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.

Recommendations for PNH sample handling

Recommendations on the source, storage and handling of samples for PNH testing^1,2

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

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Diagnosing PNH PNH testing methodologies PNH testing guidelines Recommended reagents Quality assurance