PNH-Affected RBC’s Mean Life Span Estimated by Reticulocyte-Gated Flow Cytometry Reflects In Vivo Hemolysis in PNH.
Authors: Ninomiya Haruhiko , Sato Shoko , Hasegawa Yuichi , Kojima Hiroshi , Nagasawa Toshiro 
Published: April 2020

Abstract
The most characteristic clinical feature of PNH is intravascular hemolysis of PNH-affected RBC due to deficiency in the expression of GPI-anchored membrane proteins with complement-regulatory activity, CD55 and CD59. PNH-affected (CD55- and CD59-negative) RBC with complement sensitivity should have a shortened life span due to intravascular hemolysis, although life span of PNH-RBC separately from normal RBC could not be measured, at least clinically in PNH patients. We recently developed a sensitive flow cytometric method to analyse the PNH-phenotype (CD59-negative) in reticulocyte and whole RBC (Sato S et al: Reticulocyte-gated flow cytometric analysis of red blood cells in paroxysmal nocturnal hemogobinuria. Laboratory Hematology12: 82–85, 2006). This 2-color (RNA/CD59) flow cytometry could analyse the phenotype of reticulocyte fraction in detail, that enables to assess PNH phenotype of reticulocytes in several hematological conditions such as an emergence of RBC with PNH phenotype in aplastic anemia and a minimal residual production of PNH-RBC after hematopoietic stem cell transplantation for PNH. Analyses of PNH phenotype consecutively in total 7 patients with PNH showed differences in ratios between reticulocytes and whole RBC, where % CD59-negative population in the former was always higher than the latter due to hemolysis of CD59-negative mature RBC in the circulation. On an assumption that CD59-negative reticulocytes are not destroyed in the reticulocyte maturation time and CD59-positive RBC has a mean life span (MLS) of 120 days, we proposed a formula to estimate PNH-RBC’s MLS: W/100 = R x M / [ (100-R) x 120 + R x M ], where W, % CD59-negative whole RBC; R, % CD59-negative reticulocytes; M, MLS (day) of PNH-RBC. By this formula, PNH-RBC’s MLS was estimated at 16–45 days in the patients. It showed a poor correlation with absolute reticulocyte count or hemolysis-related laboratory data (lactate dehydrogenase (LD) etc), suggesting erythropoietic ability in response to anemia was different among PNH patients due to their various hematological backgrounds underlying PNH. Comparable degree of anemia did not induce same degree of reticulocytosis in these patients. Hence, we are proposing to use an erythropoietic ability-adjusted index, red cell turnover index (RCTI), calculated from the following formula to assess the total body hemolysis of PNH-RBC: PNH-RCTI = [(absolute reticulocyte count) x (% CD59-negative retiuclocyte) / 100] / (PNH-RBC’s MLS). PNH-RCTI is assumed to correlate with turnover rate of PNH-RBC by complement-mediated lysis in the circulation. PNH-RCTI ranged 0.88–15.3 x 103 reticulocytes/μl/day (reference range, normal RCTI 0.4–0.8). Serum LD level is a non-specifc parameter being increased by in vivo hemolysis, especially intravascular hemolysis. While PNH-RBC’s MLS did not show any significant correlation with serum LD levels (r=0.306), PNH-RCTI correlated positively with serum LD levels (r=0.704, 15 points) in 7 PNH patients. These data indicate that shortened PNH-RBC’s MLS estimated from the reticulocyte-gated flow cytometry could be a reliable parameter reflecting in vivo hemolysis in PNH.