Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, potentially life-threatening acquired stem cell disorder caused by a mutation in the PIGA gene. The mutation leads to a lack of glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of blood cells, which in turn leads to an inappropriate immune response to, and hemolysis of, these cells. In addition to hemolysis, PNH is characterized by thrombosis and bone marrow failure. Although some patients may present with the nocturnal hemoglobinuria for which the disease is named, the common symptoms of PNH are nonspecific (eg, fatigue, dyspnea, anemia, abdominal pain), which presents a diagnostic challenge. A delayed or missed diagnosis prevents patients from receiving appropriate treatment and thereby decreases quality of life and impacts survival. Laboratory testing in PNH includes flow cytometry to diagnose PNH in patients with suggestive symptoms or test results. Laboratory testing is also used to classify PNH to inform treatment, and to monitor disease progression and the effects of treatment.
Quick Answers for Clinicians
Once a patient has been assessed for hemolysis (see Initial Evaluation) and paroxysmal nocturnal hemoglobinuria (PNH) is suspected, the recommended test is flow cytometry to evaluate for the presence of glycosylphosphatidylinositol (GPI)-linked antigens on blood cells. Both red blood cells (RBCs) and white blood cells (WBCs) should be analyzed. For additional details, see Flow Cytometry.
Paroxysmal nocturnal hemoglobinuria (PNH) arises due to variants in the PIGA gene, which result in the deficiency or absence of glycosylphosphatidylinositol (GPI)-anchored cell membrane proteins. This in turn leads to a partial or complete deficiency of CD55 and CD59, two important molecules in the complement cascade, on the surface of hematopoietic stem cells. These stem cells clonally expand into red blood cells (RBCs) and white blood cells (WBCs) that are susceptible to lysis by complement. The resulting hemolysis leads to a number of symptoms; however, the pathophysiology of the bone marrow failure and thromboses often observed in PNH is unknown.
Indications for Testing
Laboratory testing for PNH is used to:
- Diagnose PNH in patients with the following clinical indications :
- Unexplained hemolysis with iron deficiency, abdominal pain, thrombosis, dysphagia, or granulocytopenia/thrombocytopenia
- Coombs-negative hemolytic anemia that cannot be otherwise explained
- Thrombosis at unusual sites or with unexplained hemolytic anemia or cytopenia
- Evidence of bone marrow failure (aplastic anemia, hypoplastic anemia, myelodysplastic syndrome)
- Monitor disease progression and response to treatment in patients with confirmed PNH
A CBC with peripheral smear, reticulocyte count, and Coombs test should be performed in all cases of suspected PNH to assess anemia. Depending on whether the patient has classic PNH or PNH in the context of another disorder (see Classification of PNH table, below), leukocyte and platelet counts may be normal or low. A reticulocyte count is also recommended to assess the bone marrow response to anemia. Reticulocyte counts are generally elevated in PNH, but are lower than would be expected given the severity of anemia. A Coombs test should be performed to rule out other causes of anemia, as PNH is Coombs negative.
Serum lactate dehydrogenase (LDH), indirect bilirubin, and serum haptoglobin are useful for assessing hemolysis. Serum LDH is nearly always elevated in clinical PNH, although the level of elevation depends on the PNH classification. Indirect bilirubin may be elevated, and serum haptoglobin is generally low, although it should be noted that patients with subclinical PNH will not exhibit any evidence of hemolysis.
Flow cytometry is the preferred technique for the diagnosis of PNH and is performed to evaluate for the presence of GPI-linked antigens on blood cells. Peripheral blood is the preferred specimen, and the use of multiple specific GPI-linked reagents is recommended. Flow cytometry allows for calculation of the percentage of red blood cells (RBCs) or white blood cells (WBCs) that entirely or partially lack GPI-linked antigens compared to normal cells; this percentage is referred to as the PNH clone size.
Determination of both WBC and RBC clone size is recommended for diagnosis because there are limitations associated with either WBC or RBC testing alone. WBC analysis using fluorescently labeled aerolysin (FLAER) and CD157 as GPI-linked markers, as well as CD15 and CD64 as lineage-specific markers for granulocytes and monocytes, respectively, is the most accurate test for PNH clone size. Analysis of RBCs alone may lead to an underestimate of the overall PNH clone size, as a patient may have received a transfusion of normal RBCs. WBC testing, however, is less useful for the detection of cells that only partially lack GPI-linked antigens.
Testing for a PIGA gene variant can be used to confirm a diagnosis of PNH; however, this testing is not widely performed. Other tests for GPI-anchored proteins, such as erythrocyte acetylcholine esterase or neutrophil alkaline phosphatase assays, can be used to support a diagnosis made via flow cytometry, but are not recommended for standalone use.
|Classification||Presentation||Markers||PNH Clone Size|
|Classic PNH||Hemolysis and/or thrombosis||Reticulocytosis, high LDH, high bilirubin, low haptoglobin, normal leukocyte and platelet counts||Large (>50%)|
|PNH in the context of another disorder||Primary bone marrow disorder (eg, aplastic anemia, myelodysplastic syndrome)||Reticulocytosis, variable LDH, high bilirubin, low haptoglobin, low leukocyte and platelet counts||Varies, but generally small (<50%)|
|Subclinical PNH||No hemolysis or thrombosis||Normal or near normal LDH, bilirubin, and haptoglobin||Small (<10%)|
|Sources: Parker, 2016 ; Borowitz, 2010 ; Parker, 2005|
Bone Marrow Examination
Histologic examination of bone marrow is required for the identification of primary bone marrow disorders and thus the classification of PNH, and is indicated when pancytopenia is present to rule out other disorders or when bone marrow transplantation is considered. Cytogenetic analysis of bone marrow samples is recommended to aid in the identification of underlying disease processes associated with PNH.
Regular monitoring of clone size with flow cytometry is recommended in patients diagnosed with PNH to assess disease progression. Annual monitoring is recommended for patients with stable PNH; more frequent monitoring is suggested if the clone size is changing. Monitoring is also recommended to assess response to therapy if the patient is on eculizumab therapy and to determine transfusion needs.
WBC analysis is the most reliable test for PNH clone size (as mentioned above), but RBC analysis is the most appropriate test for monitoring subclinical PNH and response to eculizumab, particularly during stabilization of the RBC clone. RBCs also remain suitable for testing longer than WBCs, and can be used to quantify cells that are only partially deficient in GPI-anchored proteins.
Serum LDH generally decreases to near normal levels in patients treated with eculizumab. However, laboratory evidence of anemia and hemolysis may persist, regardless of the success of treatment. Iron stores and serum erythropoietin should be examined in eculizumab-treated patients with persistent anemia to determine whether additional treatment to facilitate erythropoiesis is warranted.
ARUP Lab Tests
Preferred test for initial diagnosis of PNH and quantification of PNH clones; includes high-sensitivity WBC and RBC analysis
For additional test information, refer to the Paroxysmal Nocturnal Hemoglobinuria Test Fact Sheet
Quantitative Flow Cytometry
Monitor subclinical PNH and eculizumab treatment
Quantitative Flow Cytometry
Quantify or monitor PNH clone size
Quantitative Flow Cytometry
Evaluate for hemolysis, thrombocytopenia, and leukopenia
Automated Cell Count/Differential
Assess bone marrow response to anemia
Parker CJ. Update on the diagnosis and management of paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program. 2016; 2016 (1): 208-216.PubMed
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): 211-30.PubMed
Parker C, Omine M, Richards S, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood. 2005; 106 (12): 3699-709.PubMed
Sutherland DR, Acton E, Keeney M, et al. Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection. Cytometry B Clin Cytom. 2014; 86 (1): 44-55.PubMed
Perkins S. Paroxysmal Nocturnal Hemoglobinuria. In Kjeldsberg C. Practical Diagnosis of Hematologic Disorders, 5th ed. Chicago: ASCP Press, 2006.
Merzianu M, Groman A, Hutson A, et al. Trends in Bone Marrow Sampling and Core Biopsy Specimen Adequacy in the United States and Canada: A Multicenter Study. Am J Clin Pathol. 2018;150(5):393-405.