High-sensitivity flow cytometry is the ‘gold standard’ for the testing and monitoring of paroxysmal nocturnal haemoglobinuria (PNH) clones;1-3 however, there remains no full consensus among laboratories on the best approach to the testing procedure.4 External quality-assurance data have highlighted a significant number of laboratories that reported incorrect results in samples taken both from patients known to have PNH and from others known not to have PNH.5
Standardisation and validation of PNH assays
A possible cause of erroneous results is the lack of standardisation and validation of the pre-analytical preparation and testing of samples for PNH using high-sensitivity flow cytometry. Variation can occur at every step of the process, including sample processing, instrument optimisation, optimal antibody selection, titration of reagents, gating strategy, and interpretation and reporting of results.
Although there is no fully standardised methodology for PNH testing so far, published guidelines4,6 offer recommendations on analytical procedures to enable laboratories interested in PNH testing to establish a validated procedure and to allow experienced laboratories to improve their own techniques.
Internal quality control procedures
For accurate and reliable PNH analysis, it is important to use a well-maintained, optimised and standardised flow cytometer that is checked for performance daily. The use of standard microbeads is recommended to establish appropriate cytometer settings (fluidics, laser, photomultiplier tube voltages and spectral compensation) for the detection of negative and positive target cell populations (see ‘set-up’ for 4-colour or 6-colour protocols). Ideally, application setting should be performed with non-labelled target cells (red blood cells, neutrophils, monocytes) and cells labelled with all conjugates used in the assay; however, in the majority of cases application setting is performed using compensation beads or lymphocytes (eg CYTO-COMP Cell Kits, CompBeads and Cytometer Setup & Tracking [CS&T] beads).
Instrument quality control should also include verification of optical alignment, performed using alignment microspheres with a uniform signal for each light scatter and fluorescent parameter, with the goal of obtaining a maximal signal and minimal vibration.4
Measurement of the level of carry-over from one sample to the next is also important to measure, in order to determine whether carry-over is exceeding specifications. The preferred method of assessing carry-over is to analyse a sample with high levels of PNH-positive clones, followed by a sample from a healthy volunteer. In the event that carry-over is evident, acquire a buffer tube between clinical samples.4 Instrument quality control should also include daily standardisation and compensation checks.
To validate the assay procedure, an additional assay should be performed to determine the optimal working concentration of antibody and to assess the sensitivity of the test.
External quality assessment/proficiency testing
It is essential that all clinical laboratories performing PNH testing should systematically participate in an external quality assessment/proficiency-testing programme.4 It is recommended that all laboratory staff performing PNH testing should be properly trained and competent. Currently, there are two external agencies that offer proficiency testing and accreditation for PNH clinical analysis. Please be aware that there might be a local quality-control programme in your region that you might be interested in joining.
United Kingdom National External Quality Assessment Schemes (UK-NEQAS) for leucocyte immunophenotyping
UK-NEQAS offers proficiency-testing programmes for routine and high-sensitivity testing of PNH erythrocytes and granulocytes by flow cytometry. Further details can be found at www.ukneqasli.org.uk.
College of American Pathologists (CAP)
CAP offers a flow-cytometry programme focused on PNH erythrocyte testing and has recently introduced a programme for PNH leucocytes that is applicable to a limited number of laboratories, depending on gating strategies used. Further information on the CAP programmes can be found at www.cap.org.
Interlaboratory exchange network
Besides external agencies, local quality-assurance programmes consisting of networks of laboratories are currently emerging. Interlaboratory exchange is particularly important for laboratories with limited experience in PNH analysis (laboratories that rarely perform PNH testing) and for laboratories seeking to optimise high-sensitivity testing. Interlaboratory quality-assurance programmes and databases allowing data exchange between laboratories are excellent tools for raising awareness and improving the quality of clinical testing for PNH clones;7 for example, split sample testing with other laboratories is a feasible method of demonstrating testing accuracy.4 When run formally as ring studies, exchange programmes can provide valuable information on interlaboratory-testing consensus and factors that may lead to discordant results.8
- Kelly R, et al. Ther Clin Risk Manag 2009; 5: 911-921.
- Sharma VR. Clin Adv Hematol Oncol 2013; 11: 1-11.
- Illingworth A et al. Cytometry B Clin Cytom 2018; 94: 49-66.
- Oldaker T et al. Cytometry B Clin Cytom 2018; 94: 67-81.
- Richards SJ et al. Cytometry B Clin Cytom 2009; 76B: 47-55.
- Sutherland DR et al. Cytometry B Clin Cytom 2012; 82B: 195-208.
- Marinov I et al. Clin Chem Lab Med 2013; 51: 2133-2139.
- Marinov I et al. Cytometry B Clin Cytom 2013; 84: 229-236.