Specimen considerations

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 cytometry1,2

Recommendations for PNH sample handling

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

Source Peripheral blood
Anticoagulant EDTA or heparin (no data are available to indicate a preferred agent)
Volume Minimum 1 mL; 3 mL is adequate for most testing, though more may be needed if WBC count is very low
Maximum sample age ≤7 days for RBCs; <48 h for WBCs
Sample storage 4°C after 24 h
Lysing reagent
  • No lysing required for RBCs
  • For WBCs, any commercial lysing reagent 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 granulocytes are targeted in all cases; monocytes provide confirmatory information
  • No analysis of lymphocytes
EDTA, ethylenediaminetetraacetic acid; PNH, paroxysmal nocturnal haemoglobinuria; RBC, red blood cell; WBC, white blood cell
  • References
    1. Borowitz MJ et al. Cytometry B Clin Cytom 2010; 78B: 211-230.
    2. Sutherland DR et al. Cytometry B Clin Cytom 2012; 82B: 195-208.

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