Study type: Blood collected in lavender-top [EDTA] tube and serum collected in red-top tube; related body system: Circulatory system.

PNH is a rare condition in which the patient experiences nocturnal hemoglobinuria, chronic hemolytic anemia, diminished or absent generation of new RBCs, and a tendency to thrombose. It is not considered a primary disease in and of itself but rather a secondary condition caused by an acquired defect in hematopoietic stem cells. Specifically, PNH occurs through acquired mutations in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis, class A) that eventually result in absence of glycosylphosphatidylinositol (GPI) anchor proteins on cell surfaces of damaged stem cell progeny. Normally, the GPI anchor proteins attach a numerous variety of proteins to the cell membrane so they are available when needed. It is believed that PNH is caused by complement-mediated cellular lysis that occurs in the absence of GPI-anchored complement inhibitors. The gene can have multiple mutations acquired throughout a person’s lifetime and can occur in all three cell types (RBCs, WBCs, and platelets), although the type affecting the RBCs is the easiest to identify by the presence of corresponding symptoms.

It was originally thought that the nighttime hemolysis was initiated by a state of acidosis that occurred during sleep. This theory was later disproved, and the prevailing logic is that hemolysis takes place continuously. The hematuria is more noticeable when the accumulated contents of the bladder are passed in the morning and the color of the concentrated urine is dramatically different than that seen during the day. The disease affects males and females equally because the mutations take place in somatic or body cells rather than in germ cells, which are then inherited. Symptoms of the disease usually manifest between the ages of 20 and 40 yr. PNH is frequently associated with aplastic anemia. It has also been associated with myelodysplasia, which may point to bone marrow failure syndromes as a condition that favors exposure of GPI anchor protein–deficient hematopoietic stem cells.

In patients with PNH, erythrocytes have an increased sensitivity to complement and will lyse when mixed with acidified control serum that contains complement. The patient’s RBCs are also mixed with fresh, normal serum that is ABO compatible with the patient’s cells. Some of the control serum is acidified, and some is heated to inactivate the complement. The result is positive if 10% to 50% cell lysis occurs in the samples mixed with patient and control acidified serum. No hemolysis should occur in the heated control serum.

The sugar water or sucrose hemolysis test can also be performed to investigate the presence of PNH. Low-ionic-strength isotonic sucrose will cause serum globulin to fix complement on the RBC surface. When a small amount of type-specific serum and sucrose solution is added to a sample of the patient’s washed RBCs, PNH RBCs will be lysed compared to type-specific serum and sucrose added to washed normal control RBCs. Platelet and granulocyte membranes are affected as well, but RBC hemolysis in a positive test is clear evidence of PNH. Greater than 5% hemolysis is considered positive for PNH.

Flow cytometry has replaced the Ham test as the definitive test for PNH. Levels of CD55 and CD59, membrane glycoproteins that regulate complement, on WBC and RBC surfaces are measured using flow cytometry. CD59 is more prevalent in the membrane than CD55, and the use of both is effective in identifying small and large clones of PNH cells. The findings are described as type I PNH cells, which have normal levels of both proteins; type II PNH cells, which have reduced levels; and type III PNH cells, which demonstrate an absence of CD55 and CD59 proteins. Testing is more accurately accomplished using WBCs, because the life of circulating WBCs is normal in PNH, whereas the life of PNH RBCs is considerably shortened by chronic hemolysis, especially for PNH type III RBCs. Another advantage of WBC studies over RBC is that PNH RBCs are diluted by transfusion, one of the therapeutic modalities used to treat the anemia of PNH.

The treatment strategies for PNH depend on the type and degree of affect. Treatments are limited, and there is a need for improvement in treatment options. While transfusions are effectively used to treat the anemia of PNH, iron overload is a significant consideration in the administration of serial blood cell transfusions.