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.
Once clone size has been determined, PNH can be classified according to presentation, which enables the development of an appropriate treatment plan.
Classification of PNH
||PNH Clone Size
||Hemolysis and/or thrombosis
||Reticulocytosis, high LDH, high bilirubin, low haptoglobin, normal leukocyte and platelet counts
|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%)
||No hemolysis or thrombosis
||Normal or near normal LDH, bilirubin, and haptoglobin
|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.