Paroxysmal Nocturnal Haemoglobinuria (PNH) is a stem cell disease caused by mutation of a gene involved in the synthesis of the GPI anchor of a group of surface proteins on circulating cells.
The absence of two glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that accounts for haemolysis and other PNH manifestations.
Patients with the mutated gene are deficient in all GPI-linked proteins in a clone of hematopoietic stem cells and they may develop symptoms such as (complement induced) haemolytic anaemia (destruction of red blood cells) and haemoglobinuria, where haemoglobin is found in abnormally high concentrations in the urine.
Affected cells are sensitive to complement-mediated haemolysis and this may lead to life-threatening thrombosis, chronic kidney disease, pulmonary hypertension, end organ damage, ischemic bowel disease, hepatic failure, and anaemia.
PNH can appear at any age, among men or women, and affects all races. Although global prevalence data for PNH is not available, it has an approximate rate of 1/62,500 in Britain.
How is it treated
PNH results in the death of approximately 50% of affected individuals due to thrombotic complications: allogeneic bone marrow transplantation is the only curative therapy for PNH and, until recently, there was no specific therapy.
Eculizumab, which hit the market in 2007, is a monoclonal antibody that blocks terminal complement activation and is highly effective in reducing haemolysis. It is a humanised monoclonal antibody (target complement component C5), functioning as a terminal complement inhibitor.
This can improve quality of life and reduces the risk of thrombosis in PNH patients, reducing and even eliminating the need for blood transfusions.
It is worth noting that, while Eculizumab is highly effective in reducing intravascular haemolysis in PNH; it does not stop extravascular haemolysis, and it does not treat bone marrow failure.
However, it has been so effective as a treatment that bone marrow transplants are now only considered for PNH patients with a sub-optimal response to Eculizumab.
It’s not cheap – in 2010, Soliris was the most expensive drug in the world. It cost £340,200 per year for ongoing treatment in the UK, $500,000 a year in Canada and US$409,500 a year in the United States (2010).
Traditional detection and monitoring of PNH
Before FLAER, detection of PNH clones by flow cytometry relied on fluorescently-labelled antibodies to GPI-linked proteins such as CD59 and CD55. However, there are limitations with this method which derive from the fact that these antibodies do not bind with high affinity, so small PNH clones are not detected.
In addition, they each screen for the absence of a specific protein, rather than loss of the GPI anchor, and therefore there is the risk of false-negative results.
Identifying PNH patients early in the course of their disease may offer the best opportunity for long-term management. In the past, PNH has been challenging to identify effectively. However, in recent years impressive strides have been made in the understanding of PNH pathology, accompanied by greatly improved detection techniques, including multiparametric flow cytometry using FLAER.
Detection with FLAER
Cedarlane’s FLAER, available from VH Bio in the UK, is an Alexa® fluor 488-labelled variant of aerolysin, a unique protein that binds tightly and specifically to mammalian GPI anchors. It is a sensitive and accurate test that can be used in combination with the CD55/CD59 assay for detecting PHN clones in red blood cells.
FLAER is used in a clinical laboratory setting for multi-parameter flow cytometry along with antibodies (including CD45, CD33, CD24, CD15 and CD14) to detect PNH clones (FLAER-negative cells) within the monocyte and granulocyte (cell) lineages.
FLAER binds directly to the GPI anchor, which means that only PNH cells, which lack the GPI anchor, will be negative. This results in a more accurate assessment of the GPI anchor deficit in PNH when compared with anti-CD59 or anti-CD55 – and since FLAER binds with high affinity, very small PNH populations can be detected.