The Fundamental Role of FLAER in PNH Research

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare blood disorder estimated to affect 15.9 per million people worldwide1. PNH stems from a genetic mutation in hematopoietic stem cells, specifically in the phosphatidylinositol glycan class A (PIGA) gene. This leads to a deficiency in the glycosylphosphatidylinositol (GPI) protein, which primarily helps to anchor or link proteins to the surface of a cell. In particular, CD55 and CD59 are connected to membrane surfaces and help prevent the formation of complement-mediated membrane attack complexes. When a person has PNH, there is often a lack of CD55 and CD59 surface expression, leading to chronic complement-mediated hemolysis of the cells.



Ultimately, those dealing with PNH can be affected by:

  • Hemolytic anemia – red blood cell deficiency
  • Hemoglobinuria – excess hemoglobin in urine
  • Thrombosis – blood clots that block blood vessels
  • Renal insufficiency – poorly functioning kidneys possibly due to reduced blood flow
  • Bone marrow failure – decreased production of one or more major hematopoietic cell lineages


Forty to fifty years ago, only fifty percent of patients could expect a ten-year survival of this condition. Since then, several drugs that interfere with complement complex formation, such as eculizumab, have helped to improve their prognosis, with most patients now seeing no difference in life expectancy compared to those unaffected by PNH1.


In this blog, we will cover common research methods used in the past to assess PNH and newer ones that offer more reliable results using reagents like FLAER, which we now provide.


Traditional Research Methods – the Old and the New


The very first test used for PNH was created in 1937, when Thomas Ham placed the red blood cells of PNH samples into acidified serum, where complement attached to the RBCs at the lower pH level and led to increased cell sensitivity and fragility2. Since then, the gold standard for PNH research has been flow cytometry due to its increased fidelity and reliability, particularly for its ability to detect smaller PNH cell clones (<5.0%)2.


Initial flow cytometry research assays focused on white blood cells as opposed to RBCs, as RBCs have a shortened half-life. Staining involved targeting GPI-associated proteins (GPI-AP) such as CD55 and CD59 along with the detection of non-GPI associated proteins like CD15 and CD33. However, the antibody clones designed for these assays bound to their targets with low affinity, meaning it was still difficult to detect the smallest of PNH clones.


A FLAER for Accuracy


Despite the great advancement in research for detection of PNH by flow cytometry, rare PNH clones were still difficult to detect. For instance, in one study, 42% of samples were shown to have a PNH neutrophil clone present at <1%, falling well below the 5% sensitivity threshold flow cytometry assays for CD15, CD33, CD55, or CD59 could offer3.


In 2000, Brodsky et al. created a new method of detecting PNH clones touting the ability to detect abnormal cell populations as low as 0.5%4. This new research test named FLAER (Fluorescently Labeled AERolysin) is based on the bacterial toxin Aerolysin, which selectively binds to GPI-APs.


When used for flow cytometric, normal leukocytes that express GPI-APs bind to FLAER. PNH leukocytes, on the other hand, are unable to bind FLAER due to their lack of GPI-AP expression and demonstrate absent or dim fluorescence for FLAER in a flow cytometric histogram. The FLAER assay is also unaffected by the difference in expression of GPI-AP in cells that are in earlier maturation stages. By combining FLAER with antibody markers for typical granulocyte (CD16/CD24) or monocyte (CD14) markers, it is possible to detect the presence of minimal PNH clones, such as 0.01%. BioLegend provides FLAER conjugated to the Spark Blue™ 488 fluorophore, which is suitable for PNH research, emits in the FITC channel, and can be detected on most common flow cytometers with a blue laser (488 nm) and 530/30 filter.


Technological advancements are providing more accurate and dependable research methods of studying diseases like PNH. BioLegend strives to be at the forefront of these innovations, listening to researchers to create the reagents needed to make fundamental discoveries and facilitate breakthroughs. If you have any additional questions about our reagents, please reach out to us to discuss your project further.


FLAER conjugated to the Spark Blue™ 488 fluorophore is Research Use Only (RUO) reagents.



  1. Shah, Nischay. and Harshil Bhatt. “Paroxysmal Nocturnal Hemoglobinuria.” StatPearls, StatPearls Publishing, 31 July 2023.
  2. Krauss, Jonathan S. “Laboratory diagnosis of paroxysmal nocturnal hemoglobinuria.” Annals of clinical and laboratory science vol. 33,4 (2003): 401-6.
  3. Brando, Bruno et al. “Flow Cytometric Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: Pearls and Pitfalls - A Critical Review Article.” EJIFCC vol. 30,4 355-370. 25 Nov. 2019
  4. Brodsky, R A et al. “Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.” American journal of clinical pathology vol. 114,3 (2000): 459-66. doi:10.1093/ajcp/114.3.459

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