eMedicine Specialties > Hematology > Red Blood Cells and Disorders

Paroxysmal Nocturnal Hemoglobinuria: Differential Diagnoses & Workup

Author: Emmanuel C Besa, MD, Professor, Department of Medicine, Division of Hematologic Malignancies, Kimmel Cancer Center, Thomas Jefferson University
Coauthor(s): Ulrich Woermann, MD, Consulting Staff, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland
Contributor Information and Disclosures

Updated: Mar 27, 2009

Differential Diagnoses

Hemoglobinuria, Paroxysmal Cold
Hemolytic Anemia
Mesenteric Artery Ischemia
Mesenteric Artery Thrombosis
Portal Vein Obstruction
Renal Vein Thrombosis

Other Problems to Be Considered

Which patients should be tested for paroxysmal nocturnal hemoglobinuria (PNH)?³

Patients should be tested at least once if they have:

• Evidence of hemoglobinuria
• Unexplained hemolysis (increased LDH, absent haptoglobin)
• All patients with abdominal and cerebral vein thrombosis
• All patients with thrombocytopenia, macrocytosis, or signs of hemolysis

• All PNH patients
• All aplastic anemia patients (including post bone marrow transplantation if cytopenias recur)
• MDS patients (still controversial)

Workup

Laboratory Studies

• The tests involved in establishing the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) demonstrates the presence of RBCs that are exceptionally sensitive to the hemolytic action of complement.
  • Flow cytometry – The state-of-the-art laboratory test is to send the patient's blood for flow cytometry to detect CD59 (MIRL), a glycoprotein, and CD55 (DAF) in regulation of complement action. Absence or reduced expression of both CD59 and CD55 on PNH RBCs is diagnostic. The use of flow cytometry in paroxysmal nocturnal hemoglobinuria (PNH) differs from many applications in that the diagnosis depends upon demonstrating the absence of relevant antigens. In this context, it is important that at least 2 GPI-linked antigens are studied to exclude rare congenital deficiencies of single antigens (CD55 and CD59) and polymorphism with individual antigens (CD16), which render them undetectable by some monoclonal antibody clones.
  • Fluorescent aerolysin (FLAERA)¹⁴ – A more accurate alternative reagent for paroxysmal nocturnal hemoglobinuria (PNH) screening and measuring PNH clone is the bacterial toxin aerolysin, which binds to RBCs via GPI anchor and initiates hemolysis. A modified, nonhemolytic form of a fluorescently labeled molecule has been developed that can detect PNH cells to a level of 0.5% (fluorescently labeled inactive toxin aerolysin [FLAER] binding of peripheral blood granulocytes). The advantage of this assay is that it can detect the clone in all hematopoietic cell lineages in one assay.
    
    This is the most specific test for paroxysmal nocturnal hemoglobinuria (PNH), as FLAER binds the GPI anchor specifically. So the lack of FLAER binding to granulocytes (measured by flow cytometry) is sufficient for the diagnosis of PNH. The disadvantage of the test is in measuring binding in the absence of adequate granulocytes—such as in severe aplastic anemia when the number of circulating granulocytes is extremely low.
  • Immmunotyping — Peripheral blood is the most suitable specimen for immunophenotyping for paroxysmal nocturnal hemoglobinuria (PNH), and it is important to screen both RBCs and granulocytes, because RBC transfusions are common among these patients and granulocytes may not be present in severe hypoplastic anemia patients.
  • The value of flow cytometry and monitoring serial analysis for predicting both disease course and clinical phenotype may be important, especially in assessing patients for the likelihood of thrombotic events. Studies have shown that the size of the paroxysmal nocturnal hemoglobinuria (PNH) clone correlates with the risk for venous thrombosis. Patients with less than 50% PNH granulocytes seldom develop thrombosis, whereas patients with larger clone sizes appear to be at great risk and will require anticoagulation.
• Acidified serum lysis and Ham test — If performed properly, acidified serum lysis and the Ham test (from Thomas Hale Ham) are reliable ways to diagnose paroxysmal nocturnal hemoglobinuria (PNH) (see Image 2 and below). Dr. Ham demonstrated that the RBCs in PNH were lysed by complement when normal serum was acidified or activated by alloantibodies.
  

  

  The Ham test (acidified serum lysis) establishes the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), demonstrating a characteristic abnormality of PNH red blood cells by acidified fresh normal serum. Here is a PNH patient's (Pt) red blood cells lysed by normal serum at room temperature (RT) and at 37°C compared with normal red cells (no hemolysis) (control [C]). Heated serum at 56°C inactivates complement and prevents hemolysis in PNH cells. Permission to use this image has been granted by the American Society of Hematology Slide Bank, 3rd edition.

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  The Ham test (acidified serum lysis) establishes the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), demonstrating a characteristic abnormality of PNH red blood cells by acidified fresh normal serum. Here is a PNH patient's (Pt) red blood cells lysed by normal serum at room temperature (RT) and at 37°C compared with normal red cells (no hemolysis) (control [C]). Heated serum at 56°C inactivates complement and prevents hemolysis in PNH cells. Permission to use this image has been granted by the American Society of Hematology Slide Bank, 3rd edition.

  
  
  • The serum pH is lowered to about 6.2 and the magnesium level is adjusted to 0.005 mol/L to achieve maximum sensitivity. The cells that are hemolyzed are the sensitive cells, and those that remain intact are normal cells, indicating 2-3 subpopulations of RBCs in the circulation.
  • A false-positive test result is seen in congenital dyserythropoietic anemia, type II (hereditary erythroblastic multinuclearity with positive acidified serum tests [HEMPAS]). These patients have a negative sucrose hemolysis ("sugar water test") result. Some normal serum can give a false-negative Ham test result; thus, the sucrose water test is more sensitive but less specific for paroxysmal nocturnal hemoglobinuria (PNH).
• Complement lysis sensitivity test – The complement lysis sensitivity test of Rosse and Dacie is a more precise method for diagnosing paroxysmal nocturnal hemoglobinuria (PNH). RBCs are sensitized with a potent lytic anti-I antigen and hemolyzed with limiting amounts of normal serum as a source of complement.^15,16,17 This demonstrates 3 groups of RBCs in patients with PNH including the following:
  • PNH I cells are normal in sensitivity to complement.
  • PNH II cells are moderately more sensitive to complement than normal cells.
  • PNH III cells are markedly sensitive to complement, requiring one fifteenth to one twentieth of the amount of complement for an equal degree of lysis. This group of cells is increased in patients with more severe paroxysmal nocturnal hemoglobinuria (PNH), and it is associated with a mean life span of 10-15 days.
• Sugar water or sucrose lysis test – The sugar water or sucrose lysis test uses the ionic strength of serum that is reduced by adding an iso-osmotic solution of sucrose, which then activates the classic complement pathway, and complement-sensitive cells are lysed. This test is less specific for paroxysmal nocturnal hemoglobinuria (PNH) but more sensitive than the Ham test, because some RBCs hemolyze from autoimmune hemolytic anemias, leukemia, and aplastic anemia to a minor degree. Although the tests are inexpensive and simple to perform, they are more labor intensive and less sensitive due to the short half-life of circulating PNH RBCs.
• Other tests to demonstrate intravascular hemolysis include the following:
  • Elevated serum LDH
  • Elevated reticulocyte count
  • Low-to-absent serum haptoglobin
  • Hemoglobinuria and hemosiderinuria – The presence of hemolysis may be intermittent and can be missed easily, depending on when the tests are performed.
• Evaluation of hematopoiesis should include a complete blood cell (CBC) count and a bone marrow examination. This will differentiate an erythroid and hyperplastic bone marrow during the hemolytic phase or a hypoplastic bone marrow in the aplastic phase.
• Paroxysmal nocturnal hemoglobinuria (PNH) leukocytes have a low leukocyte alkaline phosphatase (ALP) score, which is similar to that for chronic myelogenous leukemia (CML).
• PNH RBCs and other blood cells, because of the missing GPI anchor, are missing membrane proteins DAF (CD55) and MIRL (CD59). This can be detected by flow cytometry as a diagnostic test, which may replace the Ham and sugar water tests.

Imaging Studies

• Thromboses of major veins are best evaluated by radiographic means.
• Investigate hepatic vein thrombosis with a routine technetium-99m (^99m Tc) colloid scan of the liver and spleen. This study often reveals diminished function in all portions of the liver except the caudate lobe, which is spared because it is drained by the inferior vena cava rather than the hepatic vein. An MRI or ultrasonogram can demonstrate the cessation of flow through the hepatic vein or by injection or use of a dye to demonstrate a thrombus in the vein.
• MRI with contrast may demonstrate sagittal vein thrombosis.

Other Tests

PIG-A gene mutation analysis is still limited to research laboratories and, although very specific, is still not diagnostic for paroxysmal nocturnal hemoglobinuria (PNH).

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