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.

Protocols for 5-colour assays will be available soon. Please register on the portal to receive updates via newsletter

Testing considerations

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

1: Pre-analytical considerations3-6

2: Analytical considerations3,4

3: Assay validation and quality control5

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.
  • 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. Borowitz MJ et al; for Clinical Cytometry Society. Cytometry B Clin Cytom 2010; 78B: 211-230.
    4. Sutherland DR et al. Cytometry B Clin Cytom 2012; 828: 195-208.
    5. Oldaker T et al. Cytometry B Clin Cytom 2018; 94: 67-81.
    6. Illingworth A et al. Cytometry B Clin Cytom 2018; 94: 49-66.

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