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PNH with FLAER (High Sensitivity)

Test code(s) 16433

Question 1. What are the clinical and genetic features of paroxysmal nocturnal hemoglobinuria (PNH)?

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.

Question 2. Why is flow cytometry used to diagnosis PNH?

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.

Question 3. What is the sample type and flow cytometric method used by Quest Diagnostics?

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

Our test for PNH (test code 16433) 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.

Question 4. What is the sensitivity of the assay?

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.

Question 5. What is the significance of the PNH clone size at diagnosis?

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

Question 6. Following diagnosis, what interval of follow-up testing is recommended?

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.

 
References
 
  1. Borowitz MJ, Craig FE, DiGiuseppe JA, et al. Guidelines for the diagnosis and monitoring of paroxsymal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010;78:211-230.
  2. Hernandez-Campo PM, Almeida J, Sanchez ML, et al. Normal patterns ofexpression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheralblood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry. 2006;70B:71-81.
  3. Wang H, Chuhjo T, Yasue S, et al. Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria-type cells in bone marrow failure syndrome. Blood. 2002;100:3897-3902.
  4. Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355:1233-1243.
This FAQ is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.
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