Specimen considerations

Whole peripheral blood samples are recommended for evaluating both red blood cells and white blood cells. Testing should be performed on at least two different cell populations (not lymphocytes). Bone marrow samples are not appropriate outside the research setting as immature myeloid populations may express lower levels of glycosylphosphatidylinositol (GPI)-anchored proteins, which complicates interpretation.

Peripheral blood is the preferred specimen for paroxysmal nocturnal haemoglobinuria (PNH) testing by flow cytometry1

Recommendations for PNH sample handling

Recommendations on the source, storage and handling of samples for PNH testing1,2

Source Peripheral blood
Anticoagulant EDTA is the preferred/most tested agent, but heparin or ACD is also acceptable
Volume
  • For RBCs, whole anti-coagulated peripheral blood should be diluted 1:100 with PBS and a minimum of 100 µl of the diluted sample should be used
  • For WBCs, a minimum of 1 mL is required; 3 mL is adequate for most testing, though more may be needed if WBC count is very low
Maximum sample age <48 h for RBCs and WBCs
Sample storage 4°C after 24 h
Lysing reagent
  • No lysing required for RBCs
  • For WBCs, any commercial lysing/fixing agent (such as VersaLyse, FACSLyse or ImmunoPrep) or ammonium chloride
Cell populations evaluated
  • RBCs in all cases, or at least in those samples positive for a PNH clone following WBC analysis
  • WBC analysis is performed before RBC analysis and neutrophils are targeted in all cases; monocytes provide confirmatory information
  • No analysis of lymphocytes
ACD, acid citrate dextrose; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline; PNH, paroxysmal nocturnal haemoglobinuria; RBC, red blood cell; WBC, white blood cell
  • References
    1. Sutherland DR et al. Cytometry B Clin Cytom 2018; 94: 23-48.
    2. Borowitz MJ et al. Cytometry B Clin Cytom 2010; 78B: 211-230.

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