PNH, PI-Linked Antigen, Blood
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder characterized by nocturnal hemoglobinuria, chronic hemolytic anemia, thrombosis, pancytopenia, and, in some patients, acute or chronic myeloid malignancies.
PNH appears to be a hematopoietic stem cell disorder that affects erythroid, granulocytic, and megakaryocytic cell lines. The abnormal cells in PNH have been shown to lack glycosylphosphatidylinositol (GPI)-linked proteins in erythroid, granulocytic, megakaryocytic, and, in some instances, lymphoid cells. Mutations in the phosphatidylinositol glycan A gene, PIGA, have been identified consistently in patients with PNH, thus confirming the biological defect in this disorder.
A flow cytometric-based assay can detect the presence or absence of these GPI-linked proteins in granulocytes, monocytes, erythrocytes, and lymphocytes, thus avoiding the problems associated with red cell-based diagnostic methods (Ham test) in which recent hemolytic episodes or recent transfusions can give false-negative results. A partial list of known GPI-linked proteins include CD14, CD16, CD24, CD55, CD56, CD58, CD59, C8-binding protein, alkaline phosphatase, acetylcholine esterase, and a variety of high frequency human blood antigens. In addition, fluorescent aerolysin (FLAER) binds directly to the GPI anchor and can be used to evaluate the expression of the GPI linkage.
Our studies, as well as others in the literature, have shown that flow cytometry-based assays will detect all Ham-positive PNH cases, as well as some Ham-negative PNH cases. This assay replaces the sugar water test and the Ham test for the evaluation of patients with possible PNH.
Patients with PNH should be transfused with ABO-specific RBCs, which do not need to be washed. If, for some reason, they need to receive non-ABO type-specific (type O) cells, these RBC units should be washed. Since recipient antibodies to granulocyte antigens can trigger hemolytic episodes in PNH, if they have such antibodies these patients should receive leukoreduced RBCs and platelets.
Screening for and confirming the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH)
Monitoring patients with PNH
Individuals with paroxysmal nocturnal hemoglobinuria (PNH) have absent or decreased expression of all the glycosylphosphatidylinositol (GPI)-linked antigens and fluorescent aerolysin (FLAER) on peripheral blood cells derived from the PNH clone.
Recent data showed that small PNH clones can be detected in a relatively high percentage of cases of aplastic anemia and myelodysplastic syndrome. While the significance of this finding is still uncertain, it appears that these patients may benefit from immunosuppressive therapy.
This test incorporates a sophisticated technique of separating different cell populations using gating on antigen-positive cells, as well as the sensitivity to enable detection of small PNH clones. In addition, this test detects a partial loss of CD59 on RBCs (type II RBC). Patients with large proportion of type II RBC are unlikely to show high levels of hemolysis, unlike patients with complete loss of GPI-linked proteins (predominantly type III cells). While PNH is a disorder of hematopoietic stem cells and all lineages are affected, the percentage of affected cells can differ between lineages, most commonly due to RBC hemolysis and/or transfusion.
Individuals without PNH have normal expression of FLAER (neutrophils and monocytes) and normal expression of all GPI-linked antigens-CD14 (monocytes), CD16 (neutrophils and NK cells), CD24 (neutrophils), and CD59 (RBCs).
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
The sugar water test and the Ham test are no longer recommended for the evaluation of patients with possible paroxysmal nocturnal hemoglobinuria.
Recent transfusion can decrease the sensitivity of this test and interfere with accuracy.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
An interpretive report will be provided.
RED BLOOD CELLS:
PNH RBC-Partial Antigen loss: 0.00-0.99%
PNH RBC-Complete Antigen loss: 0.00-0.01%
PNH Granulocytes: 0.00-0.01%
PNH Monocytes: 0.00-0.05%
Clinical References Provides recommendations for further in-depth reading of a clinical nature
1. Miyata T, Yamada N, Iida Y, et al: Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 1994;330:249-255
2. Brodsky RA. Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria. Blood Rev 2008 Mar;22(2):65-74
3. Richards SJ, Barnett D: The role of flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria in the clinical laboratory. Clin Lab Med 2007 Sep;27(3):577-590
4. Parker C, Omine M, Richards S, et al: Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005 Dec 1;106(12):3699-3709
5. Richards SJ, Hill A, Hillman P: Recent advances in the diagnosis, monitoring and management of patients with paroxysmal nocturnal hemoglobinuria. Cytometry B Clin Cytom 2007 Sep;72(5):291-298
6. Shichishima T, Terasawa T, Hashimoto C, et al: Discordant and heterogeneous expression of GPI-anchored membrane proteins on leukemic cells in a patient with paroxysmal nocturnal hemoglobinuria. Blood 1993;81:1855-1862
7. Brodsky RA, Mukhina GL, Li S, et al: Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000;114:459-466
8. Rosse WF: Phosphatidylinositol-linked proteins and paroxysmal nocturnal hemoglobinuria. Blood 1990;75:1595-1601
9. Borowitz MJ, Craig FE, DiGiuseppe JA, et al: Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 2010 78(4):211-230
10. van der Schoot CE, Huizinga TW, van't Veer-Korthof ET, et al: Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay. Blood 1990;76:1853-1859