Laboratory findings and diagnosis

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Haemolytic Anaemia Diagnosis

CD59 FITC

CD59 FITC

leukaemic cells, one is looking for a CD59- cell population, and it is important to see a bimodal distribution in both red cells and granulocytes (a 'tail' of negative cells is not diagnostic and could be a technical artifact). In a typical patient there may be, for instance, 30% CD59- red cells and 90% CD59- granulocytes. Flow cytometry is, of course, very sensitive, and therefore very small PNH cell populations can be detected, and they are detected (especially in granulocytes), particularly in patients with non-severe aplastic anaemia. This raises the issue of what size of PNH cell population should be regarded as diagnostic of PNH. Any cut-off point is bound to be arbitrary, but we have never seen appreciable haemolytic anaemia in a patient in whom less than 5% red cells are of the PNH type. It does not seem sensible to change the diagnosis from, for example, non-severe aplastic anaemia to PNH just because 3% of the granulocytes have the PNH phenotype (in jargon this situation is sometimes referred to as 'lab PNH'), because it should not affect clinical decisions.

Pathogenesis and pathophysiology Haemolytic anaemia

Haemolytic anaemia in PNH is a due to an intracorpuscular abnormality of the red cell. In this respect, PNH is similar to inherited haemolytic anaemias, such as those caused by an enzyme defect. However, PNH is an acquired disorder, and there is no evidence that it is inherited. The abnormality of red cells in PNH is indeed an enzyme defect, but it is caused by a somatic mutation rather than by an inherited mutation. This explains the 'mosaicism' found in the blood of PNH patients, whereby cells belonging to the PNH clone, which have arisen through a somatic mutation, coexist with the remaining qualitatively normal cells.

We now know that the somatic mutation is in an X-linked gene that has been called PIG-A, for phosphatidylinositol glycan complementation group A. PIG-A encodes one of the subunits

Figure 11.2 (left) Different patterns of PNH red cells populations in several PNH patients. The top panel is from a normal individual. Patient MB has 57% red cells that lack CD59 completely (PNH III red cells). Patient CJ has only 4% normal red cells; 96% of her red cells have a partial deficiency of CD59 (PNH II red cells), indicating that virtually her entire haemopoiesis is supported by a PNH clone with a missense mutation of PIG-A. The trimodal distribution of red cells in patient RK indicates the coexistence, along with normal red cells, of a PNH III clone (22%) and a PNH II clone (12%) (modified from Dacie JV, Lewis SM, Luzzatto L etal. (1995) Laboratory methods used in the investigation of paroxysmal nocturnal haemoglobinuria (PNH). In: PracticalHaematology (JV Dacie, SM Lewis, eds), 8th edn, pp. 287-96. Churchill Livingstone, London).

Figure 11.3 The molecular basis of the cellular abnormality in PNH. In a normal cell (a) a complex biosynthetic pathway produces in the endoplasmic reticulum (ER) a glycophospholipid molecule (see inset) called glycosylphosphatidylinositol (GPI). An early step in the biosynthetic pathway is catalysed by an acetylglucosaminyl transferase; one of the subunits of this enzyme is encoded by the gene PIG-A, located on the short arm of the X chromosome (band Xp22). A number of cellular proteins become covalently linked to the GPI molecule, which serves to convey and anchor them as extracellular surface proteins to the cell membrane. The PNH cell (b) has a mutation in the PIG-A gene, causing a serious defect in the acetylglucosaminyl transferase. This in turn causes a total or partial block in the synthesis of the GPI molecule. As a result, the proteins that require a GPI anchor are unable to become membrane bound and will be lacking from the cell surface (modified from Luzzatto L, Notaro, R. (2003) Paroxysmal nocturnal hemoglobinuria. In: Blood: Principles and Practice of Hematology (RI Handin, SE Lux, TP Stossel, eds), 2nd edn, pp. 319-34. Lippincott Williams & Wilkins, Philadelphia).

Figure 11.3 The molecular basis of the cellular abnormality in PNH. In a normal cell (a) a complex biosynthetic pathway produces in the endoplasmic reticulum (ER) a glycophospholipid molecule (see inset) called glycosylphosphatidylinositol (GPI). An early step in the biosynthetic pathway is catalysed by an acetylglucosaminyl transferase; one of the subunits of this enzyme is encoded by the gene PIG-A, located on the short arm of the X chromosome (band Xp22). A number of cellular proteins become covalently linked to the GPI molecule, which serves to convey and anchor them as extracellular surface proteins to the cell membrane. The PNH cell (b) has a mutation in the PIG-A gene, causing a serious defect in the acetylglucosaminyl transferase. This in turn causes a total or partial block in the synthesis of the GPI molecule. As a result, the proteins that require a GPI anchor are unable to become membrane bound and will be lacking from the cell surface (modified from Luzzatto L, Notaro, R. (2003) Paroxysmal nocturnal hemoglobinuria. In: Blood: Principles and Practice of Hematology (RI Handin, SE Lux, TP Stossel, eds), 2nd edn, pp. 319-34. Lippincott Williams & Wilkins, Philadelphia).

of the enzyme responsible for the transfer of N-acetylgluco-samine (GlcNAc) onto phosphatidylinositol. This is the first step of the complex biosynthetic pathway that in the end produces the glycosylphosphatidylinositol (GPI) anchor required for anchoring many proteins to the cell membrane (Figure 11.3 and Table 11.3). One of these proteins, CD59 or membrane inhibitor of reactive lysis (MIRL), normally protects the red cell from being lysed by the membrane attack complex (C5-C8) that forms when complement (C) is activated. In essence, the hyper-susceptibility to C of PNH red cells is the consequence of CD59 deficiency. Chronic intravascular haemolysis in PNH is probably explained by activation of C through the 'alternative' pathway, which goes on at a low rate all the time, whereas the dramatic exacerbation of haemolysis that is characteristically associated with intercurrent viral or bacterial infection in patients with PNH is probably explained by brisk activation of C by an antigen-antibody reaction (i.e. through the 'classical' pathway).

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