Approach

Evidence of intravascular haemolysis with concomitant iron deficiency and recurrent bouts of dark urine may suggest paroxysmal nocturnal haemoglobinuria (PNH). The definitive diagnosis of PNH is made by the demonstration of cells lacking the glycosylphosphatidylinositol (GPI) anchor or proteins anchored by it to the membrane. This is best achieved by flow cytometry, using either a reagent to detect the GPI anchor (fluorescent aerolysin [FLAER]) or antibodies to such proteins as CD55, CD59, CD14 (monocytes), and CD24. Older techniques such as acidified serum lysis or sucrose lysis are no longer used.[2]

Clinical presentation

At initial presentation, about 20% of patients have dark urine. However, almost all patients have haemoglobinuria at some point in their disease course, often with infections or as paroxysms lasting 2-6 days.[1][18]

Patients may present with symptoms of anaemia (tiredness, shortness of breath, and palpitations). Other presenting symptoms that should be enquired about are abdominal pain (e.g., right upper quadrant pain in Budd-Chiari syndrome), odynophagia, dysphagia, and erectile dysfunction.[2][19]

Petechiae, ecchymoses, or frank bleeding (epistaxis, mucosal bleeding, gastrointestinal bleeding, genitourinary bleeding) may occur in the setting of concomitant marrow hypoplasia or myelodysplastic syndrome with low platelet numbers.

The physician should enquire about whether there is any history of aplastic anaemia, myelodysplasia, or recurrent infection.

On examination, the physician should look for:[20]

  • Signs of anaemia (pallor, tachycardia, and systolic heart murmur)

  • Signs of thrombosis (most commonly deep vein thrombosis, although cerebral or arterial thrombosis may be present)

  • Signs of Budd-Chiari syndrome (right upper quadrant tenderness, liver enlargement, and ascites); see  Budd-Chiari syndrome

  • Signs of elevated intracranial pressure (headache/vomiting, papilloedema, coma in severe cases), and/or frank sensorimotor deficits according to the affected site, which may be present and are related to cerebrovascular thrombotic events

Patients may also present asymptomatically with unexplained iron deficiency detected incidentally on investigation of the FBC or iron studies for another reason.

Indications for testing

Patients to be tested include those with:[2][21]

  • Haemoglobinuria or haemosiderinuria

  • Unexplained Coombs-negative haemolytic anaemia

  • Aplastic or hypoplastic anaemia (current or in the past)

  • Myelodysplasia with evidence of haemolysis (haemoglobinuria or elevated serum lactate dehydrogenase [LDH])

  • Thrombosis, especially in unusual sites (e.g., intra-abdominal) with evidence of haemolysis, or in young patients when thromboses are unexplained

  • Unexplained iron deficiency

  • Dystonic symptoms (oesophageal spasm, abdominal pain, erectile dysfunction) with signs of haemolysis

  • Renal dysfunction with signs of haemolysis

  • Thrombocytopoenia and/or granulocytopoenia with signs of intravascular haemolysis

Investigations

At presentation, full blood count and reticulocyte count, LDH, haptoglobin levels, and bilirubin are assessed to confirm intravascular haemolysis. Haemoglobin is often low, as is haptoglobin. LDH and reticulocytes are usually high, with elevated unconjugated bilirubin.

The diagnosis is supported by the presence of urine haemosiderin on microscopy and haemoglobin on dipstick. The antiglobulin Coombs test excludes autoimmune haemolytic anaemias.

The diagnostic standard and only definitive diagnostic tests are those that demonstrate the absence of the GPI anchor or proteins associated with it. The anchor can be detected by a product of a species of Aeromonas that binds directly to it. When the product bound to the GPI anchor is fluoresceinated (FLAER test), it can be used in flow cytometry to detect as little as 0.01% of granulocytes or monocytes lacking the anchor. The reagent cannot be used to examine red blood cells (RBCs), as it adheres non-specifically to glycophorin on the surface. Evidence of more than 1% to 3% of granulocytes lacking the anchor is diagnostic for PNH.[22] Fluoresceinated monoclonal antibodies to GPI-anchored proteins can also be used to detect the defective cells characteristic of PNH.[6] Immunotyping has also been used to detect missing proteins on PNH cells. Anti-CD59 is most frequently used to detect abnormal RBCs and granulocytes. Anti-CD55 can also be used to detect abnormal red blood cells and granulocytes. Anti-CD16 or anti-CD24 can be used for granulocytes and anti-CD14 for monocytes. It has been suggested that two antibodies on two cell lines should be abnormal to be certain of the diagnosis.[2] D-dimer levels can be used to assess the risk of thrombosis.

These flow cytometry tests can quantify the percentage of cells with the PNH abnormality. This should be reported for granulocytes as a measure of the size of the abnormal haematopoietic stem cell clone(s). The percentage of abnormal RBCs should also be reported as an indication of the number of cells at risk of haemolysis. The degree of abnormality of the cells in each population should also be reported. PNH II cells have about 10% of the normal amount of GPI anchor or anchored proteins, whereas PNH III cells have no detectable anchor or anchored proteins.

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