Recent Developments in Sensitive Multiparameter Flow Cytometry to detect PNH Red and White Blood Cells

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1 Recent Developments in Sensitive Multiparameter Flow Cytometry to detect PNH Red and White Blood Cells D. Robert Sutherland* Professor Dept. of Medicine University of Toronto Technical Director, Clinical Flow Cytometry Laboratory Medicine Program, Department of Pathology Toronto General Hospital/University Health Network New England Cytometry Meeting October * Consultant Alexion Pharmaceuticals

2 What is PNH?

3 Paroxysmal Nocturnal Hemoglobinuria (PNH): A Chronic, Systemic, and Life-Threatening Disease Progressive disease 2 4 Uncontrolled complement activation underlies the morbidities and mortality Prevalence: 15.9 / million 1 Diagnosed at all ages Median age early 30s 3,4 Despite best supportive care 5 year mortality: 35% 2 Quality of life severely impacted Patients Surviving (%) Actuarial Survival From the Time of Diagnosis in 80 Patients With PNH 2 Years After Diagnosis Age- and Gender- Matched Controls Patients With PNH The expected survival of an age- and gender-matched control group is shown for comparison (Hillmen et al. 1995) 1. Hill A et al. Blood 2006;108:290a. Abstract 985; 2. Hillmen P et al. N Engl J Med 1995;333: ; 3. Nishimura JI et al. Medicine 2004;83: ; 4. Socié G et al. Lancet 1996;348:

4 PNH is much more than an Anemia Normal red blood cells (RBCs) are protected from complement attack by a shield of terminal complement inhibitors Without this protective complement inhibitor shield, PNH RBCs are destroyed Complement Activation Lack of Bound CD55, CD59 Leads to Uncontrolled Complement Activation Intact RBC Reduced Red Cell Mass Free Anemia Hemoglobin 1. International PNH Interest Group. Blood 2005;106: Brodsky R Paroxysmal Nocturnal Hemoglobinuria. In: Hematology - Basic Principles and Practices. 4th ed. R Hoffman; EJ Benz; S Shattil et al. eds. Philadelphia, PA: Elsevier Churchill Livingstone;2005; Rother RP et al. JAMA 2005;293: Socie G et al. Lancet 1996;348: Hill A et al. Br J Haematol 2007;137:

5 Chronic Uncontrolled Complement Activation Leads to Devastating Consequences Thrombosis Renal Failure Complement Activation Elevated LDH Pulmonary Hypertension Abdominal Pain Chest Pain Dyspnea Significant Impact on Survival Free Hemoglobin Decreased NO Dysphagia Fatigue Hemoglobinuria Significant Impact on Morbidity Erectile Dysfunction LDH = lactate dehydrogenase. 1. International PNH Interest Group. Blood 2005;106: ; 2. Brodsky R. Paroxysmal nocturnal hemoglobinuria. In: R Hoffman et al, eds. Hematology - Basic Principles and Practices. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005; ; 3. Rother RP et al. JAMA. 2005;293: ; 4. Socie G et al. Lancet 1996;348: ; 5. Hill A et al. Br J Haematol 2007;137: ; 6. Lee JW et al. Hematologica 2010;95(s2): Abstracts 505 and 506; 7. Hill A et al. Br J Haematol 2010;149: ; 8. Hillmen P et al. Am J Hematol 2010;85:

6 MOLECULAR DEFECT IN PNH: A MUTATION OCCURS IN THE PIG-A GENE IN A HEMATOPOIETIC STEM CELL T LYMPHOCYTES STEM CELL B LYMPHOCYTES ERYTHROCYTES GRANULOCYTES MONOCYTES MUTATED PIG-A GENE PLATELETS

7 BIOCHEMICAL DEFECT IN PNH: FAILURE TO MAKE GPI-ANCHORED STRUCTURES NH 2 CD14 CD16 CD24 CD48 CD52 CD55 CD58 CD59 CD66 CD73 CD90 CD108 CD109 CD157 Man Etn GPI-linked protein Man Man Gluc P In PIG-A enzyme The biochemical defect in PNH occurs at the first stage in GPI synthesis transfer of N-acetyl glucosamine from UDP N-acetyl glucosamine to ER-associated phosphatidylinositol. Cell Membrane Modified from S. Richards

8 CURRENT UNDERSTANDING OF PNH Rare Acquired Hematopoietic Stem Cell disease Prevalence ~16 per million (new cases: 1.3 per million per year) Molecular defect in PNH Somatic mutation of the X-linked Phosphatidyl Inositol Glycan complementation class a (PIG-A) gene Mutations can be mis-sense or non-sense (or both) Biochemical defect in PNH PIG-A gene encodes protein (enzyme) involved in 1st stage of Glyco-Phosphatidyl Inositol anchor biosynthesis Mutations result in partial or absolute failure to synthesize GPI-linked proteins/glycoproteins Lack of CD59 in particular and CD55 leads to complementmediated hemolysis of RBCs

9 Loss of CD55 & CD59 on RBCs and granulocytes required to diagnose PNH BIOCYTEX KITs used up to 2004 Limitations of REDQUANT/CELLQUANT Kits 1. Total time hours per patient sample 2. Burdensome to set up (2 different cell prep. regimens, Indirect Fluorescence, 7 tubes) 3. Not very sensitive (5-10%) 4. Data difficult to interpret (manual gating/analysis to determine RBC and Gran clone sizes) 5. Cellquant must be performed within 8 Hr of sample draw 6. Redquant must be performed within 24 Hr of sample draw

10 DIAGNOSING PNH WITH FLAER - what is it? Pro-aerolysin: 52-kDa protein secreted by Aeromonas sp. After proteolytic nicking at the cell surface Aerolysin is generated that lyses cells (Howard et al, J Bacteriol 1985;163:336) Aerolysin binds to the GPI moiety of GPI-linked molecules on the cell surface (Diep et al J Biol Chem 1998; 273:2355) nicking Binding to GPI lysis

11 DIAGNOSING PNH WITH FLAER PNH cells not lysed by Aerolysin (Brodsky et al Blood 1999; 93:1749) Alexa488-labeled inactive variant of pro-aerolysin (FLAER) Binds to but does not cause lysis of cells FLAER is more sensitive than CD59 or other individual mabs to GPI-linked structures and FLAER gives a more accurate assessment of the GPI anchor deficit in PNH (Brodsky et al (Am J Clin Path 2000;114:459) (Luider et al,1st in Canada!!!)

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14 FLAER assay more reliably quantifies size of PNH clone than analysis of CD59-negative RBCs 1,2 1. Sutherland DR et al. Cytometry Part B 2007; 72B: FLAER ASSAY SUMMARY FLAER with CD33, CD45 and CD14 allows the simultaneous detection of PNH clones in monocyte and neutrophil lineages 1 1 tube (45 min), sensitive (~1-2%), inexpensive, simple data analysis, range of 4-color instruments Granulocytes could be assessed up to 48 hours post-draw if kept cold

15 Key Reasons to Test for PNH? 35% of PNH patients die within 5 years of diagnosis, even with best supportive care Early detection may impact treatment regimen and outcomes for patients with PNH and Bone Marrow failure In BM failures, presence of small PNH clone predicts response to immuno-suppressive therapy 1 Small clones can grow over time The availability of an effective PNH treatment option (eculizumab/soliris TM warrants testing in high-risk patient populations Sugimori C et al. Blood 2006:107:

16 PNH CELLS DO NOT EXPRESS GPI-LINKED STRUCTURES CD55 = DAF CD59 = MIRL While lack of CD55 and CD59 are diagnostic of PNH, hemolysis is due to lack of CD59

17 Compelling Long Term Clinical Benefits of Soliris in PNH Patients Pre-Soliris from Time of Diagnosis in 80 Patients With PNH 1 PNH Patients on Soliris Compared with Age- and Gender-Matched Controls 2* Patients Surviving (%) Age- and gendermatched controls Patients with PNH Years After Diagnosis Age- and Gender-Matched Normal Population Soliris-treated PNH Population Patients at Risk: Time (Years) Despite best supportive care - 5 year mortality: 35% 1 Hazard Ratio = 2.24 (P=0.013) *Survival after 10-years is slightly inferior to controls with causes of death related to bone marrow failure and not hemolysis or thrombosis. Please see full prescribing information for Soliris (eculizumab). Soliris (eculizumab) Summary of Product Characteristics. Alexion Europe SAS; Hillmen P et al. N Engl J Med 1995;333: Hill A et al. Presented at the 54th Annual Meeting of the American Society of Hematology (ASH). December 8-11, 2012, Atlanta, GA. Abstract #3472. Appears in Blood 2012;120 (21).

18 Two Independent Int l Groups Recommend Flow Cytometric Testing of High Risk Patients for PNH Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry Michael J. Borowitz *, Fiona E. Craig, Joseph A. DiGiuseppe, Andrea J. Illingworth, Wendell Rosse, D. Robert Sutherland, Carl T. Wittwer, Stephen J. Richards, On behalf of the Clinical Cytometry Society Borowitz MJ et al. International Clinical Cytometry Society. Part B Clin Cytometry. 2010;78B: Parker C et al. International PNH Interest Group. Blood. 2005;106:

19 Coombs-Negative Hem A Hemoglobinuria AA RA-MDS Unexplained Cytopenias Unexplained VTE/ATE Identification of Patients at High Risk for PNH Coombs-Negative Hemolytic Anemia Hemoglobinuria AA RA-MDS Unexplained Cytopenia Unexplained Thrombosis (venous or arterial) Rule PNH In or Out Using High Sensitivity Flow Cytometry and Clinical Assessment Borowitz MJ et al. for International Clinical Cytometry Society. Part B Clin Cytometry. 2010;78B: Parker C et al. for International PNH Interest Group. Blood 2005;106:

20 Thrombosis in PNH The Silent Killer Thromboembolism (TE) is the leading cause of death 1 - Thrombosis accounts for 40-67% of deaths 2 - First thrombotic event can be fatal 2,3 - First TE increases risk for death 5-10 fold - Median time to first TE was 2.3 years from diagnosis 4 Thrombosis is not adequately managed with anticoagulation 2 1. International PNH Group et al. Blood 2005; 106(12): Hillmen et al, Blood 2007, 110: Audebert HJ et al. J Neurol, 2005, 52: De Latour. Blood. 2008;112:

21 LABORATORY DIAGNOSIS OF PNH WITH FLOW CYTOMETRY Method of choice for diagnosis of PNH since mid-1990s Ability to rapidly diagnose PNH by Flow Cytometry has led to improved patient management and prognosis BUT: The disease is rare; most labs have limited experience at PNH testing Many different non-standardized Flow Cytometric approaches exist Some EQA/PT surveys showed variable ability of labs to detect abnormal PNH populations There is a need for Consensus Guidelines

22 Guidelines for the Diagnosis and Monitoring of Paroxysmal Nocturnal Hemoglobinuria and Related Disorders by Flow Cytometry Michael J. Borowitz, Fiona E. Craig, Joseph A. DiGiuseppe, Andrea J. Illingworth, Wendell Rosse, D. Robert Sutherland, Carl T. Wittwer, and Stephen J. Richards; on behalf of the Clinical Cytometry Society Cytometry Part B. 2010;78B:

23 High-Sensitivity Detection and Monitoring of PNH ICCS provided guidelines for the diagnosis and monitoring of PNH by flow cytometry 1 However, the following were not addressed: Specific reagents/reagent cocktails 2 Associated detailed analytic strategies 2 Validation of sensitive, standardized methodologies 2 The development and validation of sensitive standardized methodologies are necessary to reliably detect PNH clones <0.1% 2 1. Borowitz MJ et al. Cytometry Part B. 2010;78B: Sutherland DR et al. Cytometry Part B. 2012;82B:

24 High-Sensitivity Flow Cytometry Is Needed for Accurate Diagnosis and Monitoring 40% of PNH+ Samples Show a Clone of <1% 1 1% Clone Size >1% 1. Movalia MK et al. Poster presented at 53rd Annual meeting of the ASH, San Diego, CA

25 Practical Guidelines for the High- Sensitivity Detection and Monitoring of Paroxysmal Nocturnal Hemoglobinuria Clones by Flow Cytometry D. Robert Sutherland, Michael Keeney, and Andrea Illingworth Cytometry Part B. 2012;82B:

26 Flow Cytometric Testing for PNH RBC Analysis

27 Flow Assay Development Considerations Before you Start: General Issues Identify Target cells and Target Structures What are the target populations in PNH? RBCs, Grans, Monos (Lymphs for internal controls) Is anything known about the structural characteristics of the molecules to be targeted? Will this constrain the choice of MAb or specific conjugates? Are the MAb clones available in desired conjugated form? Will the selected conjugates/cocktails work across different platforms? Slide courtesy of A. Illingworth and D.R. Sutherland.

28 Figure 2B from Sutherland DR et al. Am J Clin Pathol 132: 2009;

29 Figure 2C from Sutherland DR et al. Am J Clin Pathol 2009; 132:

30 CD235a Antibody/Conjugate Selection: RBC Gating Structures CD235a only available target for gating RBCs Structural issues CD235a highly negatively-charged sialo-glycoprotein CD235a clone issues All CD235 antibodies cause RBCs to aggregate titration essential Unexpected Clone Issues Some CD235a conjugates are not effective in this assay regardless of titration Conjugate Issues Negatively-charged conjugates such as FITC may minimize CD235a-induced RBC aggregation Slide courtesy of A. Illingworth and D.R. Sutherland.

31 CD235a gating essential for high-sensitivity RBC assays Type I = 95.2% Type II = 4.72% Type III = 0.02% False CD59- cells in Normal sample with routine CD59FITC Assay False CD59- cells removed by CD235a Gating Slide courtesy D.R. Sutherland

32 CD59 Antibody/Conjugate Selection: RBC Target Structures (GPI-linked) CD59 for phenotyping PNH RBCs (CD55 sub-optimal) Structural Issues None known Clone Issues Some cause more RBC aggregation than others Titration essential Conjugate Issues Some CD59PE conjugates are better than others at delineating PNH Type III, Type II and Type I phenotypes Some CD59 clones function adequately as FITC conjugates but not as PE conjugates (e.g., P282PE from BD) TEST AND VALIDATE EVERYTHING!! Slide courtesy of A. Illingworth and D.R. Sutherland.

33 Variability of CD59PE Clones Clone MEM43 Clone P282 Clone MEM43 Supplemental Figure 1 from Sutherland DR et al. Cytometry Part B. 2012;82B:

34 A Good CD59PE Conjugate Out-performs a Good CD59FITC Conjugate Clone p282 Clone MEM43 Supplemental Figure 3 from Sutherland DR et al. Cytometry Part B. 2012;82B:

35 Comparison of CD55 and CD59 Staining in RBCs Type III & Type II PNH Normal CD59-PE PNH CD55 cannot separate Type II from Type III RBCs Normal CD55-PE Slide courtesy of A. Illingworth.

36 High-sensitivity RBC assay: PNH Sample 1.5 ul CD235aFITC (clone KC16) ul CD59PE (clone MEM43) Cocktail: 15uL CD235a + 5uL CD uL PBS. Use 10uL/test Sutherland DR, Illingworth A, Keeney M, Richards, S. High-sensitivity detection of PNH Red Blood Cells, Red Cell Precursors and White Blood Cells. Current Protocols in Cytometry. In press 2014.

37 The Importance of Collecting TIME Parameter Top row: air bubbles entered sample port when sample ran out By collecting TIME as a parameter, the data collected prior to sample running out can be analyzed

38 The Importance of Washing RBCs PNH Clones Can Be Missed No wash steps in RBCs No PNH? Washed x 2 PNH clone present! Slide courtesy of A. Illingworth.

39 Importance of Racking 29.5% 0.4% 24.7% 0.6% Supplemental Figure 7 from Sutherland DR et al. Cytometry Part B. 2012;82B:

40 Troubleshooting: Poorly Stained Sample Dot-plots outperform Histograms Supplemental Figure 7 from Sutherland DR et al. Cytometry Part B. 2012;82B:

41 Troubleshooting: Too much Debris Dot-plots outperform Histograms Action required: Repeat Sample!!

42 Importance of Clean Washing Media Sample 1 - PNH? Sample 2 - PNH? Contaminated washing media only CD235a staining and analysis using dot plots prevents misdiagnosis Supplemental Figure 8 from Sutherland DR et al. Cytometry Part B. 2012;82B:

43 Determine RBC Assay Sensitivity Measure Assay Sensitivity Using Spiking Make serial dilutions of PNH sample with normal blood 1 Dilution Type III RBCs Sensitivity % Undiluted 348, % 1:10 17, % 1: % 1: % 1:10, % Sensitivity on PNH samples: 20 PNH RBC phenotypes/million Sutherland DR et al. Cytometry Part B. 2012;82B:

44 Establish Background Frequency of PNH RBCs in Normal Samples 1. Stain at least 10 normal samples 2. Collect at least 1 million CD235a+ RBCs 3. Use bivariate dot plots to measure frequency of Type III RBCs 4. Rate of Type III PNH RBC phenotypes in 10 normal samples - 4 per million (range 2-6) 5. Take part in QA/PT schemes (CAP, etc.) Sutherland DR et al. Cytometry Part B. 2012;82B:

45 High-Sensitivity RBC Assay: Summary 1 RBC gating antibody Need to carefully select CD235a clone for gating No other practical alternatives Need to select a CD235aFITC conjugate to minimize aggregation RBC GPI-antibody Need to select CD59 clone, such as MEM43 or OV9A2, that retains high S:N ratio after conjugation to PE No need for CD55!! 1 Sutherland DR et al. Cytometry Part B. 2012;82B:

46 High-Sensitivity RBC Assay: Summary 2 Aggregation Need to dilute CD235a and CD59 significantly to keep aggregates below 1%. Cocktailing strongly recommended 1 Washing and Racking Need to wash twice and rack hard before acquisition 1 Validate Assay Determine frequency of PNH cells in 10 normal samples 1 Establish sensitivity with spiking experiment 1 Event Count May need to collect 1,000,000 events to detect 0.01% PNH RBCs (100 Type III cells) Sutherland DR et al. Cytometry Part B. 2012;82B:

47 Recommended Antibody Panels for PNH RBC Detection (All instruments) Red Blood Cells: CD235a-FITC CD59-PE Target cells Antibodies and Conjugates Purpose Clone and Vendor CD59-PE GPI-linked for RBC OV9A2 (ebioscience) MEM-43 (Invitrogen) RBC KC16 (BC) CD235a-FITC Gating on RBC 10F7MN (ebio) JC159 (DAKO) Sutherland DR et al. Cytometry Part B. 2012;82B:

48 Flow Cytometric Testing for PNH WBC Analysis

49 Accurate Detection of PNH WBC Clones Granulocyte PNH clone probably gives most accurate estimate of PNH clone size 1 Monocyte clones can be determined in a second tube or if instrumentation permits, the same tube 1 Monocytes confirm granulocyte result, but because monocytes are less numerous, precision can be lower 1 Type II granulocytes and monocytes can occasionally be recognized but red cells are typically better for this purpose 1 Lymphocytes are not a suitable target for PNH assessment but are useful internal controls to confirm optimal instrument setup/compensation and monitor antibody conjugate performance 2 1. Borowitz MJ et al. Cytometry Part B. 2010;78B: Sutherland DR et al. Cytometry Part B. 2012;82B:

50 Recommended Panels WBC Essential to combine two GPI-linked markers to maximize sensitivity, one of which should be FLAER 1 FLAER/CD24 (or FLAER/CD157) are the most reliable reagent combinations to detect PNH granulocytes 1.2,3 FLAER/CD14 (or FLAER/CD157) are the most reliable reagent combinations to detect PNH monocytes 1,2,3 Light scatter and CD45 gating is used for debris exclusion and pattern recognition (to identify blasts, platelets, and other cell types when present) 2,3 Lineage-specific gating is critical to accurate high-sensitivity WBC assays 2,3 CD15 is superior to CD33 for granulocyte gating 2,3 CD64 is superior to CD33 for monocyte gating 2,3 1. Borowitz MJ et al. Cytometry Part B. 2010;78B: Sutherland DR et al. Cytometry Part B. 2012;82B: Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry Part B 2014;86B:

51 What Is FLAER? FLuorescent AERolysin Aerolysin is a pore-forming toxin (lysin) from Aeromonas hydrophila Binds to GPI-anchor, not protein moiety FLAER Alexafluor 488 -conjugated inactive pro-aerolysin 1 Binds to GPI-anchor but does not cause lysis New more stable liquid formulation α-cd24 α-cd14 α-cd157 FLAER FLAER FLAER CD24 CD14 CD Brodsky RA. Blood. 2009;113: Sutherland DR et al. Cytometry Part B. 2012;82B: Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry Part B 2014;86B: FLAER: Alexa fluor 488 proaerolysin [product information]. Victoria, BC, Canada: Pinewood Scientific Services Inc; Accessed August 7, 2012.

52 Antibody clone/conjugate assessment is important CD15 clone BC 80H5 vs BD HI98 CD64 clone 22 (Coulter) conjugates

53 PNH Granulocytes with CD15 gating 4-C Granulocyte cocktail: FLAER, CD24PE, CD15PECy5, CD45PECy7 Sutherland DR et al. Cytometry Part B. 2012;82B:

54 PNH Monocytes with CD64 gating 4-C Monocyte cocktail: FLAER, CD14PE, CD64PECy5, CD45PECy7 Sutherland DR et al. Cytometry Part B. 2012;82B:

55 Validation of High-Sensitivity WBC Assays Using Spiking Dilution PNH Granulocytes Sensitivity % NEAT 51, % 1: % 1: % 1: % 1:10, % 4-Color Granulocyte Assay Sensitivity Dilution PNH Monocytes Sensitivity % NEAT 12, % 1: % 1: % 1: % 1:10,000 ND ND 4-Color Monocyte Assay Sensitivity Sutherland DR et al. Cytometry Part B. 2012;82B:

56 The Importance of Good Compensation Before Compensation Adjustment After Compensation Adjustment Slide courtesy of D.R. Sutherland.

57 Frequency of PNH Phenotypes on Normal Neutrophils and Monocytes Granulocytes CD15 gating Monocytes CD64 gating Frequency = % (n=10) Frequency = % (n=10) Sutherland DR et al. Cytometry Part B. 2012;82B:

58 Fluorescence Minus Two - Grans

59 Validation of High-Sensitivity WBC Assays Use recommended antibody clones and conjugates Use standardized reagent cocktails, staining protocols and gating strategies Stain normal samples with 4-C cocktails to verify: Assay background on 10 normal samples Antibody staining performance Stain normal samples with 2-C FMT cocktails to verify: Instrument settings (voltage and compensation) Spike PNH blood into normal blood to determine assay sensitivity Share PNH samples and compare results with another laboratory s validated PNH protocol Join recognized PNH QA/PT scheme (CAP, NEQAS, etc) Slide courtesy of A. Illingworth and D.R. Sutherland.

60 Beyond the Practical Guidelines Are there better GPI-specific antibodies available than CD24 for granulocytes? CD14 for monocytes? CD66b for granulocytes extensively used before advent of FLAER only available as FITC conjugate (incompatible with FLAER) CD16 extensively used before advent of FLAER GPI-linked on Grans, Transmembrane on NK cells very bright on mature grans, absent from eosinophils potentially problematic on MDS samples CD157 not extensively studied brightly expressed on both Grans and Monos Slide courtesy DR Sutherland

61 Comparison of CD24PE, CD66bPE and CD157PE in 4-C Granulocyte assay Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry Part B 2014;86B:

62 Correlation of PNH Gran & Mono clone sizes detected with 4-C predicate and 5-C CD157-based assays Granulocytes Monocytes

63 5-Color CD157-based assay for High Sensitivity Granulocytes and Monocytes PNH SAMPLE Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry Part B 2014;86B:

64 PNH Granulocyte and Monocyte clone sizes detected with 4-C predicate and 5-C CD157-based assays Granulocytes Monocytes Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry Part B 2014;86B:

65 Sensitivity of CD157-based Granulocyte and Monocyte assays Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection. Cytometry Part B 2014;86B: 44-55

66 Background on normal sample with 5-Color CD157-based assay Granulocytes = % Monocytes = % (n = 10) Sutherland DR et al, Cytometry Part B 2014;86B:

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68 Summary of Optimal Clones and Conjugates for High Sensitivity Granulocyte and Monocyte Assays

69 Recommended Antibody Panels for PNH WBC Testing Beckman Coulter Instruments 4-color Granulocytes: FLAER CD24-PE CD15-PC5 CD45-PC7 4-Color Monocytes (Reflex): FLAER CD14-PE CD64-PC5 CD45-PC7 5-Color Grans and Monos: FLAER CD157-PE CD64-ECD CD15-PC5 CD45-PC7 Target cells Antibodies and Conjugates Purpose Clone (Vendor) FLAER-Alexa488 GPI-linked (Grans and Monos) NA (Cedarlane) CD24-PE GPI-linked (Grans) SN3 (ebio), ALB9 (BC) WBC CD14-PE GPI-linked (Monos) 61D3 (ebio), RMO52 (BC) CD157-PE GPI-linked (Grans and Monos) SY11B5 (ebio) CD64-PC5 CD64-ECD for 5C Gating on Monocytes Gating on Monocytes 22 (BC) 22 (BC) CD15-PC5 Gating on Granulocytes 80H5 (BC) CD45-PC7 Debris exclusion and pattern recognition J33 (BC) Slide courtesy A Illingworth and DR Sutherland

70 5C CD157-based assay on 3-laser BD Canto II

71 Recommended Antibody Panels for PNH WBC Testing: BD Biosciences Instruments 4-color Granulocytes: FLAER CD24-PE CD45-PerCP CD15-APC 4-Color Monocytes (Reflex): FLAER CD14-PE CD45-PerCP CD64-APC 5-Color Grans and Monos 1 : FLAER CD157-PE CD45-PerCP CD64-APC CD15-eF450 5-Color Grans and Monos 2 : FLAER CD157-PE CD45-APC-H7 CD64-APC CD15-PerCP-eF710 Target cells Antibodies and Purpose Clone (Vendor) Conjugates FLAER-Alexa488 GPI-linked NA (Cedarlane) CD24-PE GPI-linked (Grans) SN3 (ebio), ML5 (BD) WBC CD14-PE GPI-linked (Monos) 61D3 (ebio), MOP9 (BD) CD157-PE GPI-linked (Grans and Monos) SY11B5 (ebio) CD45-PerCP Debris exclusion and pattern 2D1 (BD) recognition CD15-APC Gating for Granulocytes HI98 (BD) CD64-APC Gating for Monocytes (BD, ebio) CD15-eFluor 450 (5C) CD15-v450 (5C) CD15-PerCP-eFluor710 Gating for Granulocytes MMA (ebio) MMA (BD) Slide courtesy A Illingworth and DR Sutherland 1. For 3-laser Canto II 2. For 2-laser Canto

72 Key team Members: Acknowledgements Andrea Illingworth: Dahl-Chase Diagnostic Services, Bangor, ME Mike Keeney: London Health Sciences Centre, London, Ontario Erica Acton: University Health Network Toronto Mike Borowitz, Steve Richards and ICCS writing team Juan Azcona-Olivera: R&D Systems Angela Salazaar, Joanne Dimitrakopoulos: ebioscience Liam Whitby, Matt Fletcher, David Barnett: UK NEQAS Gerard Fernandes and colleagues: Alexion Canada Dan Hayden and colleagues: Alexion US