High-sensitivity flow cytometry is the ‘gold standard’ for the testing and monitoring of paroxysmal nocturnal haemoglobinuria (PNH) clones;1,2 however, there remains no full consensus among laboratories on the best approach to the testing procedure.3 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.4
A possible cause of erroneous results is the lack of standardisation of the pre-analytical preparation and testing of samples. 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 guidelines3,5 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.
For accurate and reliable PNH analysis, it is recommended to carry out a daily performance check of the flow cytometer using standard beads to establish appropriate cytometer settings (photomultiplier tube [PMT] 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, granulocytes, 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).
To validate the assay procedure (see ‘assay validation’ for 4-colour or 6-colour protocols), an additional assay should be performed to determine the optimal working concentration of antibody and to assess the sensitivity of the test.
It is essential that all clinical laboratories performing PNH testing should systematically participate in a proficiency-testing programme.3 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 since current external programmes do not assess this adequately.3 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.6 When run formally as ring studies, exchange programmes can provide valuable information on interlaboratory-testing consensus and factors that may lead to discordant results.7
- Kelly R et al. Ther Clin Risk Manag 2009; 5: 911-921.
- Sharma VR. Clin Adv Hematol Oncol 2013; 11: 1-11.
- Borowitz MJ et al. Cytometry B Clin Cytom 2010; 78B: 211-230.
- 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.