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Test of the Month
Laboratory tests for paroxysmal nocturnal hemoglobinuria
Article first published online: 27 FEB 2014
Copyright © 2013 Wiley Periodicals, Inc.
American Journal of Hematology
Volume 89, Issue 3, pages 339–341, March 2014
How to Cite
Preis, M. and Lowrey, C. H. (2014), Laboratory tests for paroxysmal nocturnal hemoglobinuria. Am. J. Hematol., 89: 339–341. doi: 10.1002/ajh.23612
- Issue published online: 27 FEB 2014
- Article first published online: 27 FEB 2014
- Accepted manuscript online: 11 OCT 2013 11:45AM EST
- Manuscript Accepted: 4 OCT 2013
- Manuscript Revised: 27 SEP 2013
- Manuscript Received: 16 AUG 2013
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder that is often suspected in a patient presenting with non-immune hemolytic anemia associated with pancytopenia or venous thrombosis. This disorder is a consequence of acquired somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene in the hematopoietic stem cells (HSC) of patients. The presence of these mutations leads to production of blood cells with decreased glycosyl phosphatidylinositol-anchored cell surface proteins, making red blood cells derived from the clone more sensitive to complement mediated hemolysis. The diagnosis of PNH may be difficult in some cases due a low proportion of PNH cells in the blood and occasionally due to difficulties in selecting the most appropriate diagnostic studies. The latest generation of tests allow for detection of very small populations of PNH cells, for following the natural course and response to therapy of the disease, and for helping to decide when to initiate therapy with monoclonal antibody targeting the terminal complement protein C5 (Eculizumab), anticoagulation, and in some cases allogeneic HSC transplant. In this article, we review the different diagnostic tests available to clinicians for PNH diagnosis. Am. J. Hematol. 89:339–341, 2014. © 2013 Wiley Periodicals, Inc.
Paroxysmal nocturnal hemoglobinuria (PNH) was first described as a distinct clinical entity involving intravascular hemolysis in 1882 by Dr. Paul Strübing who noted the presence of free hemoglobin in the urine of a patient . He postulated that red blood cells (RBCs) were destroyed due to sensitivity to acidic serum conditions during sleep. After 55 years, Dr. Thomas Ham tested this hypothesis by increasing hemoglobinuria in patients who were administered ammonium chloride to lower their blood pH. This led to the development of “Ham's Test” the first diagnostic test for PNH . Several decades later, the association between the alternative pathway of complement and hemolysis in PNH was demonstrated . Further studies demonstrated that platelets and neutrophils are also abnormally sensitive to complement-mediated lysis . In the early 1980s, it was found that PNH erythrocytes are deficient in decay-accelerating factor (DAF or CD55) that leads to greater C3 deposition on RBCs from PNH patients . Holguin et al. demonstrated that RBCs derived from PNH clones are also deficient in a membrane inhibitor of reactive lysis (CD59) that regulates the formation of the complement membrane attack complex . The discovery that additional cell surface proteins were deficient on PNH cells led to the hypothesis that the underlying disorder is in the cell surface glycosyl phosphatidylinositol (GPI) anchor proteins. Soon thereafter, a somatic mutation of phosphatidylinositol glycan class A (PIG-A) in the hematopoietic stem cells (HSC) was identified as the underlying genetic basis for PNH . The mutation type (missense vs. nonsense) causes a partial or absolute deficiency in the cell surface expression of GPI-anchored proteins and is associated with the disease phenotype .
The absence of CD55 and CD59 on PNH RBCs decreases resistance to complement-mediated intravascular and extravascular hemolysis. The increase in intravascular hemolysis generates free hemoglobin which acts as a potent nitric oxide (NO) scavenger . The depletion of NO has been associated with increased risk for intravascular thrombosis which is the major cause of mortality. These life-threatening thrombotic events can be seen in unusual anatomic locations such as portal veins or mesenteric vasculature. Mutant HSCs carrying the PIG-A mutation exhibit a survival advantage and tend to expand over time often leading to the development of marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome [10, 11]. The underlying mechanism for this phenomenon is still not clear.
Although PNH is a rare disease with estimated incidence of 1.3 new cases per million individuals per year, it has a profound impact on the affected patients' quality of life and survival. Patients with PNH may present with variety of signs and symptoms, some of them are common and nonspecific. Testing for PNH should be reserved for patients with clinical features, including: unexplained Coombs-negative hemolysis, hemoglobinuria, thrombosis with unusual features, and acquired bone marrow failure. The development of novel therapies including the monoclonal antibody against the terminal complement protein C5 (Eculizumab) have significantly improved the quality of life of these patients and decreased the associated morbidity . This has made accurate and efficient diagnosis of PNH a priority.
Thomas Ham developed the first diagnostic test for PNH based on the hypothesis that the nocturnal hemolysis is due to elevation of carbon dioxide and decreased blood pH during sleep . In this assay, washed RBCs are incubated with acidified serum. Under these conditions, RBCs derived from the PNH clone preferentially undergo hemolysis. The lysis of RBC is quantified using a spectrophotometric analysis of free hemoglobin. Several decades later, Hartmann and Jenkins developed the “sucrose hemolysis test” for the diagnosis of PNH . This assay is based on the hypothesis that incubating whole blood from PNH patients in hypotonic sucrose solution will trigger complement activation and in vitro hemolysis. Although these tests long served as the standards for PNH diagnosis, they lack the ability to detect low levels of PNH cells with the presence of at least 10% hemolysis required for a positive test. The specificity of the tests is also relatively low with positive results seen in other hematological disorders such as megaloblastic anemia, HEMPAS, and spherocytosis.
The development of fluorescent-labeled monoclonal antibodies to characterize cell surface proteins by flow cytometry has significantly improved the sensitivity and specificity of detecting PNH clones, thus rendering flow cytometry as the “gold standard” for PNH testing. Separate methods are available for initial screening (sensitivity of ∼1%) and for higher sensitivity assays capable of detecting PNH cells at frequencies as low as 0.01% . The lack of expression of GPI-anchored proteins such as CD55 and CD59 on the RBC was initially used as the diagnostic test for PNH. Testing the cell surface for the presence of CD55 and CD59 provides useful information about the degree of expression and can be useful in cases with partial deficiency of GPI-anchored proteins. However, analysis of PNH RBCs is not optimal as hemolysis and transfusion can change the accuracy of measuring the PNH clone size . It is now widely recognized that analyzing PNH leukocytes has an advantage in PNH clone detection and size assessment. The peripheral blood granulocytes and the monocytes are most commonly used for analysis and are preferred over lymphocytes that have variable expression of GPI anchored proteins. Based on the assays developed for RBCs, CD55, and CD59 were initially used for detection of WBCs derived from PNH clones. However, these markers have variable expression on different types of WBCs making interpretation of results problematic. For example, severe neutropenia in a patient with PNH-associated bone marrow failure may affect the ability to identify granulocytes derived from the PNH clone.
The development of the fluorescent aerolysin (FLAER) flow cytometry assay has significantly improved the sensitivity and accuracy of detecting granulocytes and monocytes derived from PNH clones compared to single marker immunophenotyping assays. This assay is based on the concept that the bacterial toxin aerolysin specifically binds to GPI-linked structures on cell surfaces. Cells derived from PNH clones lack GPI-linked proteins and therefore are not able to bind the toxin. A fluorochrome-conjugated mutant aerolysin toxin has been developed that binds to the GPI-anchored proteins but does not trigger cell lysis . When compared to the CD-59-based assay on RBCs, the sensitivity of the FLAER assay was significantly higher, especially when the tested clone was small . When compared to CD55 or CD59 analysis of PNH granulocytes, the FLAER assay demonstrated higher sensitivity and higher signal to noise ratio . The FLAER assay is generally used only with WBC, as RBCs express glycophorin, a protein that can weekly bind aerolysin and therefore affect the accuracy of the assay . When the FLAER reagent is combined with antibodies for GPI-linked protein markers on granulocytes (CD16/CD24) or monocytes (CD14), PNH clones representing as little as 0.01% can be detected. Uncovering small PNH clones in patients with bone marrow failure disorder is a challenging task and highlights the need for highly sensitive assays with the ability to detect extremely small clonal populations. The technical advancement in multiparametric flow cytometry enabled the use of immunophenotyping with non-GPI-linked protein markers for lineage characterization (e.g., CD33 and CD15) combined with FLAER reagent and GPI-linked protein markers (CD16/CD24 for granulocytes and CD14 for monocytes), to significantly improve the sensitivity and accuracy of detecting small PNH clones.[15, 18, 19](Fig. 1).
Bone marrow aspirates from patients with bone marrow failure are occasionally submitted for PNH analysis. Because marrow derived cells are at different stages of hematopoietic maturation and therefore have variable expression of GPI anchor proteins, analysis of mature cells from peripheral blood provides more homogeneous population of cells and is the preferred substrate. Bone marrow analysis has no additional diagnostic advantage compared to peripheral blood and therefore should not be used routinely for PNH diagnosis. Bone marrow analysis should be used if known PNH is suspected to be associated with a second bone marrow disorder (e.g., MDS or aplastic anemia).
PIG-A gene mutation analysis can provide final confirmation for the PNH diagnosis. However, PIG-A gene mutations are relatively common in normal hematopoiesis in healthy individuals. In PNH patients, the mutations occur in the HSC and generate a clonal population, whereas in healthy individuals the mutations are usually polyclonal and occur in differentiated progenitors. Because of this uncertainty, PIG-A gene mutation analysis should be reserved for the research setting.
Although PNH is a rare hematological disorder, patients with unexplained RBC hemolysis, thrombosis in unusual vascular locations, and bone marrow failure are frequently tested for PNH. The accurate diagnosis of PNH is imperative and has significant clinical implications for treatment and prevention of adverse events. It is widely accepted that demonstration by combined immunophenotyping and FLAER assay of at least two GPI-anchored protein deficiencies on at least two cell types (i.e., CD59 on RBC, CD24 on granulocytes, or CD14 on monocytes) is the most sensitive and informative assay available for PNH diagnosis. Positive laboratory findings should be viewed in the context of the clinical presentation and it is the clinician's role to determine both the proper classification of PNH (classical, subclinical, and PNH with another bone marrow disorder) and the appropriate treatment .
- 14Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 2010;78:211–230., , , et al.