**2.1. Red blood cells**

#### *2.1.1. Sample and reagent requirements*

Freshly drawn EDTA (preferred) or heparin anti-coagulated whole peripheral blood is used for analysis. If samples are shipped or need to be stored prior to analysis, the blood sample should be kept at 4°C and should generally be used within 48 hours of sample draw. For highsensitivity RBC analysis, the International Clinical Cytometry Society (ICCS) Guidelines recommended the use of CD235a (for RBC gating) and CD59 (to detect GPI-deficient cells) [12]. In the follow-up 'Practical Guidelines' [13], a large number of clones/conjugates to CD235a-FITC and CD59-PE were tested but only a few were found to have acceptable performance


**Table 1.** Recommended CD235a-FITC and CD59-PE conjugates for high-sensitivity PNH RBC assay.

characteristics and further validated for a variety of instrument platforms (**Table 1**). Of note, even selected conjugates required extensive titration on an individual basis to minimize aggregation prior to premixing or 'cocktailing' for this assay. Only by performing extensive titrations, we were able to identify conjugates with the best performance characteristics for this specific assay. Premixing of reagents once adequately titrated is critically important in PNH assays as both RBC and WBC assays are designed to detect GPI-deficient phenotypes, i.e., cells unstained by the GPI-specific reagents. Given the very small volumes of reagents used for the RBC assay in particular, it is usually necessary to make a dilution of the RBC cocktail such that accurately pipettable volumes of reagent can be employed.

#### *2.1.2. Staining procedure*

complementation Class A (PIG-A) gene [1]. PIG-A normally encodes an enzyme involved in the first stage of glycosylphosphatidylinositol (GPI) biosynthesis but in PNH, as a result of the mutation(s) in this gene, there is a partial or absolute inability to make GPI-anchored proteins, including complement defense structures such as CD55 and CD59 on red blood cells (RBCs) and white blood cells (WBCs) [2, 3]. Absence of CD59 in particular [4] and CD55 on RBCs is responsible for intravascular hemolysis associated with clinical PNH. Clonal expansion of the PNH population frequently occurs in patients with aplastic anemia in which normal hematopoiesis has failed, and with modern, high sensitivity assays, up to 70% of AA patients have detectable PNH clones [5]. Small populations of GPI-deficient PNH phenotypes have been reported in patients with early stage myelodysplastic syndrome (MDS) [6, 7]. Patients present with a wide range of clinical features, including intravascular hemolysis (that leads to hemoglobinuria), bone marrow failure and thrombosis, with the latter being a major cause of morbidity and mortality [5, 8]. As PNH is an acquired stem cell disease, it is important to demonstrate the loss of GPI-linked cell surface structures in at least two hematopoietic cell lineages, traditionally RBCs and neutrophils, although as more data have recently accumulated, monocytes should also be assessed as monocytes often exhibit a higher 'clone size' than is present in neutrophils. For true high-sensitivity assay design, it is critical to include carefully validated lineage-specific gating reagents such as CD235a (Glycophorin A) for RBC identification, CD15 for neutrophil identification and CD64 for monocyte identification. Examination of RBCs in the non-transfused PNH patient provides the most accurate assessment of the distribution of Type III PNH RBCs (complete CD59 deficiency), Type II PNH RBCs (partial CD59 deficiency) and normal Type I RBCs (normal CD59 expression). The distributions of these populations show a wide variation from patient to patient and delineation between the various types is not always clear-cut [9]. RBC analysis is important in PNH, as accurate determination of the distribution of Type II and III cells; patients with greater than 20% Type III RBCs almost always show clinical evidence of hemolysis [5]. While the loss of GPI-linked CD55 and CD59 was traditionally used to detect PNH RBCs [10, 11], 'routine' CD55 and/or CD59-based approaches are neither accurate nor sensitive below the 1–2% clone size, rendering them inadequate to detect small PNH clones typically found in PNH+ AA and MDS cases [5] or even in some heavily transfused PNH cases.

2 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

Freshly drawn EDTA (preferred) or heparin anti-coagulated whole peripheral blood is used for analysis. If samples are shipped or need to be stored prior to analysis, the blood sample should be kept at 4°C and should generally be used within 48 hours of sample draw. For highsensitivity RBC analysis, the International Clinical Cytometry Society (ICCS) Guidelines recommended the use of CD235a (for RBC gating) and CD59 (to detect GPI-deficient cells) [12]. In the follow-up 'Practical Guidelines' [13], a large number of clones/conjugates to CD235a-FITC and CD59-PE were tested but only a few were found to have acceptable performance

**2. Pre-analytical phase**

*2.1.1. Sample and reagent requirements*

**2.1. Red blood cells**

Blood samples are diluted 1:100 with fresh clean PBS and 100 μL is carefully pipetted using reverse pipetting techniques directly into the bottom of the staining tube taking care to avoid aerosols and blood trails on the inside of the tube. The appropriate volume of diluted CD235aFITC/CD59PE is then pipetted directly into the bottom of the tube and admixed with the diluted sample by gently up-and-down pipetting. After careful removal of the tip, the sample can be gently 'swirled' on a vortex set at a very low speed to avoid aerosol generation. After 20 minutes, the sample must be washed twice with clean phosphate buffered saline (PBS), resuspended in 1 ml of PBS and then 'racked' immediately before data acquisition to disrupt any RBC aggregates generated during the staining process [13].
