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) [neutrophils 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 2018 International Clinical Cytometry Society (ICCS)/European Society for Clinical Cell Analysis (ESCCA) consensus guidelines,1 shows tested and validated WBC antibody/reagent combinations for multicolour analysis on instrumentation platforms equipped with photomultiplier tubes (PMT) for 4- to 7-colour detection.2 Three-colour combinations are no longer recommended as these combinations can only include either CD45 or a lineage-specific gating antibody, not both.1-4 It is recommended that the antibody/reagent combinations are prepared as a cocktail, rather than adding each one individually, to minimise pipetting errors and subsequent inaccurate results. Each approach has relative advantages and disadvantages.

Suggested WBC reagent cocktails for multicolour analysis1-3

Cells Colours
1 2 3 4 5 6 7
4 colour Neutrophils FLAER CD24 CD15 CD45
4 colour Monocytes FLAER CD14 CD64 (CD33) CD45
5 colour Neutrophils + monocytes FLAER CD157 CD15 CD64 CD45
6 colour Neutrophils + monocytes FLAER CD24 CD14 CD15 CD64 CD45
6 colour Neutrophils + monocytes FLAER CD24 CD157 CD15 CD64 CD45
6 colour Neutrophils + monocytes CD157 CD24 CD14 CD15 CD64 CD45
7 colour Neutrophils + monocytes FLAER CD157 CD24 CD14 CD15 CD64 CD45
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)

 

4-colour combinations (described in detail in the 2018 consensus guidelines):1,4,5

  • Regarded as the simplest and most reliable set-up (two GPI markers, a lineage-specific marker and CD45).
    • Fluorescent aerolysin (FLAER)-CD24-CD15-CD45 (neutrophil 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:1,4,6

  • Represents a convenient option if CD157 is used to replace CD24 and CD14 as a GPI-linked antibody for both neutrophils and monocytes in addition to FLAER, as it would not be possible to include three GPI-linked antibodies along with two lineage-specific antibodies (CD15, CD64) and CD45 for debris exclusion in one tube for a 5-colour assay.
    • FLAER-CD157-CD64-CD15-CD45 (for both neutrophils and monocytes).
  • Less time consuming (single tube).
  • More cost effective.

6-colour combinations:1,4

  • Provide optimal gating through the combination of three GPI markers, two lineage-specific markers and CD45.
    • FLAER-CD24-CD14-CD15-CD64-CD45 (for both neutrophils and monocytes).1,5
    • CD157-CD24-CD14-CD15-CD64-CD45 (non-FLAER-based assay for both neutrophils and monocytes).2,3
  • Less time consuming (single tube).
  • 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.

7-colour combination1,2

  • Cross-platform assay that generates nearly identical data regardless of instrument platform.
    • FLAER-CD157-CD24-CD14-CD15-CD64-CD45
  • Less time-consuming (single tube).
  • Allows the opportunity to delineate and quantify PNH neutrophils and PNH monocytes with three different combinations of GPI-linked reagents.

The most frequently used antibody/reagent combinations for neutrophil and monocyte testing are FLAER/CD24 and FLAER/CD14.2 FLAER/CD157 is recommended for use in high-sensitivity identification of both PNH neutrophils and monocytes in single-tube 5-, 6- and 7-colour assays.1,2,6

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 most PNH assays.

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 and unlysed RBCs, as well as backgating. This is particularly important for the accurate identification of small/minor clones. CD15 is recommended for gating neutrophils and CD64 is recommended for gating monocytes. CD64 is recommended for monocyte gating instead of CD33 since basophils can be largely excluded – basophils show intermediate CD33+/decreased FLAER expression and may be misinterpreted as PNH cells.

WBC 4-colour panel

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

WBC 4-colour panel

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

The 2018 consensus 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.1 Antibody clones and conjugates for cross-platform assays using 5-, 6- and 7-colour combinations are also included in the 2018 guidelines, and are described in further detail by Sutherland et al in a more recent publication.1,2

WBC 4-colour panel

Clones recommended for Beckman Coulter1,5 Clones recommended for Becton Dickinson Biosciences1,6
Antibody/reagent Purpose Fluorochrome Clone (vendor) Fluorochrome Clone (vendor)
CD15 Gating for neutrophils PC5 80H5 (BC) APC H198 (BD)
CD45 Debris and unlysed RBC exclusion, pattern recognition PC7 J33 (BC) PerCP 2D1 (eBiosciences)
FLAER GPI-linked (neutrophils and monocytes Alexa488 NA (Cedarlane) Alexa488 NA (Cedarline)
CD24 GPI-linked (neutrophils) PE ALB9 (BC)
SN3 (eBioscience)
PE SN3 (eBioscience)
ML5 (BD)
CD64 Gating for monocytes PC5 Clone 22 (BC) APC 10.1 (BD, eBiosciences)
CD14 GPI-linked (monocytes) PE RMO52 (BC)
61D3 (eBioscience)
Tuk4 (Invitrogen)
PE MP9 (BD)
61D3 (eBiosciences)
Tuk4 (Invitrogen)
APC, allophycocyanin; BC, Beckman Coulter; BD, Becton Dickinson Biosciences; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; PC, phycoerythrin cyanine; PE, phycoerythrin; PerCP, peridinin chlorophyll; RBC, red blood cell

WBC 5-colour panel

Clones recommended for Beckman Coulter1,2 Clones recommended for Becton Dickinson Biosciences1,2 Clones recommended for cross-platform assays (Beckman Coulter and Becton Dickinson)1,2
Antibody/reagent Purpose Fluorochrome Clone (vendor) Fluorochrome Clone (vendor) Fluorochrome Clone (vendor)
CD15 Gating for neutrophils PC5 80H5 (BC) PCPeF710a

PerCPCy5.5

MMA (eBioscience)

MEM-158 (EXBIO)

PCPeF710

PerCPCy5.5

MMA (eBioscience)

MMA (EXBIO)
MEM158 (EXBIO)

CD45 Debris, unlysed RBC exclusion, pattern recognition PC7 J33 (BC) eF450

APCH7a

2D1 (eBioscience)

2D1 (BD)

eF450 2D1 (eBioscience)
CD64 Gating for monocytes ECD Clone 22 (BC) APC 10.1 (BD, eBioscience) APC 10.1 (BD)
CD157 GPI linked (neutrophils and monocytes) PE SY11B5 (eBioscience, BC, EXBIO, BD, Sysmex) PE SY11B5 (eBioscience, EXBIO, BD, Sysmex, BC) PE SY11B5 (eBioscience, EXBIO, BD, Sysmex, BC)
FLAER GPI linked (neutrophils and monocytes) Alexa-488 NA (Cedarlane) Alexa-488 NA (Cedarlane) Alexa-488 NA (Cedarlane)
aFor 2-laser FACSCanto
APC, allophycocyanin; BC, Beckman Coulter; BD, Becton Dickinson; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; PE, phycoerythrin; PerCP, peridinin chlorophyll; PC, phycoerythrin cyanine; RBC, red blood cell

WBC 6-colour panel

Clones recommended for Beckman Coulter1 Clones recommended for Becton Dickinson Biosciences1 Clones recommended for cross-platform assays (Beckman Coulter and Becton Dickinson)2
Antibody Purpose Fluorochrome Clone (vendor) Fluorochrome Clone (vendor) Fluorochrome Clone (vendor)
FLAER GPI linked Alexa488 NA (Cedarlane) Alexa488 NA (Cedarlane) Alexa488 NA (Cedarlane)
CD14 GPI linked (monocytes) APC700 RMO52 (BC) APC MφP9 (BD)
61D3 (eBio)
TuK4 (Invitrogen)
CD15 Gating for neutrophils PerCPCy5.5 80H5 PCPeF710 MEM-158 (EXBIO)
HI98 (BC)
MMA (eBioscience)
PCPeF710

PerCPCy5.5

MMA (eBioscience)

MMA (EXBIO)
MEM158 (EXBIO)

CD24 GPI linked (neutrophils) PE ALB9 (BC)
SN3 (eBioscience)
PE ML5 (BD)
SN3 (eBioscience)
APC SN3 (APC)
CD45 Debris, unlysed RBC exclusion, pattern recognition KO J33 (BC) eF450 2D1 (eBioscience) eF450 2D1 (eBioscience)
CD64 Gating for monocytes PC7 Clone 22 (BC)
10.1 (EXBIO)
PECy7 10.1 (EXBIO)
22 (BC)
PC7 10.1 (EXBIO)
22 (BC)
CD157 GPI linked (neutrophils and monocytes) PE SY11B5 (eBioscience, EXBIO, BD, Sysmex, BC)
APC, allophycocyanin; BD, Becton Dickinson; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; KO, kusabira-orange; PE, phycoerythrin; PerCP, peridinin chlorophyll; NA, not available

WBC 7-colour panel

Clones recommended for cross-platform assays (Beckman Coulter and Becton Dickinson)1,2
Antibody Purpose Fluorochrome Clone (vendor)
FLAER GPI linked (neutrophils and monocytes) Alexa488 NA (Cedarlane)
CD157 GPI linked (neutrophils and monocytes) PE SY11B5 (eBioscience, EXBIO, BD, Sysmex, BC)
CD15 Gating for neutrophils PerCPeF710
PerCPCy5.5
MMA (eBioscience)
MMA (EXBIO)
MEM158 (EXBIO)
CD64 Gating for monocytes PC7 10.1 (EXBIO)
22 (BC)
CD24 GPI linked (neutrophils) APC SN3 (APC)
CD14 GPI linked (monocytes) APCeF780 61D3 (eBioscience)
CD45 Debris, unlysed RBC exclusion, pattern recognition eF450 2D1 (eBioscience)
APC, allophycocyanin; BD, Becton Dickinson; FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol; KO, kusabira-orange; PE, phycoerythrin; PerCP, peridinin chlorophyll; NA, not available

Different fluorochrome conjugates are possible depending on the laser and detector configuration of each instrument. For optimal fluorochrome selection, see the following links:

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

RBC analysis

2-colour assay (with CD59 and CD235a):1,4

  • 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 negatively charged 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 (vendor)
RBC CD59-PE GPI linked MEM43 (Invitrogen, EXBIO, Cedarlane)
OV9A2 (eBioscience)
CD235a-FITC Gating for RBCs 10F7MN (eBioscience)
YTH 89.1 (Cedarlane)
KC16 (BC)a
JC159 (DAKO)
aRBCs stained with this conjugate must be acquired immediately as fluorescent intensity diminishes within 10 minutes
BC, Beckman Coulter; FITC, fluorescein isothiocyanate; GPI, glycosylphosphatidylinositol; PE, phycoerythrin; RBC, red blood cell

Learn more

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

Diagnosing PNH PNH testing methodologies PNH testing guidelines Specimen considerations Quality assurance
  • References
    1. Sutherland DR et al. Cytometry B Clin Cytom 2018; 94: 23-48.
    2. Sutherland DR et al. Cytometry B Clin Cytom 2018; 94: 637-651.
    3. Marinov I et al. Cytometry B Clin Cytom 2018; 94: 257-263.
    4. Marinov I et al. Cytometry B Clin Cytom 2013; 84: 229-236.
    5. Sutherland DR et al. Cytometry B Clin Cytom 2012; 82B: 195-208.
    6. Sutherland DR et al. Cytometry B Clin Cytom 2014; 86B: 44-55.

Netflow Portal


You are now leaving www.netflowconnect.com website

Alexion Pharmaceuticals, Inc. has no influence or control over these websites and shall not be held responsible in any way for any of the content held there.

Continue Cancel