Multidisciplinary clinical management of paroxysmal nocturnal hemoglobinuria

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease caused by clonal expansion of one or more hematopoietic stem cell (HSC) lines due to a somatic mutation of the phosphatidylinositol glycan anchor (PIG-A) gene located on Xp22.1. PNH incidence is 1.5-2 cases per million of the population per year. PNH can affect multiple systems in the body and requires multidisciplinary clinical management. Patients can manifest with severe pancytopenia, life-threatening thrombosis affecting the hepatic, abdominal, cerebral, and subdermal veins, and high requirements for blood transfusion due to haemolytic anemia. PNH can also be associated with bone marrow failure. Advances in diagnostic techniques and a targeted therapeutic approach for PNH have emerged in the last two decades. Eculizumab, a promising humanized monoclonal antibody against C5, is the first approved therapy for PNH.

Keywords: Paroxysmal nocturnal hemoglobinuria; diagnosis; eculizumab; treatment.

Figure 1

Pathogenesis of PNH. A somatic acquired mutation in the gene encoding PIG-A is the pathobiological mechanism underlying the development of PNH. Affected stem cells are deficient in all GPI-APs that serve as erythrocyte membrane-boundregulators of complement. Deficiency of CD55 and CD59 proteins accounts for the complement-mediated intravascular hemolysis and other clinical manifestations.

Figure 2

Clinical manifestations of PNH.

Figure 3

FLAER results of a patient with PNH. PNH testing on granulocytes using HSFC PNH testing; using SSC-CD45 graph, WBCs and Lymphocytes are gated (A). and CD45+ WBCs are selected for the next graph: SSC-CD15 in which CD15+ granulocytes are gated (B). Those granulocytes are analyzed based on their CD24, a GPI-linked molecule found on granulocytes, and FLAER expression (C). As you can see from (C), 98.7% of CD15+ Granulocytes are double negative in terms of CD24 and FLAER which shows their lack of GPI expression and called as “PNH Clone”. Lymphocytes which are gated on SSC-CD45 graph, can be evaluated also based on their CD24/FLAER pattern. Due to long spans of lymphocytes, we can see the smaller clone size (26.33% of Lymphocytes are lack of GPI) (D). PNH testing on monocytes using HSFC PNH testing; Using SSC-CD45 graph, WBCs and lymphocytes are gated (E) and CD45+ WBCs are selected for the next graph: SSC-CD64 in which CD64+ monocytes are gated (F). Those monocytes are analyzed based on their CD14, a GPI-linked molecule found on monocytes, and FLAER expression (G). 99.19% of CD64+ monocytes are double negative in terms of CD14 and FLAER which shows their lack of GPI expression and called as “PNH Clone”. Lymphocytes which are gated on SSC-CD45 graph, can be evaluated also based on their CD14/FLAER pattern. Due to long spans of lymphocytes, we can see the smaller clone size (23.18% of lymphocytes are lack of GPI) (H). PNH testing on RBCs using HSFC PNH testing; using SSC-FSC log graph, RBCs are gated (I) and those RBCs are further analyzed based on their CD235a expression (J), a molecule which is found only on RBCs. Finally CD235a+ RBCs are analyzed in terms of their CD59 expression. There are 3 types of cells present in PNH positive samples. Type 3 cells are completely negative for GPI and thus negative for CD59 expression. Type II cells partially express GPI and thus have dim-CD59 expression. Finally type I cells are healthy RBCs carrying GPI molecule and have bright CD59 expression (K and L).

Figure 4

Treatment algorithm of PNH.