This site is intended for US audiences only. If you are a resident of the European Union, please click here for the EU version of our site.

Reagents for Flow Cytometry

High-sensitivity flow cytometry aids in the accurate identification of very small numbers of PNH cells.1 Multiple antibodies/markers are needed to support analyses that are accurate and reproducible.2

  • To detect PNH clones in properly gated RBCs or WBCs, use monoclonal, fluorescently labeled antibodies against GPI-anchored proteins.1,3 Additionally, FLAER may be used to detect GPI anchors themselves in WBCs4,5
    • RBCs alone may not be sufficient due to hemolysis and the dilution effect of transfusions2,6
    • WBCs provide a more accurate analysis of PNH cells or clones1,2,7

Using FLAER to Identify PNH Granulocytes and Monocytes5

Detection of PNH cell populations by FLAER can be considered because it takes advantage of a bacterial protein called aerolysin that binds specifically to the GPI-anchored proteins on the surface of WBCs. FLAER selectively binds to WBCs expressing GPI-anchored proteins on their surface.

  • FLAER’s ability to bind selectively to GPI-anchored proteins, in contrast to using antibodies that detect GPI-anchored proteins (eg, CD48), is a way of identifying PNH cell populations in WBCs
  • Cells that are missing the GPI anchor and lacking GPI-anchored proteins (eg, CD24) are labeled as PNH blood cells or PNH clones
  • FLAER staining is useful for the identification of PNH granulocytes and monocytes, but not PNH erythrocytes
  • FLAER is not a relevant marker for erythrocytes because RBCs do not express the surface-bound proteolytic enzymes needed to process the reagent4

Recommendations for Ordering Flow Cytometry for PNH Patients8

Demonstration of attenuated or absent expression of GPI-anchored proteins and/or the GPI anchor itself on RBCs and WBCs by flow cytometry has become the standard diagnostic test to confirm the presence or absence of PNH cells. Where available, quantitative, high-sensitivity flow cytometry results on erythrocytes and granulocytes are strongly recommended. In patients with aplastic anemia/myelodysplastic syndromes (AA/MDS), high-sensitivity flow cytometry should be conducted on peripheral blood to confirm the presence of PNH cells. Flow cytometry reporting should include results reflecting the presence of PNH cells via the detected deficiency in GPI-anchored proteins or in the GPI anchor itself.

Common antibodies used in detecting PNH include:

  • CD59 and CD55 on RBCs; although the determination of PNH requires both RBC and WBC analyses
  • CD24, CD66b, and CD16 on granulocytes
  • CD14, CD48, CD55, and CD157 on monocytes

Next: Therapeutic Options for PNH

1. Richards SJ, Barnett D. Clin Lab Med. 2007;27:577-590. 2. Parker C, Omine M, Richards S, et al; for the International PNH Interest Group. Blood. 2005;106:3699-3709. 3. Richards SJ, Whitby L, Cullen MJ, et al. Cytometry Part B Clin Cytom. 2009;76B:47-55. 4. Sutherland DR, Kuek N, Azcona-Olivera J, et al. Hematopathology. 2009;132:564-572. 5. Sutherland DR, Kuek N, Davidson J, et al. Cytometry Part B Clin Cytom. 2007;72B:167-177. 6. Nebe T, Schubert J, Gutensohn K, Schrezenmeier H. J Lab Med. 2003;27:257-265. 7. Brodsky RA, Mukhina GL, Li S, et al. Am J Clin Pathol. 2000;114:459-466. 8. Richards SJ, Rawstron AC, Hillmen P. Cytometry. 2000;42:223-233.