6-colour guide

Step 1 – Set up

Instrument set-up and optimisation

For accurate and reliable paroxysmal nocturnal haemoglobinuria (PNH) analysis it is recommended to perform the following:

Daily performance checks

Daily performance checks to measure the variation from the baseline from run to run.

Beckman Coulter platform

Use Flow Check® fluorospheres, following the manufacturer’s instructions.

Becton Dickinson platform

Use Cytometer Setup and Tracking (CS&T®) beads, following the manufacturer’s instructions.

Colour compensation adjustment

Beckman Coulter platform

  • Use the VersaComp Antibody Capture Bead kit or unstained whole blood cells (granulocytes and/or monocytes) to set up photomultiplier tube (PMT) voltages and compensation to correct for spectral overlap.

Becton Dickinson platform

  • Use BD™ CompBeads or unstained whole blood cells (granulocytes and/or monocytes) and compensation to correct for spectral overlap.
  • Use single colour stained microspheres/whole blood cells with each flurochrome used in your application.

Verification of correct setting with an application control

  • Update the control protocol with the settings derived from below. Run a biological control equivalent to the application or normal whole blood.

Area scaling

  • Correction factor is set to match the area to the height measurement (shown below).
    • Use unstained whole blood cells (granulocytes, monocytes).
    • Use a forward scatter (FS) versus side scatter (SS) dot plot to gate granulocytes (monocytes).
    • Use FS-A versus FS-W and SS-A versus SS-W dot plots to define singlets.
    • Use FS-H and FS-A histograms to compare the mean channel values.
    • Set the FS area scaling factor to obtain identical values for FS-H and FS-A, respectively.

 

Scaling Adjustment
Example of area scaling adjustment for BD™ FACSCanto instrument.

PMT settings

  • Use BD™ CompBeads or unstained whole blood cells (granulocytes and/or monocytes) for optimal baseline PMT voltage setting shown in the figure below.
    • Use FS versus SS dot plot to optimize FS/SS voltages (place the population of interest on scale) and gate granulocytes (monocytes).
    • Use FS-A versus FS-W and SS-A versus SS-W dot plots to define singlets.
    • Use single parameter histograms for all six fluorochromes used in the application to optimise the PMT voltages in order to obtain values ≥2.5 robust standard deviations of the baseline setting. This step should enable differentiation of dim populations from the negative population and place positive populations entirely on scale.

 

Optimal PMT Setting
Example of optimal PMT setting for granulocytes (≥2.5 robust standard deviations of the baseline setting) on a BD™ FACSCanto instrument.

Spectral overlap compensation

  • For multicolour analysis performance of automatic spectral overlap compensation using single colour stained samples (compensation beads or normal whole blood cells) with each fluorochrome used in your application is recommended (as shown below).

 

Spectral Overlap Compensation
Spectral overlap compensation details for a 6-colour protocol for granulocyte and monocyte analysis on a BD™ FACSCanto instrument.
These protocols were developed in close collaboration with Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA, Drs Thomas Matthes and Mathieu Hauwel of the Swiss Flow Cytometry School at the University Hospital of Geneva, Switzerland, and Dr Iuri Marinov of Hematology and Blood Transfusion in Prague, Czech Republic. Images were provided with permission from the netflow Steering Committee and Swiss Flow Cytometry School.

STEP 2

Step 2 – Stain and acquire

Principle

Cells are stained with lineage-specific antibodies to clearly identify populations of interest. Glycosylphosphatidylinositol (GPI)-anchored protein deficiency is tested using specific antibodies (eg CD14, CD24, CD59, CD157) and a GPI-binding reagent (fluorescent aerolysin [FLAER]).

Methods: white blood cells (WBCs)

Single tube, 6-colour panel for simultaneous analysis of paroxysmal nocturnal haemoglobinuria (PNH) granulocytes and monocytes

The table below summarises the recommended reagents (monoclonal antibody clones) for white blood cell (WBC) analysis on Beckman Coulter and Becton Dickinson instruments. Each antibody should be titrated to determine optimal working concentration. Different fluorochrome conjugates are possible, depending on laser and detector configuration of each instrument. For optimal fluorochrome selection, see interactive wizards:

Recommended reagents/monoclonal antibody clones for 6-color WBCs analysis:

Antibodies/reagents Purpose Beckman Coulter Becton Dickinson
FLAER GPI-linked NA (Cedarlane) NA (Cedarlane)
CD14 GPI-linked RMO52
61D3
MφP9
61D3
CD15 Gating for granulocytes 80H5 HI98, MMA
CD24 GPI-linked ALB9
SN3
ML5
SN3
CD45 Backgating J33 2D1
CD64 Gating for monocytes 22 10.1

 

  1. Invert the tube with the whole blood several times to mix the sample thoroughly.
  2. Take 100 µL of blood and place it in the bottom of a 5-mL tube.
    • Be careful not to leave any blood on the edges of the tube.
  3. Add the corresponding amount of previously titrated antibodies (antibody cocktail) and mix by gentle vortexing.
  4. Incubate at room temperature in the dark for 30 minutes.
    • Be careful not to expose the antibodies to light.
  5. Add an appropriate amount of vendor-specific or any other lysing solution and follow the specific protocol.
  6. Add 2 mL phosphate-buffered saline (PBS) with albumin (PBA) and spin for 3 minutes at 300 g.
  7. Discard supernatant.
  8. Resuspend in 0.5 mL PBA.
  9. Vortex to dissociate aggregates.
  10. Acquire at least 50,000 CD15+ granulocytes, using an appropriate acquisition template.

Methods: red blood cells (RBCs)

  1. Invert the tube with the whole blood several times in order to mix the sample thoroughly.
  2. Add 1 mL PBS to the tube.
  3. Add 10 µL peripheral blood (1:100 dilution).
  4. Take 100 µL 1:100 diluted blood twice and place it in the bottom of two 5-mL fluorescence-activated cell sorting (FACS) tubes.
    • Be careful not to leave any blood on the edges of the tube
  5. Add the appropriately titrated antibody cocktail and mix by gentle vortexing.
    Antibodies/reagents Fluorochrome FITC PE
    Erythrocyte cocktail Test CD235a CD59
    Controla CD235a x
     aThis control is not required for everyday testing.
    FITC, fluorescein isothiocyanate; PE, phcoerythrin
  6. Incubate at room temperature in the dark for at least 20 minutes (up to 60 minutes).
    • Be careful not to expose the antibodies to light.
  7. Add 2 mL PBS and spin for 3 minutes at 300 g.
  8. Discard supernatant.
  9. Add 2 mL PBS and spin for 3 minutes at 300 g.
  10. Discard supernatant.
  11. Resuspend the pellet in 500 µL PBS.
  12. ‘Rack’ the tube vigorously 3–4 times to dissociate aggregates.
  13. Acquire at least 50,000 CD235a+ RBCs using an appropriate RBC acquisition template (see Step 3 – Analyse). If a small PNH clone is identified, acquire more events to increase confidence in the analysis, as 100 clustered PNH cells are typically required to verify the presence of a true PNH clone.
    • Acquire within 15 minutes as the staining intensity of CD235a fades rapidly.
These protocols were developed in close collaboration with Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA, Drs Thomas Matthes and Mathieu Hauwel of the Swiss Flow Cytometry School at the University Hospital of Geneva, Switzerland, and Dr Iuri Marinov of Hematology and Blood Transfusion in Prague, Czech Republic.

STEP 1STEP 3

Step 3 – Analyse

Dot plot layout for granulocytes and monocytes

Dot plot layout for single tube, 6-color simultaneous analysis of granulocytes and monocytes

Create a template according to the following layout:

  1. Dot plot FS lin/SS lin for elimination of debris
  2. Dot plot CD45 log/SS lin for leukocyte gating
  3. Dot plot CD15 log/SS lin for granulocyte gating
  4. Dot plot CD64 log/SS lin for monocyte gating
  5. Dot plot FLAER log/CD14 log for PNH monocyte identification
  6. Dot plot FLAER log/CD24 log (gate on lymphocytes) for compensation check
FLAER, fluorescent aerolysin; FS, forward scatter; SS, side scatter

Gating strategy for granulocytes

  • Perform analysis of the acquired sample following the described gating strategy:
Step 1: Gate granulocytes on CD15 versus SS lin and backgate on CD45 versus SS lin and FS lin versus SS lin to discriminate immature granulocytes and eosinophils.
Step 2: Identify PNH granulocytes on FLAER versus CD24.
Step 3: Check compensation on FLAER versus CD24 gated on Ly: you should discriminate B, T, NK, Ba and PNH-Ly.

 

Gating strategy for monocytes

  • Perform analysis of the acquired sample following the described gating strategy:
Step 1: Gate monocytes on CD64 versus SS lin and backgate on CD45 versus SS lin and FS lin versus SS lin to discriminate dendritic cells and apoptotic/necrotic monocytes.
Step 2: Identify PNH monocytes on FLAER versus CD14.
Step 3: Check compensation on FLAER vs CD24 gated on Ly: you should discriminate B, T, NK, Ba and PNH-Ly.

Dot plot layout for red blood cells (RBCs)

Create a template according to the following layout:

  1. Dot plot FS log/SS log for RBC gating
  2. Dot plot CD235a log/SS log for RBC gating on CD235a
  3. Dot plot FS int log/CD235a log to visualise RBC aggregates/doublets
  4. Dot plot of CD235a/CD59 and CD59 single-parameter histogram to determine CD59 expression or CD59 absence on CD235a+ RBCs
  5. Dot plot CD59 log/CD235a log to visualise co-expression
FS, forward scatter; RBC, red blood cell; SS, side scatter

Dot plot layout for RBCs

This set-up of dot plots and gating strategy arrives at the diagnostic dot plot CD235a/CD59, which allows for verification of whether the Type III gate is clean in case of a normal blood sample. There might be some events ‘straggling’ into the Type II RBC area, but this is most likely due to suboptimal staining of occasional normal RBCs. True Type II PNH RBCs have high CD235a staining (unlike in this case).
FITC, fluorescein isothiocyanate; FS, forward scatter; PE, phycoerythrin; RBC, red blood cell; SS, side scatter

Gating strategy for RBCs

  • Perform analysis of the acquired sample according the following gating strategy:

Evaluation of PNH RBCs by flow cytometry

Step 1: Gate RBCs on FS log versus SS log.
Step 2: Gate RBCs on FS log versus CD235a.

 

Step 3: Check for doublets. If >2%, rack rigorously again.

 

Step 4: Display RBCs on CD59 versus CD235a dot plot and determine Type III (CD59 negative) and Type II (CD59 partially negative) PNH RBCs.
FITC, fluorescein isothiocyanate; FS, forward scatter; PE, phycoerythrin; RBC, red blood cell; SS, side scatter
These protocols were developed in close collaboration with Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA, Drs Thomas Matthes and Mathieu Hauwel of the Swiss Flow Cytometry School at the University Hospital of Geneva, Switzerland, and Dr Iuri Marinov of Hematology and Blood Transfusion in Prague, Czech Republic. Images were provided with permission from the netflow Steering Committee and Swiss Flow Cytometry School.

STEP 2STEP 4

Step 4 – Report

Standardised terminology for PNH clones1

A glycosylphosphatidylinositol (GPI)-deficient population of cells is the terminology used to describe the absence of GPI-linked proteins on red blood cells (RBCs), granulocytes and monocytes. Paroxysmal nocturnal haemoglobinuria (PNH) clone size is determined by the size of the GPI-deficient population in the largest of the white blood cell lineages tested (ie granulocytes or monocytes). The sensitivity of the assay should be determined by performing a ‘spiking’ assay (see ‘Assay validation’ for 6-colour protocol), which describes the lower limit of detection.

GPI-deficient population Standardised terminology
>1% Population of GPI-deficient cells (granulocytes or monocytes) or ‘PNH clone’
0.1–1% Minor population of GPI-deficient cells (granulocytes or monocytes) or ‘minor PNH clone’
<0.1% Rare GPI-deficient cells or ‘rare cells with PNH phenotype’
GPI, glycosylphosphatidylinositol

Reporting PNH test results1

  • Report the proportions (%) of GPI-deficient cells in each lineage tested:
    • Type III RBCs (GPI-deficient; CD235a+, CD59-)
    • Type II RBCs (partially GPI-deficient; CD235a+, CD59-intermediate)
    • Total proportion of Type III plus Type II RBCs
    • GPI-deficient granulocytes/neutrophils (CD15+, FLAER-, CD157-)
    • GPI-deficient monocytes (CD64+, FLAER-, CD157-)
  • To avoid confusion, do not report Type I cells with normal CD59 expression.
  • Avoid the use of ambiguous language, such as ‘positive’ or ‘negative’, when reporting test results (eg ‘the PNH test was negative’ may be interpreted as that the test was negative for a marker and, therefore, positive for PNH).
    • Clear language such as ‘deficiency of GPI-linked proteins’ should be used.1 Laboratory results can then be combined with clinical findings to confirm or determine the clinical diagnosis.
  • Include details of which reagents were used to confirm that high-sensitivity flow cytometry was appropriately performed.
  • Report the level of assay sensitivity separately for RBCs and white blood cells (WBCs) to report the lower limit of detection in each lineage (ie how many PNH cells need to be present for them to be detected, compared with background levels in normal samples?).
  • Include results from current and previous assessments to allow clinicians to easily observe any trends in PNH clone size over time.

Interpretation of PNH test results

  • In the case of minor or significant GPI-deficient populations/PNH clones, further testing relating to haemolysis may be indicated to determine the next steps in patient management.
  • If supporting clinical evidence is provided indicating aplastic anaemia or myelodysplastic syndrome, a recommendation for further testing should be included on the report.

For further information on reporting, see the following links from the Canadian PNH Network:

 

These protocols were developed in close collaboration with Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA, Drs Thomas Matthes and Mathieu Hauwel of the Swiss Flow Cytometry School at the University Hospital of Geneva, Switzerland, and Dr Iuri Marinov of Hematology and Blood Transfusion in Prague, Czech Republic.

STEP 3ASSAY VALIDATION

  • References
    1. Davis BH et al. CLSI H52-A2 Red Blood Cell Diagnostic Testing Using Flow Cytometry; Approved Guideline, 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute. 2014.

Assay validation

Improper validation of flow cytometry assays is one factor responsible for reported interlaboratory variability in paroxysmal nocturnal haemoglobinuria (PNH) testing. To ensure the high-quality detection of PNH clones, assay validation should be performed as part of routine and high-sensitivity PNH testing.

The PNH assay validation process should include the proper titration of antibodies, spiking experiments to determine assay sensitivity, and the correct use of controls for confirming antibody and instrument performance.

Antibody titration assay

To produce interpretable histograms and dot plots, the proper titration of antibodies is recommended. Optimising antibody concentration reduces background noise due to non-specific binding and also significantly reduces costs. In principle, antibodies are diluted serially and used in a single-staining assay. Mean fluorescence intensity is plotted against antibody amount to draw the titration curve.

Protocol

This protocol is given for an antibody whose recommended working volume is 20 µL (for 100 µL of cells). This volume was selected as a starting point for subsequent dilutions. Adapt this protocol to any other antibody by adjusting the starting point to the manufacturer’s recommendation.

  1. Fill 10 flow cytometry measurement tubes with 100 µL cell suspension.
  2. Fill five microtubes with 10 µL phosphate-buffered saline (PBS).
  3. Dilute the commercial antibody serially in microtubes by carrying 10 µL each time to the next tube (mix well and change pipette tip). See table below.
  4. Add decreasing antibody volumes of commercial antibody stock (from 20 µL down to 6 µL) to the cell suspension.
  5. Add 8 µL diluted antibody to the cell suspension.
  6. Incubate, wash and acquire as usual.
Volumes, µL Decreasing volume Serial dilutions
Antibody 10 10a 10a 10a 10a
PBS 10 10 10 10 10
Stain added to 100 μL cell suspension 20 10 8 6 8 8 8 8 8
Final amount 20 10 8 6 4 2 1 0.5 0.25
aPipette from previous tube; dash indicates non-applicable
PBS, phosphate-buffered saline

 

PBS, phosphate-buffered saline

Determining quantity of antibody to use in assay

To determine optimal antibody quantities for flow cytometry assays, use the lowest concentration of antibody that results in at least 80% of the maximum signal intensity. By reducing the antibody concentration, background noise due to non-specific binding is reduced (as well as saving on cost).

  • CD235a (glycophorin A [GPA]): The plateau is not reached even when using 20 µL of antibody, which is the amount recommended by the manufacturer. Given the shape of the dilution curve, using less antibody is not advisable.
  • CD59: As low as 0.5 µL of antibody yields 85% of the maximum staining intensity. This would be perfectly acceptable. However, to avoid dilution effects for samples that are highly cellular, a safety margin must be applied. Between 1–2 µL of antibody should be used (eg 1.5 µL).

 


MFI, mean fluorescence intensity

RBC spiking assay

Every laboratory should perform a ‘spiking’ assay to assess the sensitivity of the PNH test

Sensitivity refers to the smallest number of cells that can specifically be detected with a particular assay. The typical acquisition number for granulocytes is 50,000 cells. The number of monocytes is typically lower and thus results in lower sensitivity than that achieved with granulocytes.

A ‘spiking’ assay, which is basically a serial dilution of PNH blood with normal blood, can verify the sensitivity of detecting PNH clones at lower dilutions.

In case PNH blood is not available, PNH clones can be mimicked by using only gating markers and not glycosylphosphatidylinositol (GPI)-linked markers in the staining solution. For example:

  • Red blood cells (RBCs), 2-colour panel: X/CD235a will show location of CD59-negative RBCs
  • Granulocytes, 4-colour panel: X/X/CD15/CD45 will show the location for fluorescent aerolysin (FLAER)/CD24-negative granulocytes
  • Monocytes, 4-colour panel: X/X/CD64/CD45 will show the location of FLAER/CD24-negative monocytes

The detailed protocol below shows how to establish RBC assay sensitivity based on a normal blood sample. In this case, sensitivity is determined by diluting RBCs stained only for GPA (therefore negative for CD59) into RBCs stained for both GPA and CD59, thus mimicking PNH clones of increasing size.

Protocol

  1. Dilute 5 µL fresh blood in 495 µL PBS or any other flow cytometry measurement (FCM)-compatible buffer.
  2. Incubate 500 µL diluted blood with 100 µL anti-GPA (CD235a fluorescein isothiocyanate [FITC] clone KC16; use at 20 µL/100 µL cells) antibody and 10 µL anti-CD59 antibody (clone MEM43; use at 2 µL/100 µL cells) for 15 minutes in the dark.
  3. Incubate 110 µL diluted blood with 22 µL anti-GPA antibody for 15 minutes.
  4. Spin at 500 g for 3 minutes and carefully discard supernatant.
  5. Resuspend in 1 mL CellFixTM.
  6. Incubate at room temperature for 30 minutes in the dark.
  7. Add 2 mL PBS; then spin at 500 g for 3 minutes and carefully discard supernatant.
  8. Resuspend in 600 µL and 110 µL PBS, respectively.
  9. Rack vigorously (or pipette) to dissociate aggregates.
  10. Pipette 100 µL single-stained RBCs in an FCM tube and acquire 250,000 events (tube 1).
  11. Distribute 90 µL double-stained RBCs in tubes 2-6 and 100 µL in tube 7.
  12. Take 10 µL single-stained RBCs and dilute serially in tubes 2-6 by carrying 10 µL each time to the next tube.
  13. Acquire 1,000,000 events.
Tube CD235a CD59 CD235a PNH clone
1 0 100 100%
2 90 10 10%
3 90 10 1%
4 90 10 0.10%
5 90 10 0.01%
6 90 10 0%
7 100 0 0%

Data analysis

The tables below show the results of a spiking experiment in which a PNH blood sample was spiked with normal blood. The same experiment can be used to check the sensitivity of detecting PNH monocytes, although the number of monocytes is typically lower and thus results in lower sensitivity than that achieved with granulocytes.

4-colour granulocyte assay sensitivity

Dilution PNH granulocytes Sensitivity, %
Neat 51,420 91.3
1:10 5799 9.4
1:100 573 0.94
1:1000 91 0.089
1:10,000 9 0.01


4-colour monocyte assay sensitivity

Dilution PNH monocytes Sensitivity, %
Neat 12,718 89.8
1:10 1227 4.1
1:100 112 0.4
1:1000 13 0.044
1:10,000 ND ND
ND, not detected

2-colour RBC assay sensitivity (based on 1,000,000 RBCs collected in data files)

Dilution Type III RBCs Sensitivity, %
Neat 348,626 34.86
1:10 17,665 1.77
1:100 1822 0.18
1:1000 203 0.02
1:10,000 18 0.0018
RBCs, red blood cells

Quality control on lymphocytes

In addition to optimising antibody concentrations and validating the sensitivity of the assay, quality controls should be put in place to confirm antibody and instrument performance.

Lymphocytes, although not recommended for the detection of PNH clones, can serve as excellent quality-control material.

Compensation check on lymphocytes

FLAER, fluorescent aerolysin; FS, forward scatter; PE, phycoerythrin; SS, side scatter

The top row shows gating dot plots for PNH testing, the middle row shows diagnostic dot plots for PNH testing, and the bottom row shows some additional ‘control’ dot plots using PNH lymphocytes (red box).

Starting from left to right, the first dot plot (FLAER/CD24) shows good signal-to-noise ratio for the FLAER-positive normal lymphocytes versus the FLAER-negative PNH lymphocytes; both populations are visibly ‘on scale’. The purpose of the PNH lymphocytes is not to assess the PNH clone (which is always much smaller than in the granulocytes), but rather to ensure that the negative cells are visible and not ‘crushed’. This dot plot also shows the FLAER+/CD24+ B-cells and includes confirmation that CD24 was added. This may be important for cases with no, or rare, PNH granulocytes, which could also be seen if the antibody were not added.

The second (FLAER/CD14-ECD) and third (FLAER/CD15-PC5) dot plots also serve as confirmation for antibody performance and instrument settings.

These protocols were developed in close collaboration with Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA, Drs Thomas Matthes and Mathieu Hauwel of the Swiss Flow Cytometry School at the University Hospital of Geneva, Switzerland, and Dr Iuri Marinov of Hematology and Blood Transfusion in Prague, Czech Republic. Images were provided with permission from the netflow Steering Committee and Swiss Flow Cytometry School.

STEP 4TESTING PITFALLS

Testing pitfalls

Sample processing pitfalls

Many of the issues encountered in paroxysmal nocturnal haemoglobinuria (PNH) testing can be remedied by careful sample preparation.

Washing

Washing is a prime example of how sample preparation can have a major impact on the results of PNH testing. Failing to wash red blood cell (RBC) samples following antibody incubation can lead to false-negative results.

Results obtained from unwashed RBCs versus those washed twice

PE, phycoerythrin; RBC, red blood cell

Washing the cells reduces the background noise and reveals a PNH population that would otherwise have gone undetected.

Andrea Illingworth explains the importance of washing RBCs (see figures above)

Racking

Incomplete re-suspension of sample cells can also lead to aggregates, which make results more difficult to interpret. Vigorous ‘racking’ (ie rubbing the tube along the top of a plastic tube rack) is required to reduce the percentage of aggregates in a sample.

Differences between samples that have been lightly vortexed versus vigorously racked

 


FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin

Light vortexing leads to 29% aggregates, while vigorous racking reduces this to 0.5% aggregates.

Andrea Illingworth explains the importance of racking (see figure above)

Filtering

It is also very important to filter reagents to avoid artefacts during PNH testing.

Normal control sample gated on GPA positivity and stained for CD59

FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin; RBC, red blood cell; SS, side scatter

The top panels show what appears to be a low (1.5%), if diffuse, population of CD59-deficient cells; however, when the gate is expanded to include intermediate glycophorin A (GPA)-stained cells (bottom panels), there appears to be a large population (14.7%) of CD59-deficient cells. These events do not represent CD59-deficient cells but are more likely the result of debris or unfiltered phosphate-buffered saline.

Other confounding factors

Andrea Illingworth discusses other confounding factors

Staining with CD59 and GPA

Importance of staining cells with CD59 and GPA

Staining samples for both CD59 and GPA allows the separation of true Type II cells from inadequately stained normal RBCs.

Analysis of a sample from a patient with a small Type III PNH clone (0.2%)

FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin

The stream of CD59-positive, intermediate GPA-stained cells seen in the top panel may be misinterpreted as a 2.0% population of PNH Type II cells.

Andrea Illingworth on how CD235a (GPA) versus CD59 provides quality control

Separating Type II from Type III RBCs

CD59 is recommended over CD55 for staining RBCs.

CD55 and CD59 staining in RBCs

PE, phycoerythrin; RBC, red blood cell

The figure clearly demonstrates that CD59 (top panels) has a better signal-to-noise ratio than CD55, allowing separation of Type II from Type III RBCs. CD55 (bottom panels) is unable to separate Type II from Type III RBCs, so only one PNH population is evident.

Andrea Illingworth talks about CD55 and CD59 staining in RBCs (see figure above)

Compensation

Importance of optimising compensation

Optimising compensation is very important.

FITC, fluorescein isothiocyanate; PE, phycoerythrin

The top-left dot plot shows a clear Type III PNH population, which is also seen on the bottom-left single-parameter histogram. Comparing the top-right dot plot and the single-parameter histogram on the bottom right, the histogram appears to show a prominent Type II population, whereas the dot plot above clearly shows a compensation issue (‘stab-like’ appearance of population in the Type II gate).

Gating

Importance of correct gating for RBC testing

Example of incorrect gate setting

FITC, fluorescein isothiocyanate; PE, phycoerythrin

At first glance, the two top graphs seem to show similar results to the dot plot and histogram below. However, the percentage of Type III cells is very different. This is due to the Type III gate (top-left dot plot) not including all negative events (it is just one channel from the baseline that excludes a large portion of the negative events). This sample also had an overcompensation issue, which pushed the negative events further to the left.

False positives

Potential false positives when testing white blood cells

Similarities between the side scatter of true PNH populations versus those of normal cells

FLAER, fluorescent aerolysin; PE, phycoerythrin; SS, side scatter

The chart on the top left shows a true PNH clone (blue) and the normal population (pink); the chart on the bottom left highlights the overlap in the side scatter of these populations. On the right-hand side, we see an immature blast cell population, with the bottom-right panel demonstrating how these cells may be misinterpreted as a PNH clone.

Andrea Illingworth explains potential false positives in white blood cells (WBCs) (see figure above)

Potential false positives caused by inappropriate gating

Gating with CD15 can be useful for excluding debris that might otherwise be misinterpreted as a PNH population.

What can happen if only CD45 gating is applied

FLAER, fluorescent aerolysin; PE, phycoerythrin; SS, side scatter

The top row illustrates a gating strategy using CD15 to remove unwanted debris from the sample. In the bottom row, where only CD45 versus side scatter has been used for gating, it can be seen that some of the population falls into the area predefined as PNH population; in reality, this population is most likely platelets and other debris.

Andrea Illingworth talks about potential false positives caused by gating (see figure above)

Lineage-specific gating

Lineage-specific gating using CD33 or CD64 instead of CD45 achieves the purest population of monocytes, improving the accuracy of PNH testing.

Importance of lineage-specific gating of monocytes

FLAER, fluorescent aerolysin; SS, side scatter

Although dual-parameter analysis with CD14 and fluorescent aerolysin (FLAER) and gating strategies using CD33 or CD64 can improve the accuracy of PNH testing in monocytes, downregulation of CD14 as a result of monocyte differentiation into dendritic cells can lead to inaccurate analysis of monocytes, where a small number of cells may look glycosylphosphatidylinositol (GPI) negative. Therefore, granulocytes are still recommended as the most appropriate white blood cells to test for PNH.

Andrea Illingworth explains the importance of lineage-specific gating in monocytes (see figure above)

Images were provided with permission from Mrs Andrea Illingworth of Dahl-Chase Diagnostic Services in Bangor, ME, USA.

ASSAY VALIDATION


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