Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia
Authors: Lim Hwee Yong , Farley Marjorie , Wittwer Carl , Parker Charles 
Published: April 2020

Abstract
PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham’s test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had &gt;5% GPI-AP− RBCs, while 18 cases (33%) had &gt;10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had &gt;10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham’s test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with &gt;10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham’s test and the sucrose lysis test as screening assays for PNH.