Sensitive measurement of fragmented red cell population using flow cytometry, and its application for estimating thrombotic microangiopathy after stem cell transplantation

Abstract

Background: Thrombotic microangiopathy (TMA) is one of the lethal complications after hematopoietic stem cell transplantation (SCT). The levels of fragmented red cells (FRCs), thrombomodulin (TM), and factor VIII-related antigen in the blood are the most important markers for estimating TMA. However, the FRC level has been measured by using microscopy and the naked eye; therefore, an improvement in technology to objectively count FRC is necessary.Methods: We established a novel technique to sensitively measure FRC as glycophorin A dull-positive small particles using a flow cytometer and estimated its reliability in patients treated with SCT. The blood level of FRC was compared with other clinical data in 257 blood samples in 16 clinical courses after SCT of 15 patients.Results: Sorted glycophorin A dull-positive small particles morphologically showed FRC. Measured FRC percentage had a weak correlation with serum levels of lactate dehydrogenase (LDH) and total bilirubin but not with TM level, whereas TM showed a weak correlation with the levels of aspartate aminotransferase and LDH. In a patient with fulminant TMA, decrement of the FRC level led to improvement in liver parameters after treatment, presumably due to the rapid clearance of FRC, and increased simultaneously with the levels of LDH and bilirubin by the TMA recurrence.Conclusions: Levels of FRC percentage and TM were independent parameters of TMA. This novel technique may be used as a standard methodology in diagnosing TMA.

The Effectiveness of Measuring for Fragmented Red Cells Using an Automated Hematology Analyzer in Patients With Thrombotic Microangiopathy

“…TMA may develop in patients after transplantation of bone marrow, 14 liver, 4 and kidney, 25 and may complicate connective tissue disease such as scleroderma. 26 There are significant differences in the FRC level between those with TMA and those without.…”

“…Recently, the FRC level has been sensitively measured using flow cytometry, in the automated hematology analyzer. [14][15][16][17] In this study, we measured the FRC level in healthy volunteers and in patients with and without TMA to find the cutoff values for the diagnosis of TMA.…”

Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

“…There is little data available regarding the clearance of the fragmented normal erythrocytes generated in patients with conditions such as microangiopathic haemolytic anaemia. These fragmented erythrocytes may account for up to 6.9% of all circulating erythrocytes [ 71 ] and thus represent a significant cellular load that requires clearance by the reticuloendothelial system. Based upon our observations, we would speculate that IgM might play a role in the recognition and clearance of damaged erythrocytes that exhibit significant disruption of cell membrane integrity ( Fig 7 ), though this will require further studies.…”