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Andrea Illingworth, MS, H(ASCP), QCYM

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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 Parameter Result Units Reference Range WBC 3.4 K/uL RBC 2.76 M/uL Hgb 9.8 g/dL Hct 31.2 % MCV 113 fl PLT 232 % Lymphocytes 52.6 10-50 % Monocytes 6.3 0-15 % Granulocytes 41.1 37.80 LDH  844  units/liter

3 Pdf Files and LMD Files Submitted for this ICCS PNH Positive Case
ICCS PNH Positive - RBC GPA-59 Tube #1 ICCS PNH Positive - WBC GM FLAER Tube #2 ICCS PNH Positive - WBC Mo FLAER Tube #3 This case (referred to and labeled as ICCS PNH Positive Tube #1, Tube #2 and Tube #3) was used in this presentation whenever possible to illustrate the typical workflow in PNH Testing and selected histograms are used in subsequent slides. LMD Files for this cases are also labeled as described

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) PNH RNCs are lysed and contents are released into the plasma Complement attack

6 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 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
Large PNH Clone (54.5%) in WBC (CD15++ Granulocytes) Smaller Type III PNH Clone (6%) in GPA+ RBCs Normal RBCs Normal Granulocytes PNH clone Note the double-negative PNH cells for CD24 and FLAER. It is important to look at the dot plot in the WBC as the single parameter histograms do not allow you to check for the absence of 2 GPI-linked antibodies/reagents. In this slide only the single parameter histogram is shown for RBCs. Both the single parameter CD59 histogram (gated on GPA+ RBCs) and the dot plot GPAvsCD59 are useful in the determination where the cursor should be set between the Type I, II and III RBCs PNH clone Difference in PNH Clones sizes between RBC and WBC may be due to hemolysis and/or transfusion

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 0o for 7 days

10 Possible WBC Reagent Combinations
Colors Cells 1 2 3 4 5 6 3 color G FLAER CD24 CD15 M CD14 CD33 4 color CD45 G+M 5 color CD33/64 6 color CD64 Adapted from the 2010 ICCS Guidelines

11 Sensitive and Specific 5-Color PNH Panel on 5 Color FC 500 in our Lab
Red Blood Cells: GPA-CD59 Granulocytes and Monocytes: FLAER - CD24 - CD14 - CD15 - CD45 Monocytes only (Reflex): FLAER - CD33 - CD14 - CD64 - CD45 Current 5C Panel 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 Monocytes Absence of FLAER and CD14 CD64 or CD33 Variable 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 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophils 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 signal 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-Alexa488 RBC: need separation compensation setting 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 increases sensitivity, e.g. GPA (CD235a) for RBCs CD15 for granulocytes/neutrophils CD64 or CD33 for monocytes This is an important slide as it demonstrates some very crucial components of a good setup for a clean and accurate PNH assay. Poor Voltage settings and compensation settings often contribute to histograms, which are hard to interpret Reagent selection: please keep in mind that not all antibody clones are the same (in our lab the CD59 MEM43 clone has worked very well) Panel design should include GPI-linked antibodies/reagents and gating antibodies in concordance with the ICCS guidelines Antibody titration and fluorochrome selection is extremely important as it affects the degree of clumping in the RBCs

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 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)
1 3 RBC gate to gate out debris GPA+ gate to gate out GPA-negative cells Dot Plot GPA-CD59 is gated on GPA+ RBCs Single Parameter histogram is also gated on GPA+ RBCs SS Log FS Log 2 4 SS Log CD59-PE GPA (CD235a) Case NL-765

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% The blue cells represent the PNH Type III cells, which show complete absence for CD59. In this patient thee are some rare events in the Type II are but they do not show up as a distinct cluster so the type II population is virtually absent The gate for aggregates should be checked as too much clumping may affect the distribution of type I, II and III RBCs. In our lab we ensure that there are no more than 1-2% aggregates. Hard racking (3-5 times) across a specimen rack typically breaks up these aggregates. Vortexing is typically not enough (see next slide) 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) Aggregates Aggregates Tubes were vortexed lightly: 29% Aggregates No Aggregates No Aggregates Tubes were racked vigorously: 0.5% Aggregates Case NL-3381

19 Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCs
Normal RBC’s with normal CD59 expression (Type I cells) PNH clone with complete CD59 deficiency (Type III cells) PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II 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) Ideally, a PNH patient with a CD59-negative PNH clone and some remaining normal CD59+ cells should be used to: optimize the voltage setting & compensation setting (a poorly compensated set up may show the type III population in the type II region) verify adequate CD59 staining on normal cells check for the location of the CD59-negative population. The option above may be helpful in the initial validation process for the PNH assay as it is hard to get a PNH+ patient. Step 3: Run suspected PNH patient with GPA / CD59 Presence of 6.2% PNH Type III RBCs Type III Type II Type I

21 The Importance of Washing RBCs Potential False Negatives in RBC’s
No wash steps in RBC’s No PNH? Washing is crucial as not washing RBCs may not allow for the identification of a PNH clone. The patient above was initially not washed and although a shoulder can be seen in the Type II area, this slightly decreased staining for CD59 may also be seen in transfused patients. After washing this sample x2, the CD59-negative Type III PNH clone can easily be identified. 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 The reason for poor staining is often due to some RBCs left on the side of the test tube when adding the diluted blood into the bottom of the test tube as they do not get stained adequately with the antibody. If this happens, set up the tube again without touching the sides when delivering the blood. This example shows the dual value of CD235a: addition of CD235a enables the lab to gate on true RBCs only resulting in a clean PNH assay (as it is important to ensure that the CD59-negative events are truly RBCs) Identifies staining artifacts in the type II area, which may otherwise be misinterpreted as Type II PNH cells

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% (25-50 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:
FLAER - CD24 - CD14* - CD15 - CD45 Monocytes only (Reflex): FLAER - CD33** - CD14 - CD64** - CD45 * Our panel includes CD14,which may be eliminated from this tube as it only allows for the identification of a PNH clone. For true estimation of size of the PNH clone in monocytes, the lineage-specific Monocyte should be run ** Our panel includes both CD64 and CD33 but either one is sufficient for lineage specific gating on monocytes

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 1: Gating out debris Step 2: Gating on CD15+ granulocytes Step 3: No PNH Clone detected in CD15++ Granulocytes

27 Normal Peripheral Blood sample WBC - Monocytes
As 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: Gating out debris Step 2: Gating on CD15+ granulocytes Step 3: Identification of PNH Clone in CD15++ Granulocytes

29 Peripheral Blood of PNH+ Patient WBC - Monocytes
Tube #2 Gating on CD45vsSS allows for determination of PNH clone in Monocytes but size of the PNH clone is not accurate (78.9%) Please note that the CD64 expression in this case was unusually dim (it typically is seen in the 3rd decade. Every patient is different and antibody expression may vary slightly! The LMD data of tube #3 also show the CD33 expression to be very dim 78.9% Tube #3 Lineage-specific gating on CD64vsSS allows for a more accurate assessment of the size of the PNH clone in Monocytes (83.3) 83.3%

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 See slide 20 as this slide follows the same principle 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 Reporting of PNH Results
Recommended: 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 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: PNH Clone detected – Yes or No 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 * Although in most PNH cases the PNH clone size in granulocyte and monocytes is similar, this case demonstrates the not infrequent finding of the PNH monocyte clone being larger than the PNH granulocyte clone. The reason and clinical significance are unknown at this time.

34 Acknowledgements Thank you!
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!

35 The Flow Cytometry Team
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

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