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ICCS Newsletter Case Study Interpretation (CSI) Identification of PNH Clones Andrea Illingworth, MS, H(ASCP), QCYM Dahl-Chase Diagnostic Services Bangor,

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Presentation on theme: "ICCS Newsletter Case Study Interpretation (CSI) Identification of PNH Clones Andrea Illingworth, MS, H(ASCP), QCYM Dahl-Chase Diagnostic Services Bangor,"— Presentation transcript:

1 ICCS Newsletter Case Study Interpretation (CSI) Identification of PNH Clones Andrea Illingworth, MS, H(ASCP), QCYM Dahl-Chase Diagnostic Services Bangor, ME

2 Clinical History – Laboratory Data 75 year old male with history of hemolytic anemia Test ordered:Peripheral Blood in EDTA was received for PNH Evaluation in WBC and RBC CBC ParameterResultUnitsReference Range WBC3.4K/uL RBC2.76M/uL Hgb9.8g/dL Hct31.2% MCV113fl PLT232K/uL % Lymphocytes52.6%10-50 % Monocytes6.3%0-15 % Granulocytes41.1%37.80 LDH 844 units/liter

3 Pdf Files and LMD Files Submitted for this ICCS PNH Positive Case ICCS PNH Positive - WBC GM FLAER Tube #2 ICCS PNH Positive - WBC Mo FLAER Tube #3 ICCS PNH Positive - RBC GPA-59 Tube #1

4 The following slides will include:  Disease Overview and important pre- analytical considerations for PNH Testing (slide 5-13)  Case discussion of PNH Positive case –RBC Testing: Setup, analysis and QC (slide 14-23) –WBC Testing: Setup, analysis and QC (slide 24-31)  Interpretation and Reporting (slide 32-33)

5 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 –WBC are not affected by the GPI-deficiency –RBCs are vulnerable to complement-mediated lysis PNH RBCs lack TCC (terminal complement inhibitor) Complement attack PNH RNCs are lysed and contents are released into the plasma

6 CCS PNH Guidelines Suggestions for PNH Testing by ICCS PNH Guidelines Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. Cytometry Part B 2010; 00B:

7 Diagnosis of PNH “Method of Choice”  Flow cytometry has become the “Method of Choice” in the detection of cells with GPI Deficiency (PNH clones) due its high accuracy and sensitivity down to 0.01%)  Some GPI-linked antibodies are CD55, CD59, CD14, CD16, CD24, CD66b and more recently FLAER  At least 2 GPI-linked antibodies must be absent for the diagnosis of PNH  Red blood cells, monocytes and granulocytes are mostly analyzed to detect PNH clones  Need for testing has become more important as there is an FDA-approved drug now to treat this disease

8 Classic Case of Clinical PNH Difference in PNH Clones sizes between RBC and WBC may be due to hemolysis and/or transfusion Large PNH Clone (54.5%) in WBC (CD15++ Granulocytes) Smaller Type III PNH Clone (6%) in GPA+ RBCs PNH clone Normal Granulocytes Normal RBCs

9 Specimen Recommendations  Peripheral blood is the preferred specimen –Bone marrow is not desirable outside of the research setting because immature myeloid populations may express lower levels of GPI-anchored proteins making interpretation difficult  No data that any specific anticoagulant is necessary, though most experience has been with EDTA  Granulocyte analysis best performed in hrs because of degranulation; RBCs may be stable at 0 o for 7 days

10 Possible WBC Reagent Combinations Cells colorGFLAERCD24CD15 3 colorMFLAERCD14CD33 4 colorGFLAERCD24CD15CD45 4 colorMFLAERCD14CD33CD45 4 colorG+MFLAERCD24CD14CD33 5 colorG+MFLAERCD24CD14CD15CD45 5 colorG+MFLAERCD24CD14CD15CD33/64 6 colorG+MFLAERCD24CD14CD15CD45CD64 6 colorG+MFLAERCD24CD14CD15CD45CD33 Colors Adapted from the 2010 ICCS Guidelines

11 Sensitive and Specific 5-Color PNH Panel on 5 Color FC 500 in our Lab What are we looking for? What are we gating on to ensure high sensitivity and specificity? How many cells are typically counted? Red Blood Cells Absence of CD59 expression GPA+ RBC’s only 50,000 or more Granulocytes Absence of FLAER and CD24 CD15+ mature granulocytes/neutrophils 50,000 or more Monocytes Absence of FLAER and CD14 CD64 or CD33 Variable Red Blood Cells:GPA-CD59 Granulocytes and Monocytes:FLAER - CD24 - CD14 - CD15 - CD45 Monocytes only (Reflex):FLAER - CD33 - CD14 - CD64 - CD45 Current 5C Panel If a laboratory has 6 color capability, the following panels is suggested: FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33) as it involves FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33) as it involves FLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophils FLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophils FLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytes FLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytes

12 Pre-analytical Considerations  Instrument Optimization –Appropriate Voltage adjustment Cells positive for the antibody should show bright signalCells positive for the antibody should show bright signal Cells negative for the antibody need to be “on scale”Cells negative for the antibody need to be “on scale” –Optimize compensation settings WBC: setting may be similar to Leukemia/lymphoma evals but need to be tweaked if using FLAER-Alexa488WBC: setting may be similar to Leukemia/lymphoma evals but need to be tweaked if using FLAER-Alexa488 RBC: need separation compensation settingRBC: need separation compensation setting  Reagent and Panel Selection  Reagent and Panel Selection – it is important to select the most specific reagents with the best signal/noise ratio, e.g. –CD59 is preferred over CD55 for RBCs –FLAER/CD24 for WBC-Granulocytes –FLAER/CD14 for WBC-Monocytes –Antibodies should titered  Lineage specific gating  Lineage specific gating increases sensitivity, e.g. –GPA (CD235a) for RBCs –CD15 for granulocytes/neutrophils –CD64 or CD33 for monocytes

13 Pre-analytical Considerations – Quality Control  Validation of PNH Assay –Several normal peripheral blood samples should be run To verify adequate staining of antibodies in normal cells To determine the background and sensitivity of the Assay  PNH Surveys –NEQAS (UK) –CAP RBC and WBC Survey  Inter-laboratory comparisons  Inter-laboratory comparisons of PNH+ samples (containing larger and smaller PNH clones) may help to improve confidence levels in the detection of PNH clones

14 PNH Testing - RBC Panel: CD235a(GPA)-FITC / CD59-PE (MEM43 clone)

15 RBC Testing Procedure  Make 1:100 Dilution of peripheral blood (EDTA)  Pipette microliters of this dilution into bottom of the test tube (make sure no blood is smeared on the side of the tube!)  Add appropriately titered CD59-PE  Add appropriately titered GPA-FITC (CD235a) –Do not use GPA-PE –See Sutherland et al (AJCP 2009:132: )  Incubate in the dark at RT for 20 minutes  Wash twice with PBS!  Resuspend in 0.5-1ml PBS  Rack vigorously!  Run on the flow cytometer using your PNH-RBC panel

16 RBC - Normal Control (PB) Case NL-765 SS Log GPA (CD235a) CD59-PE FS Log RBC gate to gate out debris 2.GPA+ gate to gate out GPA- negative cells 3.Dot Plot GPA- CD59 is gated on GPA+ RBCs 4.Single Parameter histogram is also gated on GPA+ RBCs

17 Tube #1: RBCs showing PNH Type III Clone Dot Plot verifies that PNH cells (blue) show same level of GPA staining as normal cells (red). Doublets (aqua) should not be >2% Single parameter histogram of CD59 expression (gated on GPA+ RBCs) can be used together with dot plot to establish cursor setting (for Type I, II and III RBCs)

18 Importance of “Racking” (Dragging tube hard several times across a specimen rack to break up clumping) Case NL-3381 Tubes were vortexed lightly: 29% Aggregates Tubes were racked vigorously: 0.5% Aggregates Aggregates Aggregates No Aggregates

19 Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCs PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells) PNH clone with complete CD59 deficiency (Type III cells) Normal RBC’s with normal CD59 expression (Type I cells) Gating on GPA+ RBC’s

20 Alternate Options for PNH QC in RBCs Step 1:Run normal RBCs with GPA and with CD59 to determine the position of normal RBCs (Type I cells) Step 2:Run normal RBCs with GPA and without CD59 to determine the position of RBCs with complete CD59 Deficiency (Type III cells) Type III Type II Type I Step 3:Run suspected PNH patient with GPA / CD59 Presence of 6.2% PNH Type III RBCs

21 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!

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

23 Summary - RBC  CD235a-FITC/CD59 –PE is the preferred antibody combination with best signal/noise ratio –select most sensitive and specific clone (e.g. MEM43 and p282) –use CD59-PE preferably –addition of GPA (preferably FITC conjugate) results in higher sensitivities and cleaner RBC assays  Washing twice and “racking” is important!  Analysis of 50,000 RBCs can result in sensitivity of 0.05%-0.1% ( PNH cells) in a clean assay  Difference in Clone size between RBC and WBC is important to determine hemolysis (RBC clone usually lower than WBC clone)  Report both Type II and Type III as the total PNH Clone if there is a separate Type II RBC population present

24 PNH Testing – WBC Panel Granulocytes and Monocytes: Granulocytes and Monocytes: FLAER - CD24 - CD14* - CD15 - CD45 Monocytes only (Reflex): Monocytes only (Reflex): FLAER - CD33** - CD14 - CD64** - CD45

25 WBC Testing Procedure  Pipette microliters of peripheral blood (EDTA) into test tube  Add appropriately titered antibodies (rinse out antibody thoroughly)  Incubate in the dark at RT for 30 minutes  Lyse with your laboratory’s lysing reagent (e.g. Immunoprep, Optilyse, FACS Lyse, Ammonium Chloride etc.  Wash once with PBA  Resuspend in 0.5-1ml of PBA  Run on the flow cytometer using your WBC-PNH panel

26 Normal Peripheral Blood sample WBC – Granulocytes/Neutrophils Step 2: Step 2: Gating on CD15+ granulocytes Step 3: No PNH Clone detected in CD15++ Granulocytes Step 1: Step 1: Gating out debris

27 Normal Peripheral Blood sample WBC - Monocytes FLAER14As the panel contains FLAER-CD , the CD45vsSS histogram allows some gating on the monocytes (green) to check for FLAER-CD14 Deficiency. Please note that for accurate assessment of PNH monocytes, lineage-specific gating on CD64+ or CD33+ monocytes is preferred

28 Tube #2: Peripheral Blood of PNH+ Patient WBC (Granulocytes/Neutrophils) Step 1: Step 1: Gating out debris Step 2: Step 2: Gating on CD15+ granulocytes Step 3: Identification of PNH Clone in CD15++ Granulocytes

29 Peripheral Blood of PNH+ Patient WBC - Monocytes Gating on CD45vsSS allows for determination of PNH clone in Monocytes but size of the PNH clone is not accurate (78.9%) Lineage-specific gating on CD64vsSS allows for a more accurate assessment of the size of the PNH clone in Monocytes (83.3) 83.3% 78.9% Tube #3 Tube #2

30 Alternate Options for PNH QC in WBCs Step 1:Run normal WBCs with gating antibodies and with GPI-linked antibodies to determine the position of normal WBCs Step 2:Run normal WBCs with gating antibodies and without GPI-linked antibodies to determine the position of WBCs with complete GPI-Deficiency Step 3:Run suspected PNH patient with gating antibodies and GPI-linked antibodies Presence of 54.4% PNH Granulocytes

31 Summary - WBC  FLAER/CD24 appears to be the preferred antibody combination to detect PNH clones in granulocytes  FLAER/CD14 is most tested combination to detect PNH clones in monocytes  Lineage-specific gating results in higher sensitivities and cleaner WBC assays –CD15 for granulocytes –CD64 and/or CD33 –CD45 can be very useful for pattern-recognition (back-gating to see what the populations are, e.g. blasts, platelets, other immature cells, debris)  Analysis of 50,000 granulocytes can result in sensitivity of 0.05%-0.1% (25-50 PNH cells) in a clean assay  Report both Type II and Type III granulocytes and monocytes as the total PNH Clone if they are present

32  Report clone sizes in all populations tested (RBCs, granulocytes and possibly monocytes)  Report Type II and Type III RBCs as well as Type II and Type III granulocytes (even though their significance is not established)  Repeat samples on same patient should comment on change in size of PNH clone  Provide histograms if possible  Report level of sensitivity Reporting of PNH Results Recommended: Avoid:  Reporting reactivity with each individual marker  Avoid ambiguous (“positive versus negative”) language –e.g. “CD59 test is Negative” may mean to some that CD59 is negative and therefore positive for PNH  Don’t over-interpret small clones as evidence of hemolytic PNH

33 Reporting – ICCS PNH Positive Case Key information: 1.PNH Clone detected – Yes or No 2.PNH Clone size in WBC in granulocytesin granulocytes in monocytes*in monocytes* 3.PNH Clone size in RBC with distribution of Type II cellsType II cells Type III cellsType III cells Total PNH Clone sizeTotal PNH Clone size 4.Flow cytometry graph of PNH Clone is provided

34 Acknowledgements  To all the PNH Experts who have helped us along on this journey towards Best Practices in PNH Testing –Dr. Robert Sutherland –Dr. Stephen Richards –Dr. Michael Borowitz –Dr. Wendell Rosse –Dr. Bruce Davis –And many others…… Thank you! Thank you!

35 Back: Medical Technologists Ashley B, Tony K, Brie S, Neisha B and Monica G Front: Medical Director M Movalia MD, Operational Director A Illingworth and S DuFresne MD The Flow Cytometry Team


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