Testing protocols

Performing a high-sensitivity paraoxysmal nocturnal haemoglobinuria (PNH) flow cytometry assay is complex and requires special attention when setting it up in the laboratory. The set-up of a good PNH protocol will facilitate:

• Delivery of reliable and consistent results over time.
• Performance of high-sensitivity testing and avoidance of common mistakes.
• Improved efficiency in the laboratory.

This section provides step-by-step instructions on how to establish optimal PNH assays in the most efficient way. These protocols are applicable to the wide range of instruments capable of analysis using 4–7 colours.^1,2 In addition to guidance on assay validation and the accurate reporting of results, our experts share advice on PNH testing pitfalls that frequently lead to suboptimal PNH diagnosis or misdiagnosis. The links below will take you to guides 4- and 6-colour assay protocols, with guides for 5- and 7-colour protocols coming soon.

Have you selected the appropriate reagents for your assay and instrument? Please follow the link to view the recommended reagents for 4-colour and 6-colour assays.

Guides

4-colour guide 6-colour guide

Testing considerations

Before and during PNH analysis, the following conditions should apply:

1: Pre-analytical considerations^3-6

• The sample was received in good condition and has been prepared in compliance with a standardised protocol.
• The optimal panel with the most suitable antibody clones, conjugates and cocktails (glycosylphosphatidylinositol [GPI]-linked reagents and lineage-specific gating antibodies) has been selected.
• The instrument has been optimised (adequate voltages and compensation settings and standardisation) [see ‘set up’ for 4-colour or 6-colour protocols].

2: Analytical considerations^3,4

• A standardised protocol has been established, based on published PNH guidelines.
• Appropriate lineage-specific gating strategies (eg CD235a for red blood cells [RBCs], CD15 for neutrophils, CD64 for monocytes) have been selected (see ‘set up’ for 4-colour or 6-colour protocols).
• A suitable number of cells have been acquired in order to verify the presence of a true PNH clone (see ‘stain and acquire’ for 4-colour or 6-colour protocols).
• An assessment has been performed using internal control cells to check reagent and instrument performance.

3: Assay validation and quality control⁵

• The assay has been validated.
  • Verification of antibody performance (expected antigen expression on normal blood) [see ‘assay validation’ in 4-colour or 6-colour protocols].
  • Verification of ‘clean PNH gate’ (normal samples do not show PNH phenotypes) [see ‘set up’ for 4-colour or 6-colour protocols].
  • Verification of PNH populations in the expected location (PNH-positive blood) [see ‘set up’ for 4-colour or 6-colour protocols].
  • Spiking experiments to determine assay sensitivity (PNH-positive blood) [see ‘assay validation’ in 4-colour or 6-colour protocols].
• The laboratory has joined a quality-assurance/proficiency-testing programme and exchanged PNH samples with other laboratories.

4: Clear reporting⁶

• The report clearly states whether a PNH clone is present or absent (see ‘report’ in 4-colour or 6-colour protocols).
• The report includes the PNH clone size in RBCs and white blood cells (WBCs; include data for both neutrophils and monocytes, if possible) [see ‘report’ in 4-colour or 6-colour protocols].
• Histograms/dot plots are included (see ‘report’ in 4-colour or 6-colour protocols).
• The antibodies tested and assay sensitivity have been specified (see ‘report’ in 4-colour or 6-colour protocols).
• The report does not contain misleading or ambiguous terminology (see ‘report’ in 4-colour or 6-colour protocols).
• The report documents any recommendations for re-testing.