PNH testing protocols
- Step 1 – set up – 4 colour
- Step 1 – set up – 6 colour
- Step 2 – stain and acquire – 4 colour
- Step 2 – stain and acquire – 6 colour
- Step 3 – analyse – 4 colour
- Step 3 – analyse – 6 colour
- Step 4 – report
- Assay validation
Morado et al recently published data supporting the indications for paroxysmal nocturnal haemoglobinuria (PNH) testing:
- Morado M et al. Diagnostic screening of paroxysmal nocturnal hemoglobinuria: prospective multicentric evaluation of the current medical indications. Cytometry B Clin Cytom 2016. DOI: 10.1002/cyto.b.21480.
Reflex flow cytometry screening for PNH in patients with myelodysplastic syndrome (MDS) was recommended in the guidelines of the British Committee for Standards in Haematology:
- Killick SB et al. Guidelines for the diagnosis and management of adult myelodysplastic syndromes. Br J Haematol 2014; 164: 503-525.
The first guidelines for the diagnosis and management of PNH were published by the International PNH Interest Group in 2005:
- Parker C et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005; 106: 3699-3709.
More recently, specific guidelines on the accurate identification and monitoring of PNH clones using flow cytometry were published by the International Clinical Cytometry Society (ICCS) in 2010:
- Borowitz MJ et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 2010; 78B: 211-230.
The ICCS guidelines were followed in 2012 by a supporting document outlining concise, practical protocols for the high-sensitivity detection of PNH clones:
- Sutherland DR et al. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom 2012; 82B: 195-208.
Confirmation of the reliability and robustness of the practical protocols published by Sutherland et al was reported in a multicentre study carried out by Marinov et al, published in 2013:
- Marinov I et al. Intra- and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing. Cytometry B Clin Cytom 2013; 84B: 229-236.
Movalia et al analysed the distribution of PNH-clone sizes within high-risk patient groups, highlighting the need to continue actively testing for PNH in these populations:
- Movalia MK et al. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. 54th Annual Meeting of the American Society of Hematology, 8-11 December 2012, poster 1271.
Cannizzo et al published a follow-up assessment of patients with PNH clones, demonstrating the change in clone size over time:
- Cannizzo E et al. Cellular composition and clonal evolution of 392 type III PNH clones: an Italian multi-laboratory study. 18th Congress of the European Hematology Association, 13-16 June 2013, poster 177-P.
Results from a study carried out in collaboration with the United Kingdom National External Quality Assessment Service (UK NEQAS), published in 2009, describe the development of an external quality-assessment programme using stabilised whole-blood PNH samples in an attempt to improve the quality of diagnostic testing for PNH:
- Richards SJ et al. Development and evaluation of a stabilized whole-blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by flow cytometry. Cytometry B Clin Cytom 2009; 76B: 47-55.
Work on the standardisation of assays for PNH testing, in collaboration with UK NEQAS for Leucocyte Immunophenotyping (LI), was published in 2014, highlighting reduced variability in PNH testing using standardised protocols and conjugates compared to in-house methods, in laboratories with extensive experience in PNH testing:
- Fletcher M et al. Standardizing leucocyte PNH clone detection: an international study. Cytometry B Clin Cytom 2014; 86B: 311-318.
Confirmation of the reliability of standardised approaches to PNH testing was reported by Sutherland et al, demonstrating good assay performance within and among laboratories using standardised reagent cocktails, including laboratories with limited experience in PNH testing:
- Sutherland DR et al. High-sensitivity detection of PNH red blood cells, red cell precursors, and white blood cells. Curr Protoc Cytom 2015; 72: 6.37.1-6.37.29.
Data published from a genetic analysis of patients with PNH reveal insights into the molecular mechanisms underlying PNH clonal expansion:
- Shen W et al. Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria. J Clin Invest 2014; 124: 4529-4538.