ICCS NewsletterCase Study Interpretation (CSI)

Identification of PNH Clones

Andrea Illingworth, MS, H(ASCP), QCYM

Dahl-Chase Diagnostic ServicesBangor, ME

aillingworth@dahlchase.comwww.dahlchase.com

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

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 ParameterCBC Parameter ResultResult UnitsUnits Reference RangeReference Range

WBCWBC 3.43.4 K/uLK/uL 4.6-10.94.6-10.9

RBCRBC 2.762.76 M/uLM/uL 4.69-6.134.69-6.13

HgbHgb 9.89.8 g/dLg/dL 14.1-18.114.1-18.1

HctHct 31.231.2 %% 43.5-53.743.5-53.7

MCVMCV 113113 flfl 80.0-97.080.0-97.0

PLTPLT 232232 K/uLK/uL 142-424142-424

% Lymphocytes% Lymphocytes 52.652.6 %% 10-5010-50

% Monocytes% Monocytes 6.36.3 %% 0-150-15

% Granulocytes% Granulocytes 41.141.1 %% 37.8037.80

LDHLDH  844 844  units/liter units/liter 313-618313-618

Pdf Files and LMD Files Submitted for this ICCS PNH Positive Case

ICCS PNH Positive - WBC GM

FLAER-24-14-15-45

Tube #2Tube #2

ICCS PNH Positive - WBC GM

FLAER-24-14-15-45

Tube #2Tube #2

ICCS PNH Positive - WBC Mo

FLAER-33-14-64-45

Tube #3Tube #3

ICCS PNH Positive - WBC Mo

FLAER-33-14-64-45

Tube #3Tube #3

ICCS PNH Positive - RBC

GPA-59

Tube #1Tube #1

ICCS PNH Positive - RBC

GPA-59

Tube #1Tube #1

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)

Paroxysmal Nocturnal Hemglobinuria (PNH)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 RBCThis deficiency can be seen in both the WBC and RBC– WBC are not affectedWBC are not affected by the GPI-deficiency by the GPI-deficiency– RBCs are vulnerableRBCs are vulnerable to complement-mediated lysis to complement-mediated lysis

PNH RBCs lack TCC (terminal complement inhibitor)

PNH RBCs lack TCC (terminal complement inhibitor)

Complement attackComplement attackPNH RNCs are lysed and contents are released into the plasma

PNH RNCs are lysed and contents are released into the plasma

Suggestions for PNH Testing by ICCS PNH GuidelinesCCS 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: 000-000

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: 000-000

Diagnosis of PNH

Flow cytometry has become the “Method of Choice”“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

Classic Case of Clinical PNHClassic Case of Clinical PNH

Difference in PNH Clones sizes between RBC and WBC may be due to hemolysis and/or transfusion

Difference in PNH Clones sizes between RBC and WBC may be due to hemolysis and/or transfusion

Large PNH Clone (54.5%) in Large PNH Clone (54.5%) in WBC (CD15++ Granulocytes)WBC (CD15++ Granulocytes)Large PNH Clone (54.5%) in Large PNH Clone (54.5%) in WBC (CD15++ Granulocytes)WBC (CD15++ Granulocytes)

Smaller Type III PNH Clone Smaller Type III PNH Clone (6%) in GPA+ RBCs(6%) in GPA+ RBCsSmaller Type III PNH Clone Smaller Type III PNH Clone (6%) in GPA+ RBCs(6%) in GPA+ RBCs

PNH PNH cloneclonePNH PNH cloneclone PNH PNH

cloneclonePNH PNH cloneclone

Normal Normal GranulocytesGranulocytesNormal Normal GranulocytesGranulocytes

Normal Normal RBCsRBCsNormal Normal RBCsRBCs

Specimen RecommendationsSpecimen Recommendations

Peripheral bloodPeripheral blood is the preferred specimen is the preferred specimen– Bone marrow is not desirable outside of the research setting Bone marrow is not desirable outside of the research setting

because immature myeloid populations may express lower levels of because immature myeloid populations may express lower levels of

GPI-anchored proteins making interpretation difficultGPI-anchored proteins making interpretation difficult

No data that any specific anticoagulant is necessary, No data that any specific anticoagulant is necessary,

though most experience has been with though most experience has been with EDTAEDTA

Granulocyte analysis best performed in Granulocyte analysis best performed in 24-4824-48 hrshrs because because

of degranulation; RBCs may be stable at 0of degranulation; RBCs may be stable at 0oo for 7 days for 7 days

Possible WBC Reagent CombinationsPossible WBC Reagent Combinations

Cells 1 2 3 4 5 6

3 color G FLAER CD24 CD15

3 color M FLAER CD14 CD33

4 color G FLAER CD24 CD15 CD45

4 color M FLAER CD14 CD33 CD45

4 color G+M FLAER CD24 CD14 CD33

5 color G+M FLAER CD24 CD14 CD15 CD45

5 color G+M FLAER CD24 CD14 CD15 CD33/64

6 color G+M FLAER CD24 CD14 CD15 CD45 CD64

6 color G+M FLAER CD24 CD14 CD15 CD45 CD33

Colors

Adapted from the 2010 ICCS GuidelinesAdapted from the 2010 ICCS Guidelines

Sensitive and Specific 5-Color PNH Panel Sensitive and Specific 5-Color PNH Panel on 5 Color FC 500 in our Labon 5 Color FC 500 in our Lab

What are we looking for?What are we looking for? What are we gating on to ensure high What are we gating on to ensure high sensitivity and specificity?sensitivity and specificity?

How many cells are How many cells are typically counted?typically counted?

Red Blood CellsRed Blood Cells Absence of CD59 expressionAbsence of CD59 expression GPA+ RBC’s onlyGPA+ RBC’s only 50,000 or more50,000 or more

GranulocytesGranulocytes Absence of FLAER and CD24Absence of FLAER and CD24 CD15+ mature CD15+ mature granulocytes/neutrophilsgranulocytes/neutrophils 50,000 or more50,000 or more

MonocytesMonocytes Absence of FLAER and CD14Absence of FLAER and CD14 CD64 or CD33CD64 or CD33 VariableVariable

Red Blood Cells:Red Blood Cells: GPA-CD59GPA-CD59

Granulocytes and Monocytes:Granulocytes and Monocytes: FLAER - CD24 - CD14 - CD15 - CD45FLAER - CD24 - CD14 - CD15 - CD45

Monocytes only (Reflex):Monocytes only (Reflex): FLAER - CD33 - CD14 - CD64 - CD45FLAER - CD33 - CD14 - CD64 - CD45

Current Current 5C Panel5C PanelCurrent Current 5C Panel5C Panel

If a laboratory has If a laboratory has 6 color capability6 color capability, the following panels is suggested: , the following panels is suggested:

FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33)FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33) as it involves as it involves

• FLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophilsFLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophils

• FLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytesFLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytes

If a laboratory has If a laboratory has 6 color capability6 color capability, the following panels is suggested: , the following panels is suggested:

FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33)FLAER / CD24 / CD14 / CD15 / CD45 / CD64 (or CD33) as it involves as it involves

• FLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophilsFLAER/CD24 to determine PNH cells (absence of both) in CD15++ granulocytes/neutrophils

• FLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytesFLAER/CD14 to determine PNH cells (absence of both) in CD64++ (CD33++) monocytes

Pre-analytical Considerations Instrument OptimizationInstrument Optimization

– Appropriate Voltage adjustmentAppropriate 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 settingsOptimize compensation settings• WBC: setting may be similar to Leukemia/lymphoma evals but need WBC: setting may be similar to Leukemia/lymphoma evals but need

to be tweaked if using FLAER-Alexa488to be tweaked if using FLAER-Alexa488• RBC: need separation compensation settingRBC: need separation compensation setting

Reagent and Panel SelectionReagent 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 gatingLineage specific gating increases sensitivity, e.g.– GPA (CD235a) for RBCs– CD15 for granulocytes/neutrophils– CD64 or CD33 for monocytes

Pre-analytical Considerations – Quality Control

Validation of PNH AssayValidation 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 SurveysPNH Surveys– NEQAS (UK)– CAP RBC and WBC Survey

Inter-laboratory comparisonsInter-laboratory comparisons of PNH+ samples (containing larger and smaller PNH clones) may help to improve confidence levels in the detection of PNH clones

PNH Testing - RBC

Panel: CD235a(GPA)-FITC / CD59-PE (MEM43 clone)Panel: CD235a(GPA)-FITC / CD59-PE (MEM43 clone)Panel: CD235a(GPA)-FITC / CD59-PE (MEM43 clone)Panel: CD235a(GPA)-FITC / CD59-PE (MEM43 clone)

RBC Testing Procedure

Make 1:100 Dilution of peripheral blood (EDTA)

Pipette 50-100 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 2009:132:564-572)See Sutherland et al (AJCP 2009:132:564-572)

Incubate in the dark at RT for 20 minutes

Wash twice with PBS!Wash twice with PBS!

Resuspend in 0.5-1ml PBS

Rack vigorously!Rack vigorously!

Run on the flow cytometer using your PNH-RBC panel

RBC - Normal Control (PB)

Case NL-765Case NL-765

SS LogSS Log

SS LogSS Log

GPA (CD235a)GPA (CD235a) CD59-PECD59-PE

FS LogFS Log

1111 3333

2222 4444

1. 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

1. 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

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%

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)

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)

Importance of “Racking”Importance of “Racking”(Dragging tube hard several times across a specimen rack to break up clumping)(Dragging tube hard several times across a specimen rack to break up clumping)

Case NL-3381Case NL-3381

Tubes were vortexed lightly: Tubes were vortexed lightly: 29% Aggregates29% AggregatesTubes were vortexed lightly: Tubes were vortexed lightly: 29% Aggregates29% Aggregates

Tubes were racked vigorously:Tubes were racked vigorously: 0.5% Aggregates 0.5% AggregatesTubes were racked vigorously:Tubes were racked vigorously: 0.5% Aggregates 0.5% Aggregates

AggregatesAggregatesAggregatesAggregatesAggregatesAggregatesAggregatesAggregates

No AggregatesNo AggregatesNo AggregatesNo Aggregates No AggregatesNo AggregatesNo AggregatesNo Aggregates

Normal Expression of CD59 (Type I) and Abnormal Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCsExpression 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’sGating on GPA+ RBC’s

Alternate Options for PNH QC in RBCs

Step 1:Step 1: Run normal RBCs with Run normal RBCs with GPA and GPA and with CD59with CD59 to to determine the position determine the position

of of normal RBCs (Type I normal RBCs (Type I cells)cells)

Step 1:Step 1: Run normal RBCs with Run normal RBCs with GPA and GPA and with CD59with CD59 to to determine the position determine the position

of of normal RBCs (Type I normal RBCs (Type I cells)cells)

Step 2:Step 2: Run normal RBCs with GPA Run normal RBCs with GPA and and without CD59without CD59 to to

determine the position of determine the position of RBCs with complete CD59 RBCs with complete CD59 Deficiency (Type III cells)Deficiency (Type III cells)

Step 2:Step 2: Run normal RBCs with GPA Run normal RBCs with GPA and and without CD59without CD59 to to

determine the position of determine the position of RBCs with complete CD59 RBCs with complete CD59 Deficiency (Type III cells)Deficiency (Type III cells)

Type IIIType IIIType IIIType III Type IIType IIType IIType II Type IType IType IType I

Step 3:Step 3: Run suspected PNH patient Run suspected PNH patient with GPA / CD59with GPA / CD59

Step 3:Step 3: Run suspected PNH patient Run suspected PNH patient with GPA / CD59with GPA / CD59

Presence of 6.2% PNH Type III RBCsPresence of 6.2% PNH Type III RBCs Presence of 6.2% PNH Type III RBCsPresence of 6.2% PNH Type III RBCs

The Importance of Washing RBCsThe Importance of Washing RBCsPotential False Negatives in RBC’sPotential False Negatives in RBC’s

No wash steps in RBC’s

No PNH?No PNH?

Washed x 2

PNH Clone present!PNH Clone present!

CD235a (GPA) vs CD59 provides Quality Control

Separates true Separates true Type II PNH cells Type II PNH cells

from poorly from poorly stained normal stained normal

RBCsRBCs

Summary - RBCSummary - RBC

CD235a-FITC/CD59 –PE isCD235a-FITC/CD59 –PE is the preferred antibody combination with the preferred antibody combination with best signal/noise ratiobest signal/noise ratio– select most sensitive and specific clone (e.g. MEM43 and p282)select most sensitive and specific clone (e.g. MEM43 and p282)– use CD59-PE preferablyuse CD59-PE preferably– addition of GPA (preferably FITC conjugate) results in higher addition of GPA (preferably FITC conjugate) results in higher

sensitivities and cleaner RBC assays sensitivities and cleaner RBC assays

WashingWashing twice and “racking” is important! twice and “racking” is important!

Analysis of 50,000 RBCs can result in Analysis of 50,000 RBCs can result in sensitivitysensitivity of 0.05%-0.1% (25- of 0.05%-0.1% (25-50 PNH cells) in a clean assay50 PNH cells) in a clean assay

Difference in Clone size between RBC and WBCDifference in Clone size between RBC and WBC is important to is important to determine hemolysis (RBC clone usually lower than WBC clone)determine hemolysis (RBC clone usually lower than WBC clone)

Report both Type II and Type III as the Report both Type II and Type III as the total PNH Clonetotal PNH Clone if there is a if there is a separate Type II RBC population presentseparate Type II RBC population present

PNH Testing – WBC Panel

• Granulocytes and Monocytes:Granulocytes and Monocytes:

FLAER - CD24 - CD14* - CD15 - CD45FLAER - CD24 - CD14* - CD15 - CD45

• Monocytes only (Reflex):Monocytes only (Reflex):

FLAER - CD33** - CD14 - CD64** - CD45FLAER - CD33** - CD14 - CD64** - CD45

• Granulocytes and Monocytes:Granulocytes and Monocytes:

FLAER - CD24 - CD14* - CD15 - CD45FLAER - CD24 - CD14* - CD15 - CD45

• Monocytes only (Reflex):Monocytes only (Reflex):

FLAER - CD33** - CD14 - CD64** - CD45FLAER - CD33** - CD14 - CD64** - CD45

WBC Testing Procedure

Pipette 50-100 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

Normal Peripheral Blood sampleWBC – Granulocytes/Neutrophils

Step 2:Step 2: Gating on CD15+ granulocytes

Step 3:Step 3: No PNH Clone No PNH Clone detected in CD15++ detected in CD15++ GranulocytesGranulocytes

Step 1:Step 1: Gating out debris

Normal Peripheral Blood sampleWBC - Monocytes

• As the panel contains FLAERFLAER-CD24-1414-15-45, 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

• As the panel contains FLAERFLAER-CD24-1414-15-45, 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

Tube #2: Peripheral Blood of PNH+ PatientWBC (Granulocytes/Neutrophils)

Step 1:Step 1: Gating out debris

Step 2:Step 2: Gating on CD15+ granulocytes

Step 3:Step 3: Identification Identification of PNH Clone in of PNH Clone in CD15++ GranulocytesCD15++ Granulocytes

Peripheral Blood of PNH+ PatientWBC - Monocytes

Gating on CD45vsSS Gating on CD45vsSS allows for determination allows for determination of PNH clone in of PNH clone in Monocytes but size of Monocytes but size of the PNH clone is not the PNH clone is not accurate (78.9%)accurate (78.9%)

Gating on CD45vsSS Gating on CD45vsSS allows for determination allows for determination of PNH clone in of PNH clone in Monocytes but size of Monocytes but size of the PNH clone is not the PNH clone is not accurate (78.9%)accurate (78.9%)

Lineage-specific gating Lineage-specific gating on CD64vsSSon CD64vsSS allows for allows for a more accurate a more accurate assessment of the assessment of the size size of the PNHof the PNH clone in clone in Monocytes (83.3)Monocytes (83.3)

Lineage-specific gating Lineage-specific gating on CD64vsSSon CD64vsSS allows for allows for a more accurate a more accurate assessment of the assessment of the size size of the PNHof the PNH clone in clone in Monocytes (83.3)Monocytes (83.3)

83.3%83.3%83.3%83.3%

78.9%78.9%78.9%78.9%

Tube #3Tube #3

Tube #2Tube #2

Alternate Options for PNH QC in WBCs

Step 1:Step 1: Run normal WBCs with Run normal WBCs with gating antibodies and gating antibodies and

with with GPI-linked antibodiesGPI-linked antibodies to to determine the position determine the position of of normal WBCsnormal WBCs

Step 1:Step 1: Run normal WBCs with Run normal WBCs with gating antibodies and gating antibodies and

with with GPI-linked antibodiesGPI-linked antibodies to to determine the position determine the position of of normal WBCsnormal WBCs

Step 2:Step 2: Run normal WBCs with Run normal WBCs with gating antibodies and gating antibodies and without GPI-linked without GPI-linked antibodiesantibodies to determine to determine

the the position of WBCs with position of WBCs with complete GPI-complete GPI-

DeficiencyDeficiency

Step 2:Step 2: Run normal WBCs with Run normal WBCs with gating antibodies and gating antibodies and without GPI-linked without GPI-linked antibodiesantibodies to determine to determine

the the position of WBCs with position of WBCs with complete GPI-complete GPI-

DeficiencyDeficiency

Step 3:Step 3: Run suspected PNH Run suspected PNH patient with gating patient with gating antibodies andantibodies and GPI-GPI-

linked linked antibodiesantibodies

Step 3:Step 3: Run suspected PNH Run suspected PNH patient with gating patient with gating antibodies andantibodies and GPI-GPI-

linked linked antibodiesantibodies

Presence of 54.4% PNH GranulocytesPresence of 54.4% PNH GranulocytesPresence of 54.4% PNH GranulocytesPresence of 54.4% PNH Granulocytes

Summary - WBCSummary - WBC FLAER/CD24FLAER/CD24 appears to be the preferred antibody combination to detect appears to be the preferred antibody combination to detect

PNH clones in granulocytesPNH clones in granulocytes

FLAER/CD14FLAER/CD14 is most tested combination to detect PNH clones in is most tested combination to detect PNH clones in monocytesmonocytes

Lineage-specific gatingLineage-specific gating results in higher sensitivities and cleaner WBC results in higher sensitivities and cleaner WBC assays assays – CD15 for granulocytes CD15 for granulocytes – CD64 and/or CD33CD64 and/or CD33– CD45 can be very useful for pattern-recognition (back-gating to see CD45 can be very useful for pattern-recognition (back-gating to see

what the populations are, e.g. blasts, platelets, other immature cells, what the populations are, e.g. blasts, platelets, other immature cells, debris)debris)

Analysis of 50,000 granulocytes can result in Analysis of 50,000 granulocytes can result in sensitivitysensitivity of 0.05%-0.1% of 0.05%-0.1% (25-50 PNH cells) in a clean assay(25-50 PNH cells) in a clean assay

Report both Type II and Type III granulocytes and monocytes as the Report both Type II and Type III granulocytes and monocytes as the total total PNH ClonePNH Clone if they are present if they are present

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 ResultsReporting of PNH Results

Recommended:Recommended:Recommended:Recommended:

Avoid:Avoid:Avoid: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

Reporting – ICCS PNH Positive CaseReporting – ICCS PNH Positive Case

Key information:Key information:

1.1. PNH Clone detected PNH Clone detected – – Yes or NoYes or No

2.2. PNH PNH Clone size in Clone size in WBCWBC• in granulocytesin granulocytes• in monocytesin monocytes**

3.3. PNH PNH Clone size in Clone size in RBCRBC with distribution of with distribution of • Type II cellsType II cells• Type III cellsType III cells• Total PNH Clone Total PNH Clone

sizesize4.4. Flow cytometry Flow cytometry graph graph

of PNH Cloneof PNH Clone is is provided provided

  

  

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!

Back: Medical Technologists Ashley B, Tony K, Brie S, Neisha B and Monica GFront: Medical Director M Movalia MD, Operational Director A Illingworth and S DuFresne MD

The Flow Cytometry TeamThe Flow Cytometry Team