Paroxysmal Nocturnal Hemoglobinuria (PNH)

At a Glance

PNH is a rare, acquired stem cell disorder that results in episodic intravascular hemolysis, hemoglobinuria, hemolysis, and venous thrombosis. A somatic mutation causes loss of cell surface linkage proteins, which results in loss of complement regulatory proteins on the red blood cell surface. PNH may occur de novo, but it is also associated with aplastic anemia and may evolve into aplastic anemia after an indeterminate amount of time.

PNH is a rare clinical presentation, estimated to occur in the range of 1-10 cases per million. It is primarily a disease of adults and may have an insidious onset. All patients, however, are anemic (usually seriously so). They may describe dark urine after sleeping. However, this is actually a less likely presentation than the anemia. Hemolysis may occur chronically and throughout the day. Chronic renal failure and iron deficiency anemia may occur. Chronic urinary iron loss, expressed as hemosiderinuria, is a constant feature. Venous thrombosis is present in approximately one-third of patients and is related to abnormal platelet function due to the loss of surface linkage proteins on platelets. Thrombosis is one of the more likely serious clinical scenarios for PNH patients, causing gastrointestinal pain, Budd-Chiari syndrome, headaches, and pulmonary thromboembolism.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The predominant features of presentation somewhat determine the testing procedure. Evaluate first for the common causes of a disease presentation. For example, if a patient presents with iron deficiency anemia, other causes of iron deficiency anemia need ruled out. If the patient presents with acute, delayed, or chronic hemolytic anemia, other causes, such as drug-related hemolytic anemia and extrinsic transfusion-related hemolytic anemia, are important reviews. Pancytopenia may require evaluation for aplastic anemia. Hemoglobinuria can be due to a variety of causes from urinary tract calculi to neoplasia. Once all common causes of the initial presentation have been ruled out, a more explicit diagnostic approach for PNH can be implemented.

As with all anemias, a complete blood count (CBC) and differential should be performed. The degree of anemia and hemoglobin levels can vary widely from very low (<6 g/dL) to within reference range. The mean corpuscular volume (MCV) can also vary from microcytic (if presenting as iron deficiency anemia) to macrocytic (if reticulocytosis and folate/vitamin B12 deficiency occur because of chronic hemolysis). No pathologically characteristic findings are seen on the peripheral smear. Polychromatophilia may accompany macrocytosis. There may also be variable leukopenia and thrombocytopenia. Bone marrow examination also produces variable results with variable cellularity. Bone marrow may be hypocellular or aplastic in the aplastic anemia-associated presentation. Stainable storage iron is usually absent. Dyserythropoiesis is often present but may range from mild to moderate.

Standard tests are available for evaluation of PNH. The sucrose lysis test is the most commonly used screening test for PNH. Blood is collected in heparinized tubes, and red cells are separated and washed. An isotonic sucrose solution allows complement to aggregate on the red blood cell surface in the presence of type-compatible serum. After 1 hour of incubation, lysis greater than 5% of the red blood cells is compatible with the diagnosis of PNH.

An additional test to consider for PNH is urine hemosiderin. Urine hemosiderin is almost always present in PNH and serves as a good screening test for the disorder. Chronic hemolytic anemia depletes haptoglobin, and hemoglobin is reabsorbed by renal tubules and excreted as hemosiderin granules. A positive sucrose lysis test should be confirmed with an acidified serum test (sometimes referred to as Ham's test). Again, red blood cells are collected in heparinized tubes and washed. Type-compatible serum is adjusted to pH 6.8, and the red blood cells are incubated with activated and heat-inactivated serum (complement). Hemolysis is observed with PNH cells, but no hemolysis is observed for control red blood cells.(Table 1)

Table 1

Test Results Indicative of the Disorder
Osmotic Fragility Urine Hemosiderin Acidified Serum Test (Ham's Test)
Hemolysis with Sucrose Positive Positive for Hemolysis

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

Use of the wrong collection tube, for example EDTA, can block complement activation and produce false-negative results in the sucrose lysis test. Most methods now use buffered sucrose solutions, since unbuffered sucrose solutions can also produce false-negative results. EDTA can also block complement activation in the acidified serum test, producing a false-negative result. Accurate pH adjustment is required to pH 6.78 +/- 0.1 for reliable results. Use of a reference laboratory may be required if sucrose lysis and acidified serum tests are not routine at your institution. Urine hemosiderin does not have to be intracellular to be considered positive, given that normal urine should be negative for hemosiderin granules.

What Lab Results Are Absolutely Confirmatory?

A definitive diagnosis of PNH is available through flow cytometry. PNH patients have decreased expression of a protein called glycophosphatidylinositol (GPI) anchor proteins on red blood cells. Consequently, there will be decreased binding of CD55 and CD59, which modulate complement activity and primarily bind to the cell surface GPI. Both CD55 and CD59 can be decreased on red blood cell surfaces; however, since CD55 is more highly expressed than CD59, it is more likely to provide the definitive result.

Flow cytometry is very sensitive and can measure small cellular populations or clones affected by the disease (to the level of 1-5%). It can also detect the emergence of these clones in aplastic anemia or hypoplastic bone marrow patients. As with most flow cytometry, blood should be collected in heparinized tubes and assessed within 24 hours of collection for optimal testing.

What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?

The gene responsible for PNH is the phosphatidylinositolglycan A (PIG-A) gene. It is found on the X chromosome. The somatic mutations associated with PNH can be highly variable. Most mutations consist of small base pair deletions or insertions, but base substitutions can also occur. The mutations typically lead to a DNA frameshift and consequently result in reduced transcription and translation of protein product.

The mutations can also occur at multiple locations within the PIG-A gene. Mutations in aplastic-anemia associated PNH tend to be larger and/or more frequent as compared to patients with PNH only. In addition to complement modulating proteins (CD55 and CD59), GPI binds many other proteins that may variably affect membrane stability and complement activation. One of the more perplexing problems in identifying PNH is the variability in GPI expression, resulting in patients that range from mild to severe suppression of GPI synthesis. The use of flow cytometry for detection of reduced CD55 binding is one way to sort out this mosaicism or expression. Ultimately, routine gene and whole genome sequencing may lead to important information regarding PNH and its association with aplastic anemia.

What Factors, If Any, Might Affect the Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?

Because of the rarity of PNH, many errors are related to testing patients for PNH who have not been thoroughly evaluated for the more common etiologies of their presentation, whether it be chronic hemolytic disease or iron deficiency anemia. Most errors result from this incomplete clinical analysis and occur at the early stages of clinical review. All patients with the appearance of iron deficiency anemia, for example, should be thoroughly reviewed for bleeding sources and evaluated using standard CBC, differential, and screening tests for iron deficiency anemia before pursuing PNH testing. Likewise, a patient presenting with chronic hemolytic disease should be thoroughly reviewed for previous transfusions, drug-induced hemolysis, and other chronic causes of hemolysis before pursuing more specific PNH testing

Since the screening tests for PNH (sucrose lysis test, Ham's test) rely on biological materials from the patient, as compared to normal red blood cells, the tests of necessity are less precise and are potentially less sensitive when improperly performed. These test results are qualitative and based on visual inspection. It is important to adequately evaluate control tubes for lack of hemolysis in both the sucrose lysis test and Ham's test. Thus, it is worthwhile to have such testing performed at a reference laboratory with experience in standardizing these tests.

Flow cytometry is probably the most precise of the tests available for PNH; however, even flow cytometry has several issues. One issue is the measurement for the "absence" of the CD55 marker. Proving negative results is always a more difficult interpretation for the flow cytometry pathologist. Also, the fact that CD59 is expressed at levels eight-fold less than CD55 makes CD59 an inadequate "back-up" marker for PNH.

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