PNH with FLAER (High Sensitivity)

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder characterized by chronic hemolysis, increased risk for venous thrombosis, and bone marrow failure. The intravascular hemolysis results in free hemoglobin, which, in turn, can contribute to pain, fatigue, esophageal dysmotility, and erectile dysfunction.

PNH is also characterized by mutation in the PIGA gene and deficiency in GPI-linked proteins. The glycophosphoinositol (GPI) moiety allows a number of different proteins to attach to the cell membrane. There are more than 20 genes required for synthesis of the GPI anchors, one of which is the PIGA gene located on the X chromosome. An acquired mutation in the single PIGA gene in a male, or in one copy in a female due to X inactivation (lyonization), is sufficient to produce deficiencies in the expression of GPI-linked proteins. If this mutation occurs in a self-replicating hematologic stem cell, expansion of that PNH “clone” can lead to a GPI-deficient cell population, potentially in all hematologic lineages.

Flow cytometry is sensitive and more informative than mutation analysis. By simultaneously evaluating different cell populations, flow cytometry can assess the size of the PNH clone in each of the commonly affected hematopoietic cell lineages (erythrocytes [RBCs], granulocytes, and monocytes).^1,2 Thus, flow cytometry can determine whether the patient has reduced (partial) expression of GPI proteins (ie, PNH type II) or complete absence of GPI proteins (ie, PNH type III). Patients with type III are more likely to have severe hemolysis, since the absence of GPI-linked CD55 renders RBCs more sensitive to complement-mediated lysis.

Peripheral blood is the only appropriate sample for flow cytometry PNH testing. Bone marrow samples are not recommended.

Our test for PNH (test code 94148) is a high sensitivity assay that assesses the GPI-linked CD59 on erythrocytes. It uses a combination of several GPI-linked markers and the pan-GPI ligand, FLAER (fluorescent aerolysin), to assess granulocytes and monocytes. Additional transmembrane antigens are used to distinguish the different cell populations.

The analytical sensitivity of our assay is 0.01%; it can detect 1 GPI-deficient cell in 10,000 cells. However, sufficient hypocellularity, as may occur in aplastic cases, may limit the sensitivity. When this happens, it will be indicated on the report.

At presentation, most patients with PNH will have a dominant PNH clone detectable in 3 analyzed lineages. However, the degree of hemolysis, the particular PIGA mutation, and the presenting symptoms may influence the clone size. Small PNH clones (below 5%) can also be associated with myelodysplastic syndromes (MDS) and aplastic anemia, so clinical correlation is essential in those studies showing a small PNH clone.³

Guidelines recommend monitoring the PNH clone size at regular intervals.¹ If the patient is clinically stable, flow cytometric evaluation once a year may be sufficient. However, if symptoms recur or results of other laboratory tests demonstrate progression, more frequent testing is recommended. Serial flow cytometry is particularly useful for determining the response after initiation of eculizumab immunotherapy.⁴

In patients with aplastic anemia or low grade MDS and an initial negative PNH test, follow-up testing to monitor for emergence of a PNH clone may be indicated.