New Reagents for Flow Cytometry for PNH1
A new method for flow cytometry testing for PNH is gaining popularity because it takes advantage of a fluorescent bacterial protein called recombinant aerolysin that binds specifically to the GPI anchor of proteins on the surface of a cell. This new reagent, FLAER (Fluorescent Labeled Inactive Toxin Aerolysin) will only bind cells if they express GPI-linked proteins on their surface.2
- The anchoring mechanism of binding to the GPI-anchored proteins helps recognize all proteins that are attached to the surface of cells
- Cells that are missing the GPI anchor and proteins (e.g., CD59) are labeled as PNH blood cells or PNH clones
- The percentage of negative cells from this test highly correlates with the percentage of cells that do not express GPI-linked proteins such as CD59 and CD24
Identifies PNH granulocytes
- FLAER staining is useful for the identification of PNH granulocytes but not PNH red blood cells. FLAER is not a relevant marker for erythrocytes3
Recommendations for ordering flow cytometry for PNH patients
Demonstration of a decreased expression of GPI-anchored proteins on erythrocytes and granulocytes by flow cytometry has become the standard diagnostic test to confirm the presence or absence of a PNH clone. Where available, quantitative flow cytometry results on erythrocytes and granulocytes are strongly recommended. In patients with AA, MDS, or unexplained cytopenias, conduct high-sensitivity flow cytometry on peripheral blood in addition to bone marrow aspirate to confirm the presence of PNH.
Flow cytometry should be performed on peripheral blood, requesting a quantification of GPI-anchored proteins on the cell surfaces of both erythrocytes and granulocytes.
Most commonly used antibodies include:
- CD59
- CD55
- Other GPI-anchored antigens (CD14, CD16, CD24) can also be studied on leukocytes
Given the short life span of granulocytes, flow cytometry on peripheral blood must be performed within 1 to 2 days of obtaining the sample
PNH clone sizes of 1% or greater should lead clinicians to suspect the diagnosis of PNH. Many laboratories may have varying threshold values for a positive test based on the results of normal donor cells. A cutoff of several standard deviations above the mean of values from normal cells is typically chosen as the lower limit of a positive test. However, results that are marginally elevated above an established cutoff value may not indicate a true PNH clone and the assay should be repeated to rule out a false positive test. A common threshold value in laboratories with expertise in the identification of PNH cells is 1%.
Normal histogram vs PNH histogram4†
†Adapted from Richards et al, 2000.
Panel (a) above shows the erythrocyte population of a healthy subject where 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 three erythrocyte populations.4
