ICCS Newsletter
Case Study Interpretation (CSI)
Identification of PNH Clones
Andrea Illingworth, MS, H(ASCP), QCYM
Dahl-Chase Diagnostic Services
Bangor, ME
aillingworth@dahlchase.com
www.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
CBC Parameter
Result
Units
Reference Range
WBC
3.4
K/uL
4.6-10.9
RBC
2.76
M/uL
4.69-6.13
Hgb
9.8
g/dL
14.1-18.1
Hct
31.2
%
43.5-53.7
MCV
113
fl
80.0-97.0
PLT
232
K/uL
142-424
% Lymphocytes
52.6
%
10-50
% Monocytes
6.3
%
0-15
% Granulocytes
41.1
%
37.80
LDH
844
units/liter
313-618
Pdf Files and LMD Files
Submitted for this ICCS PNH Positive Case
ICCS PNH Positive - RBC
ICCS PNH Positive - WBC GM ICCS PNH Positive - WBC Mo
GPA-59
FLAER-24-14-15-45
FLAER-33-14-64-45
Tube #1
Tube #2
Tube #3
The following slides will include:
 Disease Overview and important preanalytical 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)
 Rare Hematopoietic stem cell defect (2-6 cases /million)
 Somatic mutation of the PIG-A gene prevents the assembly of the GPIanchor 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
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: 000-000
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
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
PNH
clone
Difference in PNH Clones sizes between RBC and WBC
may be due to hemolysis and/or transfusion
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 24-48 hrs because
of degranulation; RBCs may be stable at 0o for 7 days
Possible WBC Reagent Combinations
Colors
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
Sensitive and Specific 5-Color PNH Panel
on 5 Color FC 500 in our Lab
Current
5C Panel
Red Blood Cells:
GPA-CD59
Granulocytes and Monocytes:
FLAER - CD24 - CD14 - CD15 - CD45
Monocytes only (Reflex):
FLAER - CD33 - CD14 - CD64 - CD45
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
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
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
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
PNH Testing - RBC
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)
 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
RBC - Normal Control (PB)
1
1. RBC gate to
gate out debris
3
2. GPA+ gate to
gate out GPAnegative cells
SS
Log
FS Log
2
3. Dot Plot GPACD59 is gated
on GPA+ RBCs
4. Single
Parameter
histogram is
also gated on
GPA+ RBCs
SS
Log
GPA (CD235a)
4
CD59-PE
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)
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
Tubes were racked vigorously: 0.5% Aggregates
No Aggregates
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)
Gating on GPA+ RBC’s
PNH clone with
complete CD59
deficiency (Type III
cells) and partial
CD59 deficiency (Type
II cells)
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)
Step 3: Run suspected PNH patient
with GPA / CD59
Presence of 6.2% PNH Type III RBCs
Type III Type II
Type I
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
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% (2550 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
PNH Testing – WBC Panel
• Granulocytes and Monocytes:
FLAER - CD24 - CD14* - CD15 - CD45
• Monocytes only (Reflex):
FLAER - 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 sample
WBC – Granulocytes/Neutrophils
Step 1: Gating out debris
Step 2: Gating on CD15+
granulocytes
Step 3: No PNH Clone
detected in CD15++
Granulocytes
Normal Peripheral Blood sample
WBC - Monocytes
• As the panel contains FLAER-CD24-14-15-45, 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+ Patient
WBC (Granulocytes/Neutrophils)
Step 1: Gating out debris
Step 2: Gating on CD15+
granulocytes
Step 3: Identification
of PNH Clone in
CD15++ Granulocytes
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%)
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%
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
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
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
Reporting – ICCS PNH Positive Case
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
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!
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