PNHSource

Reagents for Flow Cytometry¹

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 red blood cells (RBCs) or white blood cells (WBCs), use monoclonal, fluorescently labeled antibodies against GPI-anchored proteins.^1,3 Additionally, fluorescently labeled inactive toxin aerolysin (FLAER) may be used to detect GPI anchors themselves in WBCs^4,5
  • RBCs alone may not be sufficient due to hemolysis and the dilution effect of transfusions ^2,6
  • WBCs provide a more accurate analysis of PNH clones^1,2,7

Using FLAER to identify PNH granulocytes and monocytes⁵

A new technology used in the detection of PNH cell populations by flow cytometry is gaining popularity because it takes advantage of a bacterial protein called aerolysin that binds specifically to the GPI-anchored proteins on the surface of WBCs. This new reagent, 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 more direct and accurate in 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 a PNH clone. 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. 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, RBC analysis is most useful in detecting PNH type II cells (see below)
• CD24, CD66b, and CD16 on granulocytes
• CD14, CD48, CD55, and CD157 on monocytes

Normal histogram vs PNH histogram^8*

^*Adapted from Richards et al. Cytometry (Comm Clin Cytometry). 2000.

Panel (a) above shows the erythrocyte population of a healthy subject in which all erythrocytes are of the normal type (PNH type I). Panel (b) shows the erythrocyte population of a PNH patient that has a combination of normal erythrocytes and erythrocytes completely deficient in GPI-anchored proteins (PNH type III). Panel (c) shows a PNH patient with a combination of normal erythrocytes and erythrocytes partially deficient in GPI-anchored proteins (PNH type II). Panel (d) shows a PNH patient with a combination of all 3 erythrocyte populations.⁸