A Flaer for PNH
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder. The basic defect in PNH is an expansion of hematopoietic stem cells carrying a mutation in the phosphatidylinositol glycan class A (PIG-A) gene. Because of this mutation, certain types of proteins are unable to be expressed on the surface of the cell due to defective assembly of the glycosyl-phosphatidylinositol (GPI) anchor, a necessary step in surface attachment of certain proteins. Proteins affected by this defect include CD55 and CD59 (regulators of complement activation), CD52, CD14, and others.
The clinical picture of PNH is characterized by a triad of - varying degrees of - intravascular hemolysis, thrombosis and bone marrow failure. Red blood cells lacking (partially or completely) GPI-anchored CD55 and CD59 are unable to regulate and terminate complement activation, thereby making the cells susceptible to lysis by the terminal complement complex. This causes intravascular hemolysis, the primary clinical manifestation in PNH. Hemolysis is chronic with periodic exacerbations (paroxysms), which can lead to the need for frequent blood transfusions. The main signs and symptoms of PNH are: fatigue, hemoglobinuria, abdominal pain, dysphagia, dyspnea, jaundice, thrombosis and development of pulmonary hypertension. Bone marrow failure leads to — combinations of— anemia, neutropenia and thrombocytopenia. The clinical course of PNH varies from patient to patient. Some patients are affected only by mild symptoms while others display a more severe clinical phenotype.
Treatment for PNH remains largely supportive with supplements to aid in the regeneration of erythrocytes (e.g. folate and iron). Recently, a humanized anti-C5a monoclonal antibody has shown promise in the treatment of hemolysis.
The reason that patients with PNH develop bone marrow failure is not completely understood. It has been proposed that PNH constitutes a permissive state, in which GPIdefective clones may have a survival advantage over normal stem cells that are targeted by an autoimmune destructive process. Alternatively, (abnormal) GPI-negative stem cells in the bone marrow may pose a target for immune recognition and subsequent destruction. Bone marrow failure manifestations can be treated by similar immunosuppressive therapy as used in other scenarios of bone marrow failure.
Published guidelines recommend that patients with aplastic anemia, refractory anemia-myelodysplastic syndromes, and patients with any one of the following - venous thrombosis involving unusual sites, hemoglobin in the urine, intravascular hemolysis without antibodies, or episodic abdominal pain, dysphagia and hemolysis - should be screened for PNH. Diagnosis of PNH in the "pre-Flow Cytometry" era was accomplished with the Ham's test and sucrose lysis assay. However, as has become clear with the development of flow-based methods, the latter assays were sensitive enough to detect subclinical PNH, or small numbers of GPI deficient cells.
Currently, the diagnosis of PNH, as performed in the Diagnostic Immunologic Laboratories, depends on the flow cytometric detection of cell surface expression of molecules that are dependent on the GPI structure, such as CD52, CD55 and CD59 on leukocytes and erythrocytes. This method is particularly useful in the quantification of abnormal cells ("PNH clones"). Thus, it is possible to follow progression or regression of the GPI-negative clones amongst the different cell populations, in the context of the clinical phenotype and independent of recent blood transfusions (if leukocytes are analyzed).
Moreover, it has been determined that GPInegative cell populations can often be detected in patients with bone marrow failure syndromes, who lack the typical clinical manifestations of PNH. Lastly, the presence of these clones in patients with myelodysplastic syndromes, complicating bone marrow failure, has been linked to a more favorable response to immunosuppressive therapy.
Our Laboratory has recently adapted the PNH assay to include a reagent that directly identifies the GPI anchor, independent of the cell surface structure it is attached to. Aerolysin is a toxin derived from the Aeromonas hydrophila bacterium. Proteolytic cleavage by cellular proteases converts inactive pro-aerolysin into active aerolysin. The introduction of 2 mutations that disrupts the lytic potential, but retains its GPIbinding capacity, together with the coupling to a fluorescent marker created the FLAER reagent. FLAER is highly sensitive and specific in the detection of GPI-deficient cells, regardless of the cell surface receptor it is part of. Due to a lack of proteases, FLAER is not a relevant marker for erythrocytes.
As can be seen in the figure on this page, the fluorescence intensity difference between positive and negative cells is considerably greater with FLAER than with anti-CD59. Moreover, there are conditions in which there is down-regulation (loss) or lack of CD59 (and CD55) expression, unrelated to PNH. An example is illustrated in the figure, showing a lymphocyte population with reduced CD59 expression, but no difference in FLAER staining. This pattern has been observed in a variety of patients with immune activation, predominantly, but not exclusively, affecting lymphocytes. Further exploration in this patient (with DiGeorge syndrome and autoimmune cytopenias) has revealed that the CD59-negative fraction constitutes a T-cell population.
Thus, a combination of FLAER and surface expression of CD59 (and other molecules) provides a reliable method to diagnose PNH, to enumerate the size of the PNH clones (as small as <1% GPI-negative cells) and to distinguish between CD59-negative (or CD52, after treatment with anti-CD52 [e.g. campath] monoclonal antibodies) and GPInegative cell populations as part of PNH.
