Flow Cytometry

Flow cytometry — a simple, inexpensive, peripheral blood test — is the standard diagnostic for PNH

Flow cytometry: the most sensitive, informative assay for PNH diagnosis

Detection of PNH clones by flow cytometry, the standard diagnostic test for PNH, provides the most consistent and sensitive method of testing for PNH. Click here to view the diagnostic tools and techniques brochure. This test has replaced older diagnostic tools, such as the Ham and sucrose lysis tests, because it has better sensitivity and specificity for detecting and measuring PNH clones.1-3

  • Use monoclonal antibodies against GPI-anchored proteins (CD59 and CD55) or FLAER (fluorescent-labeled inactive toxin aerolysin)1-3
  • Order quantitative results expressing clone size of both erythrocytes and granulocytes2
    • Erythrocytes alone may not be sufficient due to hemolysis and the dilution effect of transfusions
  • Test both granulocytes and erythrocytes
    • Erythrocytes alone may not be sufficient due to hemolysis and the dilution effect of transfusions
  • Sample should be sent to a high-sensitivity lab with the ability to detect clones as low as 0.01%
  • Flow cytometry is noninvasive and inexpensive

Detection of the PNH clone using flow cytometry

This test can use either monoclonal antibodies, directed against GPI-anchored proteins (such as CD59) on the surface of blood cells, or fluorescent-labeled inactive toxin aerolysin (FLAER), directed against GPI anchors themselves. Sometimes multiple antibodies are needed to support a result that is accurate and that can be reproduced.

Monoclonal antibodies

  • Each binds to a specific GPI-anchored protein4
  • Identifies PNH erythrocytes and PNH granulocytes — which is a more difficult process, but can be done4

Erythrocyte testing

  • PNH red blood cells are the easiest to analyze, but the proportion of these cells relative to the proportion of other PNH cell types is often misleading because they are easily destroyed by terminal complement. Therefore the PNH red blood cell proportion may not reflect the true PNH stem cell clone size
  • PNH patients frequently receive packed red blood cell transfusions, which serve to dilute the PNH red blood cell clone, further contributing to a confounded result that does not reflect the true PNH clone size

Granulocyte testing

  • Granulocyte numbers sufficient for flow cytometry can be difficult to obtain in some patients, and the results from a single antibody staining can be suspect
    • Several antibodies (e.g., antibodies against CD59, CD55, CD16, CD24, and CD66) may be used simultaneously to ensure accuracy of the assay
    • Analysis of granulocytes provides the most accurate assessment of the true PNH clone size because these cells are not sensitive to complement-mediated destruction and are not subject to dilution during the frequent red blood cell transfusions that many patients receive


References: 1. Parker C, Omine M, Richards S, et al; for the International PNH Interest Group. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood. 2005;106:3699-3709. 2. Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood. 1996;87:5332-5340. 3. Richards SJ, Barnett D. The role of flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria in the clinical laboratory. Clin Lab Med. 2007;27:577-590. 4. Brodsky RA. Paroxysmal nocturnal hemoglobinuria. In: Hoffman R, Benz EJ Jr, Shattil SJ, et al, eds. Hematology: Basic Principles and Practice. 4th ed. Philadelphia, PA: Elsevier Churchill Livingstone; 2005:419-427.