Recommended reagents

Reliable testing for paroxysmal nocturnal haemoglobinuria (PNH) includes the detection and quantification of PNH clones by a high-sensitivity flow cytometry assay. This method requires the selection of suitable monoclonal antibodies/reagents for target cell gating and at least two different antibodies/reagents recognising surface glycosylphosphatidylinositol (GPI)-linked proteins. When testing for PNH clones, white blood cells (WBCs) [granulocytes and monocytes] and red blood cells (RBCs) should be analysed. Lymphocytes are not a suitable target population due to their long lifespan but are helpful as an internal control.

WBC analysis

The following table, adapted from the 2010 International Clinical Cytometry Society (ICCS) guidelines, shows suggested WBC antibody/fluorochrome combinations for multicolour analysis on instrumentation platforms equipped with photomultiplier tubes (PMT) for 3- to 6-colour detection. Each approach has relative advantages and disadvantages.

Suggested WBC reagent combinations for multicolour analysis1

Cells Colours
1 2 3 4 5 6
3 coloura Granulocytes FLAER CD24 CD15
3 coloura Monocytes FLAER CD14 CD33
4 colour Granulocytes FLAER CD24 CD15 CD45
4 colour Monocytes FLAER CD14 CD64 (CD33) CD45
4 colour Granulocytes + monocytes FLAER CD24 CD14 CD33
5 colour Granulocytes + monocytes FLAER CD24 CD14 CD15 CD45
5 colour Granulocytes + monocytes FLAER CD24 CD14 CD15 CD64 (CD33)
5 colour Granulocytes + monocytes FLAER CD157 CD15 CD15 CD64 (CD33)
6 colour Granulocytes + monocytes FLAER CD24 CD14 CD15 CD45 CD64
6 colour Granulocytes + monocytes FLAER CD24 CD14 CD15 CD45 CD33
aNo longer recommended
Markers in bold red detect PNH cells (PNH cells are the negative population); the remainder are used for target cell gating
Fluorochrome choice can vary (see recommendations below)
FLAER, fluorescent aerolysin; WBC, white blood cell

3-colour combinations:2

  • No longer recommended as these combinations can only include either CD45 or a lineage-specific gating antibody, not both

4-colour combinations (described in detail in the 2012 Practical Guidelines):2,3

  • Regarded as the simplest and most reliable set-up (two GPI markers, a lineage-specific marker and CD45)
    • Fluorescent aerolysin (FLAER)-CD24-CD15-CD45 (granulocyte tube)
    • FLAER-CD14-CD64-CD45 (monocyte tube)
  • Less demanding concerning instrumentation requirement and spectral overlap compensation
  • CD64 is assumed to be superior to CD33 for monocyte gating since basophils can be largely excluded (basophils show intermediate CD33+/decreased FLAER expression and may be misinterpreted as PNH cells)
  • Broad availability of commercial gating monoclonal antibodies conjugated with bright fluorochromes (better discrimination and gating of target populations)

5-colour combinations:2,4

  • Cannot include the three necessary GPI-linked antibodies/reagents (FLAER, CD24, CD14), two lineage-specific antibodies (CD15, CD64) and CD45 for debris exclusion in one tube
  • Represents an option if CD157 is used instead of CD24 and CD14 as the second GPI-linked antibody in addition to FLAER
    • FLAER-CD157-CD64-CD15-CD45 (for both monocytes and granulocytes)
  • Less time consuming (single tube)
  • More cost effective

6-colour combinations:2

  • Provide optimal gating through the combination of three GPI markers, two lineage-specific markers and CD45
    • FLAER-CD24-CD14-CD15-CD64-CD45 (for both monocytes and granulocytes)1,3
  • Less time consuming
  • More cost effective
  • Voltage set-up/compensation is often more challenging when compared to 4-colour combinations
  • Availability of antibody clones labelled with recommended fluorochromes can be limited
  • Require the selection of antibodies/fluorochromes with good signal/noise ratio and accurate verification of compensation settings to ensure the correct interpretation of dot plots

The most frequently used antibody/reagent combinations for granulocyte and monocyte testing are FLAER/CD24 and FLAER/CD14. CD157 is an additional promising reagent for high-quality identification of both PNH granulocytes and monocytes in a single-tube 5-colour assay.4

Other GPI-specific antibodies (such as CD16 and CD66b) are not recommended, as CD16 deficiency is also observed in eosinophils while CD66b is only available as the FITC conjugate, thus precluding the use of FLAER, which is considered mandatory for PNH testing.

Lineage-specific gating is crucial; the choice of gating antibody determines the level of sensitivity. CD45 should always be used to gate out CD45-negative debris as well as backgating. This is particularly important for the accurate identification of small/minor clones.

To produce interpretable histograms and dot plots, it is advisable for all antibodies to be properly titrated.

Please see the example of an antibody titration assay for 4-colour or 6-colour assays.

The 2012 Practical Guidelines published by Sutherland et al provide more detailed guidance concerning optimal antibody clone and conjugate selection, specific reagent combinations for the most common instrument platforms and analytical strategies for high-quality detection of PNH RBCs and WBCs.3

WBC 4-colour panel for Beckman Coulter platforms2

Target cells Antibodies and fluorochromes Purpose Clone (vendor)
WBC CD15-PC5 Gating for granulocytes 80H5 (BC)
CD45-PC7 Backgating J33 (BC)
FLAER GPI linked NA (Cedarlane)
CD24-PE GPI linked ALB9 (BC)
SN3 (eBioscience)
CD64-PC5 Gating for monocytes 22 (BC)
CD14-PE GPI linked RMO52 (BC)
61D3 (eBioscience)
BC, Beckman Coulter; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; PC, phycoerythrin cyanine; PE, phycoerythrin; WBC, white blood cell

WBC 4-colour panel for Becton Dickinson platforms2

Target cells Antibodies and fluorochromes Purpose Clone (vendor)
Granulocytes (neutrophils) CD15-APC Gating for granulocytes H198 (BD)
CD45-PerCP Backgating 2D1 (BD)
FLAER GPI linked NA (Cedarlane)
CD24-PE GPI linked SN3 (eBioscience)
ML5 (BD)
Monocytes CD64-APC Gating for monocytes 10.1 (BD)
CD45-PerCP Backgating 2D1 (BD)
CD14-PE GPI linked MφP9 (BD)

61D3 (eBioscience)

FLAER GPI linked NA (Cedarlane)
APC, allophycocyanin; BD, Becton Dickinson; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; PE, phycoerythrin; PerCP, peridinin chlorophyll

WBC 6-colour panel1,4

Antibodies / reagents Purpose Clones recommended for Beckman Coulter Clones recommended for Becton Dickinson
FLAER GPI linked NA (Cedarlane) NA (Cedarlane)
CD14 GPI linked RMO52 (BC)
61D3 (eBioscience)
MφP9 (BC)
61D3 (eBioscience)
CD15 Gating for granulocytes 80H5 (BC) HI98 (BC)
MMA (eBioscience)
CD24 GPI linked ALB9 (BC)
SN3 (eBioscience)
ML5 (BC)
SN3 (eBioscience)
CD45 Backgating J33 (BC) 2D1 (BC)
CD64 Gating for monocytes 22 (BC) 10.1 (BC)
FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; NA, not available

Different fluorochrome conjugates are possible depending on laser and detector configuration of each instrument. For optimal fluorochrome selection see interactive wizards:

To see examples of these antibodies in action, please visit the netflow case studies.

RBC analysis

2-colour assay (with CD59 and CD235a):2

  • CD235a provides optimal lineage-specific gating on RBCs.
    • CD235a can also serve as a quality-control indicator to validate optimal RBC staining (eg blood drops on the sides of tubes that have not been stained with antibody may cause artefactual decrease of CD235a and CD59).
    • CD235a causes significant aggregation if not appropriately titrated and fluorescein isothiocyanate (FITC) is the preferred conjugate (rather than phycoerythrin [PE], which is prone to higher agglutination).
  • CD59 shows the best signal/noise ratio (there is some variation across CD59 clones: MEM43 and OV9A2 work well).
    • PE is the recommended conjugate.
    • CD55 is not recommended due to its low staining intensity.
Target cells Antibodies and fluorochromes Purpose Clone
RBC CD59-PE GPI linked MEM43 (Invitrogen/eBioscience)
OV9A2 (eBioscience)
CD235a-FITC Gating for RBCs KC16 (BC)
BC, Beckman Coulter; FITC, fluorescein isothiocyanate; GPI, glycosylphosphatidylinositol; PE, phycoerythrin; RBC, red blood cell
  • References
    1. Borowitz MJ et al. Cytometry B Clin Cytom 2010; 78B: 211-230.
    2. Marinov I et al. Cytometry B Clin Cytom 2013; 84: 229-236.
    3. Sutherland DR et al. Cytometry B Clin Cytom 2012; 82B: 195-208.
    4. Sutherland DR et al. Cytometry B Clin Cytom 2014; 86B: 44-55.

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