the data as a PNH clone-containing sample. Furthermore, in keeping with current state-of-the-art guidelines [12, 13], the RBC lineage should also be analyzed on every sample tested for the presence of PNH WBCs. As these rare CD157-negative non-PNH samples only contain normal (Type I) RBCs, there is even less chance of misinterpretation. An example of a CD157-negative case is shown in **Figure 4**. The sample was stained with a 7-color combination Accurate and High Sensitivity Identification of PNH Clones by Flow Cytometry http://dx.doi.org/10.5772/intechopen.71286 9

**Figure 3.** Sequence of bivariate gating and diagnostic dot-plots for FLAER/CD157- based analysis of PNH neutrophils and monocytes.

of FLAER, CD157, CD24, CD14, CD15, CD64 and CD45. While the CD157 failed to stain normal neutrophils in this sample, FLAER and CD24 stained the neutrophils in the expected manner. Similarly, while CD157 failed to stain normal monocytes in this sample, FLAER and CD14 stained monocytes in the expected manner.

#### **3.3. Reporting**

smallest lymphocytes. The CD45 versus SS plot is then gated through Boolean gating on Time and "not debris" with a gate drawn around the CD45+ cells. The CD45 versus SS plot is useful not only for pattern recognition but also for excluding any unlysed RBCs and other debris not removed by the debris exclusion gate. The CD15 versus SS plot is gated on the CD45+ populations and includes a gate drawn around the CD15++ neutrophils excluding as well as possible the CD15 dim + eosinophils visible to the left of the neutrophil/granulocyte population. The diagnostic FLAER/CD24 plot is gated on the CD15++ neutrophils and a region is drawn to encompass the FLAER-negative/CD24-negative cells, which represent the PNH neutrophils. The CD64 versus SS plot is also gated on the CD45+ cells and a region is drawn around the CD64++ monocytes. The FLAER versus CD14 dot plot is gated on the CD64++ monocytes and a region is drawn to delineate the FLAER-negative/CD14-negative cells, which represent the PNH monocytes. The lymphocytes gated on the CD64-negative/low SS plot are not a suitable target population for the PNH clone quantification due to their long lifespan. However, they serve as internal controls for verification of antigen expression and compensation settings. Plotting FLAER versus CD24, CD14, CD15 and CD64 verifies the instrument voltage and compensation settings as visible and clustered populations in the "correct" location. Plot FLAER versus CD24 shows B-cells (FLAER+/CD24+) verifying that both reagents were added, FLAER+/CD24-

8 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

negative NK and T-cells and no dual-negative cells as this is a PNH-negative sample.

ADP-ribosyl cyclase 2 (CD157) is a GPI-anchored cell surface enzyme encoded by the bone marrow stromal cell antigen-1 gene, which plays a role in pre-B cell growth [21]. Within the hematopoietic system, CD157 is highly expressed on both mature neutrophils and monocytes [22] leading to the possibility that CD157 could replace both CD24 and CD14, allowing the development of a single tube, high sensitivity 5C assay to identify and quantify both PNH neutrophils and PNH monocytes on cytometers with five or more PMTs [14–16]. The ability to perform simultaneous evaluation of both PNH neutrophils and PNH monocytes is particularly attractive to laboratories equipped with 5-C instruments such as the FC500 due to the major cost and time savings involved over running two separate 4-color assays for neutrophils and monocytes [14–16]. The gating and analysis strategies are similar to the ones used for the above described single-tube 6-color assay, except for the diagnostic FLAER/CD157 dot plots gated on CD15++ neutrophils and CD64++ monocytes (**Figure 3**). Three control lymphocyte plots (FLAER/CD15, FLAER/CD64 and FLAER/CD157) are also shown to monitor

It is important to note that several CD157-negative, non-PNH cases have been observed in the authors' laboratories (unpublished data). For these rare cases, the inclusion of the second GPI reagent (FLAER) as part of the built-in robustness of the assay prevents the misinterpretation of the data as a PNH clone-containing sample. Furthermore, in keeping with current state-of-the-art guidelines [12, 13], the RBC lineage should also be analyzed on every sample tested for the presence of PNH WBCs. As these rare CD157-negative non-PNH samples only contain normal (Type I) RBCs, there is even less chance of misinterpretation. An example of a CD157-negative case is shown in **Figure 4**. The sample was stained with a 7-color combination

*3.2.2.2. FLAER/CD157-based assay*

instrument setup and compensation.

The following components are recommended for a PNH report:


and monocytes may also show the presence of Type II populations but the clinical and biological significance of these populations has not been established at this time. It is therefore recommended to report only the total PNH clone size in the neutrophils and monocytes.

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**b.** PNH population 0.1–1%: "minor population of PNH cells" or "minor PNH clone."

**6.** Levels of the limit of quantification (LOQ) for the neutrophil assay and the RBC assay, stating the recommended LOQ of 0.05% or better for RBCs (100,000 gated cells) and 0.1% or better for neutrophils (50,000 gated cells). It is important to include this information to the provider as an LOQ of 1% means that the possibility of a minor clone (less than 1%) cannot

**7.** Histograms or dot plots if possible because the dot plots may provide powerful visual supportive evidence of the PNH clone and also provide evidence of the quality of the assay.

The results of PNH testing by flow cytometry are usually reported as percentage of type II and III PNH cells from the total gated neutrophils, monocytes and red blood cells. Assays reporting numeric data are considered as semi-quantitative, therefore the post-analytical validation process should comprise confirmation of accuracy, specificity, sensitivity, repeatabil-

The accuracy of a measurement is described by its trueness, which refers to the closeness of agreement between the average value of a large number of test results and the true or accepted reference value [25]. For PNH assays, we do not have cellular reference standard, therefore accuracy cannot be determined directly. Alternatively, interlaboratory comparison and/or external quality assessment represent the only available option for assay validation

The analytical specificity of PNH testing assays reflects the choice and validation of all anti-

**c.** PNH population < 0.1%: "rare cells with GPI deficiency" or "rare cells with PNH

**4.** Interpretive terminology of reporting PNH clones based on CSLI H52-A2 [23]:

**5.** List of all gating and diagnostic markers used for the PNH assay.

**a.** PNH population > 1%: "PNH clone."

phenotype.

be excluded based on this LOQ.

ity, reproducibility and stability [24, 25].

and mandatory step for ISO accreditation [26].

bodies/reagents and corresponding fluorochromes (**Tables 1**–**3**).

**4.1. Accuracy of PNH assays**

**4.2. Specificity of PNH assays**

*4.2.1. Analytical specificity*

**4. Post-analytical phase and assay validation**

**Figure 4.** Example of CD157-negative, non-PNH case, 7-color FLAER/CD24/CD14/CD157-based protocol.

Type III RBCs. Type I RBCs are normal red blood cells with bright CD59 expression and a lifespan of approximately 120 days. Type III PNH RBCs have complete CD59 deficiency, which results in no protection from complement-mediated lysis and a shortened lifespan of 10–15 days. Type II PNH RBCs have partial CD59 deficiency resulting in partial protection from complement-mediated lysis. Just as the expression of CD59 on Type II RBCs varies considerably from patient to patient, the lifespan of Type II cells reflects this being intermediate between Type I normal RBCs and Type III PNH RBCs. Since the clinical significance of Type II PNH RBCs and Type III PNH RBCs is well established, it is recommended to report them separately and combined as the total PNH RBC clone.

**3.** PNH clone size in both lineages for the WBCs (neutrophils and monocytes). The PNH monocyte clone is often larger than the neutrophil PNH clone and reporting only the PNH neutrophil/granulocyte clone may underestimate the PNH clone size in the WBCs. Neutrophils and monocytes may also show the presence of Type II populations but the clinical and biological significance of these populations has not been established at this time. It is therefore recommended to report only the total PNH clone size in the neutrophils and monocytes.

	- **a.** PNH population > 1%: "PNH clone."
	- **b.** PNH population 0.1–1%: "minor population of PNH cells" or "minor PNH clone."
	- **c.** PNH population < 0.1%: "rare cells with GPI deficiency" or "rare cells with PNH phenotype.
