Practical Guidelines and Tips for Sensitive and Accurate Identification of PNH Clones

Practical Guidelines and Tips for Sensitive and Accurate Identification of PNH Clones AFCG Meeting Nov 28 Dec 1, 2013 Wellington, New Zealand Andrea Illingworth, MS, H(ASCP), QCYM Dahl-Chase Diagnostic Services, USA

Scope of Presentation Evolution of PNH testing Prevalence of PNH Clones PNH Guidelines ICCS 2010 Practical Guidelines 2012 High-Sensitivity PNH Assay RBC WBC Assay Validation Reporting PNH Testing Results

Paroxysmal Nocturnal Hemglobinuria (PNH) Rare Hematopoietic stem cell defect (2-6 cases /million) Somatic mutation of the PIG-A gene prevents the assembly of the GPI-anchor and results in partial or complete deficiency of Glycosylphosphatidylinositol (GPI) PNH is characterized by continuous destruction of PNH RBCs, it often occurs in bone marrow failures (e.g. AA and MDS) This deficiency can be seen in both the WBC and RBC WBCs are not affected by the GPI-deficiency RBCs are vulnerable to complement-mediated lysis PNH RBCs lack TCC (terminal complement inhibitor) Complement attack PNH RBCs are lysed and contents are released into the plasma

Patient Testing for PNH Using High Resolution Flow Cytometry at Dahl-Chase Dec 2007 Nov 15, 2013 Patient Selection based on Diagnostic Pathway 9,289 Total screening tests (including follow-up cases) 8,836 Total patients screened 572 PNH positive patients 6.5% 337 Patients w/ PNH Clones > 1% in WBC 3.8% 236 Patients w/ minor PNH Clones <1% in WBC 2.7% 134 of these PNH+ patients had follow-up studies Using the Diagnostic Pathway, every 26th Patient has shown a PNH Clone greater than 1%

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. 2011.

Patient Initially Submitted With Hx of AA/MDS Test# PNH Gran PNH Mono PNH RBC 1 6.7 6.8 1.4 2 12.8 11.8 0.82 3 20.8 13.5 0.43 4 15.6 20.2 0.12 5 15.4 21.6 0.2 6 17.8 24.9 0.2 7 32.4 24.8 0.22 8 23.2 21.3 0.25 9 24.5 24.5 0.72 Increase in PNH clone size from 6.7% to 24.5% within 12 months.

Classic Case of Clinical PNH Large PNH Clone (51.5%) in WBC (Granulocytes) Smaller Type III PNH Clone (4.7%) in RBCs PNH clone Normal Granulocytes PNH clone Normal RBCs Difference in PNH Clones sizes between RBC and WBC is due to hemolysis and/or transfusion

Effects of Eculizumab: Protecting PNH Red Blood Cells from complement-mediated lysis Patient had a 95% PNH Clone in the WBCs Initial visit 5% PNH cells Visit 2 19% PNH cells Visit 3 48% PNH cells Visit 4 91% PNH cells This process typically happens over the course of 26 weeks (NEJM Vol 355:1233-1243, Sept 21, 2006)

The Need for a Consensus Guideline for PNH Immunophenotyping The disease is rare and most labs have limited experience in PNH testing 1 The International PNH Interest Group (IPIG) recommended testing, including high-sensitivity testing, without specifying how this should be done 1 Flow cytometry is the method of choice to screen for PNH, but many different approaches exist 1 Some external quality assessment proficiency surveys have shown a wide range in ability of labs to detect abnormal PNH populations 1 The correct diagnosis of PNH is essential for effective patient management 2 1. Sutherland DR et al. Cytometry Part B. 2012;82B:195-208. 2. Borowitz MJ et al. Cytometry Part B. 2010;78B:211-230.

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:211-230.

High-Sensitivity Detection and Monitoring of PNH ICCS provided guidelines for the diagnosis and monitoring of PNH by flow cytometry. 1,2 However, the following were not addressed: Specific reagent cocktails 1 Associated detailed analytic strategies 1 Validation of sensitive, standardized methodologies 1 The development and validation of sensitive standardized methodologies are necessary to reliably detect PNH clones <1% 2 1. Borowitz MJ et al. Cytometry Part B. 2010;78B:211-230. 2. Sutherland DR et al. Cytometry Part B. 2012;82B:195-208.

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:195-208.

Recommended RBC Panel for BC & BD Platforms Sutherland DR, Keeney M, Illingworth A: Practical Guidelines for the high-sensitivity detection and monitoring of Paroxysmal Nocturnal Hemoglobinuria (PNH) clones by flow cytometry. Cytometry Part B 2012; 82B: 195-208

Recommended WBC Panels for BC Platforms Sutherland DR, Keeney M, Illingworth A: Practical Guidelines for the high-sensitivity detection and monitoring of Paroxysmal Nocturnal Hemoglobinuria (PNH) clones by flow cytometry. Cytometry Part B 2012; 82B: 195-208

Recommended WBC Panels for BD Platforms Sutherland DR, Keeney M, Illingworth A: Practical Guidelines for the high-sensitivity detection and monitoring of Paroxysmal Nocturnal Hemoglobinuria (PNH) clones by flow cytometry. Cytometry Part B 2012; 82B: 195-208

Important Considerations for Optimization of PNH Testing PNH is rare most labs have never seen a positive case 1 Step #1: Preanalytical Considerations 1 Instrument optimization (adequate voltages and compensation settings) Selection of reagents and panels Step #2: Assay Quality Control 1 Validation of PNH assay Survey materials and exchange of PNH samples with other labs Step #3: Analytical Considerations 2 Procedure (eg, wash vs no wash in RBCs) Cell lineage-specific gating Step #4: Clear Reporting 2 Avoid misleading terminology (eg, negative for GPI-linked antibody means positive for PNH clone) 1. Sutherland DR et al. Cytometry Part B. 2012;82B:195-208. 2. Borowitz MJ et al. Cytometry Part B. 2010;78B:211-230.

High-Sensitivity PNH Testing RBC Assay Based on published PNH Guidelines

RBC Panel and Sample Requirements Specimen Peripheral Blood in EDTA most tested but Heparin or ACD acceptable Bone marrow not recommended GPI-linked antibody CD59 shows best signal/noise ratio MEM43 and OV9A2 most tested and validated clones CD55 too dim Gating antibody CD235a most tested and validated antibody FITC shows least aggregation

RBC Assay - Testing Procedure Sample Preparation Make 1:100 Dilution of peripheral blood (EDTA) Pipette 50-100 microliters of this dilution into test tube Add appropriately titered CD59-PE Add appropriately titered CD235a-FITC (GPA) and vortex gently Incubate in the dark at RT for 20 minutes (up to 60 minutes) Wash twice with PBS! Resuspend in 0.5-1ml PBS Rack vigorously! Analyze within 15 minutes to avoid fading of CD235a-FITC Analysis use PNH-RBC panel with assay specific voltage and compensation settings Event Count Most cases: Acquisition of 50,000 CD235a+ RBCs is enough for sensitivity of 0.1%)

RBC Assay Instrument Setup Forward and Side Scatter Adjustment Use Log FS versus Log SS (not linear) Place RBC population so it is clearly visible Make sure that discriminator does not cut off cells) Voltage Adjustment of PMTs Adjust voltage of PMTs specific for the RBC assay RBCs should be on-scale in both FL1 (CD235a-FITC) and FL2 (CD59-PE) parameters* Compensation Set two-color compensation specific for the RBC assay Validate with stained and unstained normal samples *If FC500 or later in use DO NOT use negative offset

RBC - Normal Sample SS Log Check for aggregates (0.67) FS Log Dotplot CD235a-FITC / CD59-PE FS Log SS Log CD235a(GPA)-FITC Single Parameter CD59-PE Case NL13-2770

RBC - PNH Positive Sample Dotplot CD235a-FITC / CD59-PE Check for aggregates (0.49) Single Parameter CD59-PE Case PNH13--2733

CD59 Expression Type I, II and III RBCs Normal RBC s with normal CD59 expression (Type I cells) Life span 120 days Normal RBCs PNH clone with complete CD59 deficiency (Type III cells) Life span 10-15 days Type III PNH RBCs PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells) Type II PNH RBCs Life span: intermediate

Validation of high sensitivity RBC Assay Measure Assay Sensitivity using Spiking - make serial dilutions of PNH sample in normal Dilution Type III RBCs Sensitivity % Undiluted 346,626 34.86 1:10 17,665 1.77%* 1:100 1,822 0.18% 1:1000 203 0.02% 1:10000 20 0.002% Sensitivity on PNH samples: 20 PNH RBC phenotypes/million Sutherland DR, Keeney M, Illingworth A. Practical Guidelines for the high-sensitivity detection and monitoring of Paroxysmal Nocturnal Hemoglobinuria (PNH) clones by flow cytometry. Cytometry B Clin Cytom epub April 24 2012.

The Importance of Washing RBCs Potential False Negatives in RBC s No wash steps in RBC s No PNH? Washed x 2 PNH Clone present!

CD235a (GPA) vs CD59 provides Quality Control Separates true Type II PNH cells from poorly stained normal RBCs

Normal Sample or PNH? Gating on RBC by FS/SS only Gating on RBC by CD235a (GPA)

High-Sensitivity PNH Testing WBC Assay Based on Published PNH Guidelines

Detection of PNH Clones in WBC Granulocytes are most typically used to assess PNH clone size in WBC occasionally Type II granulocytes can be seen Monocytes should also be analyzed to confirm the granulocyte PNH clone Monocyte clone size often higher than granulocyte clone (reason unclear) precision is lower due to lower cell number occasionally Type II monocytes can be seen Lymphocytes are not a suitable target population for testing due to long life span But they serve as excellent internal controls for antibody QC (FLAER and CD24) voltage and compensation settings

WBC Assay - Reagents FLAER/CD24 appears to be the preferred antibody combination to detect PNH clones in granulocytes (CD157 looks promising) FLAER/CD14 is most tested combination to detect PNH clones in monocytes (CD157 looks promising) Lineage-specific gating results in higher sensitivities and cleaner WBC assays CD15 for granulocytes (not CD33) CD64 for monocytes (CD33 is second choice) CD45 can be very useful for debris exclusion and patternrecognition (back-gating to see what the populations are, e.g. blasts, platelets, other immature cells)

WBC Assay Instrument Setup Forward and Side Scatter Adjustment Use FS and SS in linear mode (not log) Place WBC populations on scale as separate leukocyte clusters Ensure FSC threshold is not excluding lymphocytes (used as internal controls) Voltage Adjustment of PMTs Optimize voltage of PMTs for good separation between antigen-positive and antigen-negative cells (signal/noise ratio) Ideally: use PNH case (showing FLAER/CD24-negative and positive cells)* Other option: use antibodies which show negative and positive staining (e.g. CD3)* Compensation (set up specific for PNH assay) Set compensation specific for this WBC assay (FLAER Alexa488 needs less compensation than FITC-conjugated antibody) Set up control dot plots more 2-parameter dot plots and less single parameter histograms check for compensation issues and ensure that all populations are on-scale Verification of settings with normal sample and/or PNH+ sample *If FC500: DO NOT use negative offset

WBC Assay - Testing Procedure Sample preparation Pipette 50-100 microliters of peripheral blood (EDTA) into test tube Add appropriately titered antibodies Incubate in the dark at RT for 20-30 minutes Lyse with your laboratory s lysing reagent (e.g. Immunoprep, FACS Lyse, Optilyse C, Ammonium Chloride etc.) Wash once with PBA Resuspend in 0.5-1ml of PBA Analysis on the flow cytometer using PNH-WBC panel with assay specific voltage and compensation settings Event Count: Most cases: 50,000 cells are enough for Granulocytes (resulting in sensitivity of 0.1%)

Check Voltage Settings in WBCs! Step 1: Run normal WBCs - with gating antibodies (CD45 and CD15) - without GPI-linked antibodies (FLAER and CD24) - shows unstained Grans on-scale in both PMTs Step 2: Run normal WBCs - with gating antibodies (CD45 and CD15) - with GPI-linked antibodies (FLAER and CD24) - shows stained FLAER+/CD24+ Grans Step 3: Run Test sample - with CD45, CD15, FLAER and CD24 Presence of 54.4% PNH Granulocytes

Case 1 - Normal 45 Year old Female with Pancytopenia Initial WBC tube FLAER-24-14-15-45 No PNH Clone in CD15++ Grans detected No Reflex mono tube with CD64 gating necessary (monocytes gated on CD45vsSS provide additional confirmation that no PNH clone is present

Case 2 PNH Clone 55 Year old Female with Aplastic Anemia and Hemoglobinuria Normal Grans Antibody and Compensation Check WBC tubes FLAER-24-15-45 FLAER-14-64-45 PNH Grans 91.81% PNH Clone in CD15++ Grans detected 97.0% PNH Clone in CD64++ Monos detected PNH Monos Normal Monos (13-2733)

Compensation Check on lymphocytes Appropriate Compensation Poor Compensation

11-2544 Patient with Aplastic Anemia/PNH Granulocyte Clone: 13.3% RBC Clone: 64.0% Monocyte Clone: 68.9% Sometimes there is a significant Difference in PNH Clone size between Granulocytes and Monocytes!

Validation of High Sensitivity WBC assays using Spiking Dilution PNH Granulocytes Sensitivity % NEAT 51,420 91.3% 4-Color Granulocyte Assay Sensitivity 1:10 5,799 9.4% 1:100 573 0.94% 1:1000 91 0.089% 1:10000 9 0.01% Dilution PNH Monocytes Sensitivity NEAT 12,718 89.8% 4-Color Monocyte Assay Sensitivity 1:10 1227 4.1% 1:100 112 0.4% 1:1000 13 0.044% 1:10000 ND ND Sutherland DR, Keeney M, Illingworth A. Practical Guidelines for the high-sensitivity detection and monitoring of Paroxysmal Nocturnal Hemoglobinuria (PNH) clones by flow cytometry. Cytometry B Clin Cytom epub April 24 2012.

Assay Validation - Summary Antibody performance Check expected antigen expression on normal blood Instrument optimization Ensure visibility of all populations in various dot plots Background check Check that normal samples do not show any events in the PNH gate Verification of PNH populations in the expected location use PNH blood or FMT setup Analytical sensitivity spiking experiments (PNH+ blood in normal blood) Accuracy of results Interlaboratory exchange and Proficiency Testing

Significance of Minor PNH Clones Do we really need to care about these small populations of PNH cells?

Minor PNH Clone (less than 1%) WBC RBC Normal Granulocytes Normal RBCs PNH clone PNH clone 0.57% PNH cells in CD15++ Granulocytes/Neutrophils 0.06% Type III PNH cells in CD235a+ RBC s (308 cells/500,000 total RBCs)

PNH cells less than 0.1% WBC RBC Special Requirements for reporting these very minor PNH populations No background events in normal samples Ideally 50-100 cells with PNH phenotype Cells need to be clustered and in the right location Presence of these PBH populations in both RBC and Granulocytes/neutrophils

Event count for PNH Assay RBC Assay 50,000 CD235a+ cells WBC Granulocytes/Neutrophil Assay 50,000 CD15++ cells WBC Monocyte Assay Approximately 5,000 CD64++ cells or more Most cases: 50,000 cells are enough (detecting 50 PNH cells - 0.1%) Poisson statistics suggests that if you find zero PNH RBCs in 50K events, the probability is 99% that the level of PNH RBCs will be <0.01%. May need to collect more events for small PNH clones 100,000 events showing a cluster of 50 Type III cells (0.05% PNH RBCs) 500,000 events showing a cluster of 50 Type III cells (0.01% PNH RBCs)

Follow-Up Testing of Patients with PNH Clones Dec 2007 May 2012 113 PNH Clone+ patients with follow-up testing (ranging from 5 months up to 29 months) Patients categorized based on PNH clone size in neutrophils/granulocytes < 0.1% 0.11-1% 1.01 10% 10.01 100% The goal was to determine if there was a change in PNH clone size category within 12 months (increase, decrease or same)

Results of 113 Patients with Follow-up May 26, 2012 <0.1% 0.11-1.0% 1.01-10% 10.01-100% Increased 0 2 (16%) 9 (38%) 0 Decreased 0 2 (16%) 5 (21%) 1 No Change 33 8 (67%) 10 (41%) 43 Totals (113) 33 12 24 44 Monitoring recommendations 6-12 months 3-6 months 3-6 months As indicated Based on ESCCA, Oral presentation of results of 89 patients, Sept 2011, accepted poster at EHA June 2012, updated May 26, 2012 for submitted poster to ESCCA to reflect additional follow-up results

Patient with Unexplained Cytopenia and Hemolysis 472478 80 70 60 50 Grans Test # PNH Gran PNH Mono PNH RBC 40 30 20 Monos RBCs 1 6.1 6.6 0.23 2 21.0 29.3 0.95 10 0 3 58 67 3.8 2009 2010 2011 Increase of PNH Clone from 6.1% to 58% within 2 years and 8 months

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

5-Color CD157-based Assay for High Sensitivity Granulocytes and Monocytes FLAER-Alexa488 - CD157-PE - CD64-ECD - CD15-PC5 - CD45PC7 Gating on CD15++ Grans Gating on CD64++ Monos FLAER/CD157- negative PNH Granulocytes FLAER/CD157- negative PNH Monocytes Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry B epub, August 2013

Correlation of 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 B epub, August 2013

Background on normal sample with 5-Color CD157-based assay FLAER CD157PE CD64ECD CD15PC5 CD45PC7 Sutherland DR, Acton E, Keeney M, Davis BH, Illingworth A. Cytometry B epub, August 2013

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 B epub, August 2013.

Clear Reporting is Important Flow Cytometry can identify the presence of a PNH Clone but does not diagnose PNH!

Example of Positive PNH Result Key information: 1. PNH Clone detected yes or no 2. PNH Clone size in WBC in granulocytes in monocytes 3. PNH Clone size in RBC with distribution of Type II cells Type III cells Total PNH Clone size 4. Flow cytometry graph of PNH Clone is provided

Example of negative PNH Result Provide level of sensitivity in negative reports

Standardized Terminology in our Lab PNH Population Greater than 1% 0.1%-1% Type PNH Clone Minor PNH Clone or population of PNH Cells 0.1% and Below Rare cells with GPI-deficiency It is important to not over-interpret small PNH clones as Clinical PNH!

Acknowledgements To all the PNH Experts who have helped along on this journey towards Best Practices in PNH Testing particularly Rob Sutherland from Toronto General Hospital All other participants from the ICCS Guidelines and Practical Guidelines To all the Flow Laboratories across the World who have helped to understand the complexities and challenges of state-of-the-art, yet practical PNH assay design To the staff at the Dahl-Chase flow cytometry laboratory who has mastered the many challenges of optimizing this assay Thank you!