High-Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells
UNIT 6.37
D. Robert Sutherland,1,5 Andrea Illingworth,2 Michael Keeney,3 and Stephen J. Richards4 1
Laboratory Medicine Program, University Health Network/Toronto General Hospital, Toronto, Ontario, Canada 2 Dahl-Chase Diagnostic Services, Bangor, Maine 3 Pathology and Laboratory Medicine, London Health Sciences Centre, London, Ontario, Canada 4 Haematological Malignancy Diagnostic Service, Department of Clinical Haematology, St. James University Hospital, Leeds, United Kingdom 5 Contact author.
Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and interlaboratory performance characteristics even in laboratories with little prior C 2015 by John Wiley & Sons, Inc. experience performing PNH testing. Keywords: PNH r high-sensitivity flow r RBCs r WBCs
How to cite this article: Sutherland, D.R., Illingworth, A., Keeney, M., and Richards, S.J. 2015. High-sensitivity detection of PNH red blood cells, red cell precursors, and white blood cells. Curr. Protoc. Cytom. 72:6.37.1-6.37.29. doi: 10.1002/0471142956.cy0637s72
INTRODUCTION Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol glycan complementation Class A (PIG-A) gene (Takeda et al., 1993). In normal individuals, this gene encodes an enzyme involved in the first stage of glycophosphatidyl-inositol (GPI) biosynthesis. In PNH, as a result of the mutation(s) in the PIG-A 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 Phenotypic Analysis Current Protocols in Cytometry 6.37.1-6.37.29, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142956.cy0637s72 C 2015 John Wiley & Sons, Inc. Copyright
6.37.1 Supplement 72
(RBCs) and white blood cells (WBCs) (Nicholson-Weller et al., 1983; Holguin et al., 1989). Absence of CD59, in particular (Yamashina et al., 1990), 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 or hypoplastic anemia in which normal hematopoiesis has failed, and, with modern, high-sensitivity assays, up to 70% of AA patients have detectable PNH clones (Parker et al., 2005). Small populations of GPI-deficient PNH phenotypes have been reported in patients with early-stage myelodysplastic syndrome (MDS), particularly the refractory cytopenias with unilineage dysplasia [RCUD] variant (Dunn et al., 1999; Raza et al., 2014). 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 (Hillmen et al., 1995; Parker et al., 2005). As PNH is an acquired stem cell disease, it is important to demonstrate deficiency of GPI-linked cell surface structures in at least two hematopoietic cell lineages, traditionally RBCs and neutrophils, although monocytes can also be used. 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 wide variation from patient to patient, and delineation between the various types is not always clear-cut (Richards et al., 2000). RBC analysis is important in PNH, as accurate determination of the distribution of Type I, II, and III cells can predict clinical phenotype in that patients with greater than 20% Type III RBCs almost always show clinical evidence of hemolysis (reviewed in Parker et al., 2005). There have been reports in the past in which neither neutrophil nor red cell assays could be performed in situations where patients were severely neutropenic or where patients had received multiple red cell transfusions for severe anemia and/or hemolysis (van der Schoot et al., 1990). However, with the recent introduction of highly sensitive flow assays for neutrophils, monocytes, and red cells, it is unlikely that PNH clones would go undetected in such patients. With the advent of complement blockade therapy for patients with hemolytic PNH and the subsequent improved outcome, transfusion requirements, quality of life, and reduction in thrombotic risk, flow cytometry monitoring of PNH populations in patients has become increasingly important (Hillmen et al., 2004). One of the consequences of blocking complement C5 in these patients is that deposition/opsonization of C3d occurs on PNH red cells and can lead to extravascular hemolysis in some patients (Hill et al., 2010). A method (Alternate Protocol 1) is included that allows simultaneous demonstration of PNH among immature red cells and complement deposition on RBCs. This assay forms an essential part of monitoring patients on Eculizumab therapy in Stephen J. Richards’ laboratory, but is not recommended as a general screening test for PNH clones, where the use of Basic Protocol 1 is strongly recommended. Protocols are provided for high-sensitivity detection of PNH RBCs (Basic Protocol 1), PNH reticulocytes (Alternate Protocol 1), PNH neutrophils (Basic Protocol 2), and PNH monocytes (Basic Protocol 3), as well as both PNH neutrophils and PNH monocytes (Alternate Protocol 2). All WBC assays take advantage of the use of internal control populations of lymphocytes to monitor instrument, reagent, and assay performance. Detecting PNH by Flow Cytometry
6.37.2 Supplement 72
Current Protocols in Cytometry
HIGH-SENSITIVITY DETECTION OF PNH RED BLOOD CELLS While the loss of GPI-linked CD55 and CD59 was traditionally used to detect PNH RBCs (van der Schoot et al., 1990; Hall and Rosse, 1996), ‘routine’ CD55 and/or CD59based approaches are neither accurate nor sensitive below the 1% to 2% clone size, rendering them inadequate to detect small PNH clones typically found in PNH+ AA and MDS cases (Parker et al., 2005) or even in some heavily-transfused PNH cases. For high-sensitivity RBC analysis, the ICCS Guidelines recommended the use of CD235a (for RBC gating) and CD59 (to detect GPI-deficient cells) (Borowitz et al., 2010). Due to a lack of published empirical evidence, it was problematic to identify conjugates of CD235a and CD59 that 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 (Sutherland et al., 2009). In the follow-up ‘Practical Guidelines’ (Sutherland et al., 2012), a number of clones/conjugates to CD235a-FITC and CD59-PE were tested and validated for a variety of instrument platforms. Of note, selected conjugates required extensive titration on an individual basis to minimize aggregation prior to admixing or ‘cocktailing’ for this assay.
BASIC PROTOCOL 1
Red blood cells are analyzed by a series of bivariate dot plots of TIME versus side scatter (SS), forward (FS) versus side scatter with detectors set in logarithmic mode, CD235a versus FS (to gate RBCs, and to quantify and exclude any remaining RBC aggregates), and finally CD59 versus CD235a. TIME is collected as a parameter (as is recommended for all clinical flow assays), so that if fluidics problems are encountered during data acquisition, the sample can be re-acquired if possible, or if not, data acquired prior to the fluidics issue can be ‘gated’ and only that portion of the data file subsequently analyzed. Bivariate dot plots are recommended over single-parameter histograms, especially for samples containing small numbers of bona fide PNH phenotypes, for identifying poorlystained samples that need to be re-stained, and for detecting media contamination and troubleshooting instrumentation issues (Sutherland et al., 2012 and supplementary data).
Materials EDTA (preferred) or heparin anti-coagulated peripheral blood sample
UNIT 6.37
D. Robert Sutherland,1,5 Andrea Illingworth,2 Michael Keeney,3 and Stephen J. Richards4 1
Laboratory Medicine Program, University Health Network/Toronto General Hospital, Toronto, Ontario, Canada 2 Dahl-Chase Diagnostic Services, Bangor, Maine 3 Pathology and Laboratory Medicine, London Health Sciences Centre, London, Ontario, Canada 4 Haematological Malignancy Diagnostic Service, Department of Clinical Haematology, St. James University Hospital, Leeds, United Kingdom 5 Contact author.
Flow cytometry is the method of choice to ‘diagnose’ paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and interlaboratory performance characteristics even in laboratories with little prior C 2015 by John Wiley & Sons, Inc. experience performing PNH testing. Keywords: PNH r high-sensitivity flow r RBCs r WBCs
How to cite this article: Sutherland, D.R., Illingworth, A., Keeney, M., and Richards, S.J. 2015. High-sensitivity detection of PNH red blood cells, red cell precursors, and white blood cells. Curr. Protoc. Cytom. 72:6.37.1-6.37.29. doi: 10.1002/0471142956.cy0637s72
INTRODUCTION Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol glycan complementation Class A (PIG-A) gene (Takeda et al., 1993). In normal individuals, this gene encodes an enzyme involved in the first stage of glycophosphatidyl-inositol (GPI) biosynthesis. In PNH, as a result of the mutation(s) in the PIG-A 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 Phenotypic Analysis Current Protocols in Cytometry 6.37.1-6.37.29, April 2015 Published online April 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142956.cy0637s72 C 2015 John Wiley & Sons, Inc. Copyright
6.37.1 Supplement 72
(RBCs) and white blood cells (WBCs) (Nicholson-Weller et al., 1983; Holguin et al., 1989). Absence of CD59, in particular (Yamashina et al., 1990), 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 or hypoplastic anemia in which normal hematopoiesis has failed, and, with modern, high-sensitivity assays, up to 70% of AA patients have detectable PNH clones (Parker et al., 2005). Small populations of GPI-deficient PNH phenotypes have been reported in patients with early-stage myelodysplastic syndrome (MDS), particularly the refractory cytopenias with unilineage dysplasia [RCUD] variant (Dunn et al., 1999; Raza et al., 2014). 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 (Hillmen et al., 1995; Parker et al., 2005). As PNH is an acquired stem cell disease, it is important to demonstrate deficiency of GPI-linked cell surface structures in at least two hematopoietic cell lineages, traditionally RBCs and neutrophils, although monocytes can also be used. 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 wide variation from patient to patient, and delineation between the various types is not always clear-cut (Richards et al., 2000). RBC analysis is important in PNH, as accurate determination of the distribution of Type I, II, and III cells can predict clinical phenotype in that patients with greater than 20% Type III RBCs almost always show clinical evidence of hemolysis (reviewed in Parker et al., 2005). There have been reports in the past in which neither neutrophil nor red cell assays could be performed in situations where patients were severely neutropenic or where patients had received multiple red cell transfusions for severe anemia and/or hemolysis (van der Schoot et al., 1990). However, with the recent introduction of highly sensitive flow assays for neutrophils, monocytes, and red cells, it is unlikely that PNH clones would go undetected in such patients. With the advent of complement blockade therapy for patients with hemolytic PNH and the subsequent improved outcome, transfusion requirements, quality of life, and reduction in thrombotic risk, flow cytometry monitoring of PNH populations in patients has become increasingly important (Hillmen et al., 2004). One of the consequences of blocking complement C5 in these patients is that deposition/opsonization of C3d occurs on PNH red cells and can lead to extravascular hemolysis in some patients (Hill et al., 2010). A method (Alternate Protocol 1) is included that allows simultaneous demonstration of PNH among immature red cells and complement deposition on RBCs. This assay forms an essential part of monitoring patients on Eculizumab therapy in Stephen J. Richards’ laboratory, but is not recommended as a general screening test for PNH clones, where the use of Basic Protocol 1 is strongly recommended. Protocols are provided for high-sensitivity detection of PNH RBCs (Basic Protocol 1), PNH reticulocytes (Alternate Protocol 1), PNH neutrophils (Basic Protocol 2), and PNH monocytes (Basic Protocol 3), as well as both PNH neutrophils and PNH monocytes (Alternate Protocol 2). All WBC assays take advantage of the use of internal control populations of lymphocytes to monitor instrument, reagent, and assay performance. Detecting PNH by Flow Cytometry
6.37.2 Supplement 72
Current Protocols in Cytometry
HIGH-SENSITIVITY DETECTION OF PNH RED BLOOD CELLS While the loss of GPI-linked CD55 and CD59 was traditionally used to detect PNH RBCs (van der Schoot et al., 1990; Hall and Rosse, 1996), ‘routine’ CD55 and/or CD59based approaches are neither accurate nor sensitive below the 1% to 2% clone size, rendering them inadequate to detect small PNH clones typically found in PNH+ AA and MDS cases (Parker et al., 2005) or even in some heavily-transfused PNH cases. For high-sensitivity RBC analysis, the ICCS Guidelines recommended the use of CD235a (for RBC gating) and CD59 (to detect GPI-deficient cells) (Borowitz et al., 2010). Due to a lack of published empirical evidence, it was problematic to identify conjugates of CD235a and CD59 that 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 (Sutherland et al., 2009). In the follow-up ‘Practical Guidelines’ (Sutherland et al., 2012), a number of clones/conjugates to CD235a-FITC and CD59-PE were tested and validated for a variety of instrument platforms. Of note, selected conjugates required extensive titration on an individual basis to minimize aggregation prior to admixing or ‘cocktailing’ for this assay.
BASIC PROTOCOL 1
Red blood cells are analyzed by a series of bivariate dot plots of TIME versus side scatter (SS), forward (FS) versus side scatter with detectors set in logarithmic mode, CD235a versus FS (to gate RBCs, and to quantify and exclude any remaining RBC aggregates), and finally CD59 versus CD235a. TIME is collected as a parameter (as is recommended for all clinical flow assays), so that if fluidics problems are encountered during data acquisition, the sample can be re-acquired if possible, or if not, data acquired prior to the fluidics issue can be ‘gated’ and only that portion of the data file subsequently analyzed. Bivariate dot plots are recommended over single-parameter histograms, especially for samples containing small numbers of bona fide PNH phenotypes, for identifying poorlystained samples that need to be re-stained, and for detecting media contamination and troubleshooting instrumentation issues (Sutherland et al., 2012 and supplementary data).
Materials EDTA (preferred) or heparin anti-coagulated peripheral blood sample
