Shoko Sato

Enhanced expression of CD71, transferrin receptor, on immature reticulocytes in patients with paroxysmal nocturnal hemoglobinuria.

Erythroid cells in the marrow express CD71, transferrin receptor, and reticulocytes released from the marrow lose their expression during maturation. The immature reticulocyte fraction, the proportion of reticulocytes with the highest content of RNA, has been determined by hematology analysis. In the present study, we examined CD71 expression on immature reticulocytes by flow cytometry (FCM) in paroxysmal nocturnal hemoglobinuria (PNH) patients with reticulocytosis. We modified ‘reticulocyte‐gated FCM’ to multi‐color FCM, i.e. RNA/CD71, RNA‐CD59 or CD59/CD71/CD45. In PNH, in addition to the increased number of immature reticulocytes (%CD71‐positive), a more immature phenotype in regard to both CD71 intensity and RNA content levels was demonstrated. In seven PNH patients studied, %CD71‐positive reticulocytes were significantly increased at 32.2 ± 11.9% (n = 10, normal 10.4 ± 3.5%, P = 0.002). RNA content levels (assessed by mean fluorescence index, MFI) in CD71‐positive reticulocytes were significantly increased at 812.0 ± 215.2 MFI in PNH (n = 10, normal 508 ± 86.1 MFI, P = 0.002). These data indicate that stimulated erythropoietic conditions induce the release of more immature reticulocytes to the peripheral blood than ordinary erythropoietic conditions. CD71 intensity on immature reticulocytes was well correlated with their RNA content levels, indicating the usefulness of CD71 as an immature reticulocyte marker.

Pseudoreticulocytosis in a patient with hemoglobin Köln due to autofluorescent erythrocytes enumerated as reticulocytes by the Cell-Dyn 4000.

Pseudoreticulocytosis in a 25-year-old female patient with hemoglobin Köln is reported. The abnormal hemoglobin, hemoglobin Köln (beta chain, Val98-->Met), had previously been confirmed in the patient at the age of 21 years, as well as in her mother, by polymerase chain reaction-based direct sequence analysis of the beta globin gene. The patient underwent splenectomy at the age of 22 years. On her admission to our hospital for treatment of an immunoglobulin A nephropathy, an analysis by an automated hematology analyzer, the Abbott Cell-Dyn 4000 (CD4000), reported a marked reticulocytosis. Staining by the Brecher method with new methylene blue indicated a moderate reticulocytosis (5.7%) of a lesser extent than that indicated by the CD4000 (51.1%). The frequencies of red blood cells (RBC) with Pappenheimer bodies (13.8%), Heinz bodies (32.7%), and Howell-Jolly bodies (0.3%) were increased. The CD4000 detects RBC with RNA fluorescently stained with CD4K530 as reticulocytes. Autofluorescence of RBC with hemoglobin Köln, as we demonstrated by flow cytometry and fluorescent microscopy, was considered to have caused the pseudoreticulocytosis on the fully automated reticulocyte enumeration by the CD4000.

Reticulocyte-gated flow cytometric analysis of red blood cells in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins in the affected blood cell membranes. Analysis of blood cells by flow cytometry is useful to identify the affected blood cells with PNH-specific phenotypes. Because PNH-affected red blood cells (RBC) have shortened life-spans in the circulation, ratios of PNH-affected populations analyzed by flow cytometry in whole RBC are lower than those in PNH-affected erythropoiesis. We developed a reticulocyte-gated 2-color flow cytometry of RBC and revealed that the percentages of PNH-affected, CD59--populations in reticulocytes were higher than in whole RBC in patients with PNH. A serial analysis of a patient with PNH who underwent peripheral blood stem cell transplantation confirmed the usefulness of this method to evaluate PNH-affected RBC populations with high sensitivity; ie, the presence of CD59- reticulocytes in the circulation could be a sensitive marker for PNH-affected erythropoiesis.

Early heparin administration attenuates tissue factor-mediated thrombin generation during simulated cardiopulmonary bypass.

We tested the hypothesis that heparin administration prior to the emergence of tissue factor (TF) would increase plasma TF pathway inhibitor (TFPI) and attenuate TF‐mediated thrombin generation during simulated cardiopulmonary bypass (CPB).

Shortened lifespan of paroxysmal nocturnal haemoglobinuria-affected RBC estimated from differences in ratios of CD59-negative populations between reticulocytes and whole RBC.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haemolytic disease characterized by complement‐sensitive red blood cells (RBC). PNH‐affected RBC (PNH‐RBC) should have a shortened mean lifespan (MLS); however, direct measurement is difficult. We have recently developed a sensitive flow cytometric assay to analyse PNH‐affected reticulocytes that may closely correspond to the PNH clone‐derived erythropoiesis. Naturally, the CD59‐negative populations in reticulocytes were larger than those in whole RBC in PNH. We estimated the MLS of PNH‐RBC in six PNH patients from the differences in the ratios of CD59‐negative populations between reticulocytes and whole RBC. The MLS of PNH‐RBC was calculated using the following formula: W/100 = R × M/[(100 − R) × 120 + R × M], where W, percentage CD59‐negative whole RBC; R, percentage CD59‐negative reticulocytes; M, MLS (days) of CD59‐negative RBC. The MLS of PNH‐RBC, estimated as 16–45 days in the PNH patients, showed a weak positive and a weak negative relation with RBCs and percentage reticulocytes, respectively, among the patients. The MLS, in individual patients, altered irrespective of RBC and percentage reticulocytes. The MLS calculated from our methods may be a parameter that evaluates the haemolytic conditions in PNH.