Performing a paroxysmal nocturnal haemoglobinuria (PNH) 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 constant 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 a minimum of 4 and up to 6 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.
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Before and during PNH analysis, the following conditions should apply:
1: Pre-analytical considerations1,2
- The sample was received in good condition.
- The cells have 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) [see ‘set up’ for 4-colour or 6-colour protocols].
2: Analytical considerations1,2
- 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 granulocytes, 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 control1,2
- 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 reporting1,2
- The report includes the PNH clone size in RBCs and white blood cells (WBCs; include data for granulocytes 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 terminology (see ‘report’ in 4-colour or 6-colour protocols).
- Borowitz MJ et al; for Clinical Cytometry Society. Cytometry B Clin Cytom 2010; 78B: 211-230.
- Sutherland DR et al. Cytometry B Clin Cytom 2012; 82B: 195-208.