Reticulocyte Maturation Process — Experimental Demonstration of RET Channel Using Anemic Mice

• Reticulocyte Maturation Process — Experimental Demonstration of RET Channel Using Anemic Mice

AUTHOR(S)

Mari KONO, Yuri TAKAGI, Tamiaki KONDO, Takuji HANAMURA and Keiji FUJIMOTO

Cell analysis center, Scientific Affairs, Sysmex Corporation

SUMMARY

The RET channel on the XT2000i  and XE series of Sysmex automated blood analyzers is a reticulocyte measurement channel. By staining the residual RNA with the florescent dye " RETSEARCH II " it divides reticulocytes into three fractions, high-, medium- and low- fluorescence intensity reticulocytes ( HFR, MFR, LFR respectively ). Counting the cells is a useful tool for understanding the status of erythropoiesis and is widely used in the diagnosis of for example anemia and bone marrow suppression. It is believed that these fractions represent reticulocytes of different maturation stages, ranging form very early and immature ( HFR ) to more mature reticulocytes ( LFR ), but individual features of the different subpopulations have not been characterized to date.

Our objectives were to isolate cells of the different stages and to analyze them for differences in morphology and RETSEARCH II staining pattern. Furthermore, we want to compare this to the expression of the well known reticulocyte marker CD71 ( Transferrin Receptor ) and the new methylene blue staining and classification system.

We induced anemia in mice through intraperitoneal phenylhydrazine injection, which leads to severe reticulocytosis in the animals after five days. Anemic murine blood was collected and fractionated into reticulocyte subpopulations by a cell sorting system. The cells were fixed and examined by transmission electron microscopy ( TEM ) or stained and examined for specific morphology and antigen expression.

By double labeling with RETSEARCH II and CD71-Alexa488 and subsequent FACS analysis we created a simulated Ret channel scattergram. We found that CD71 was expressed only on the more immature cells and rapidly disappeared at the differentiation step from HFR to MFR. Through TEM and confocal microscopy we confirmed that immature reticulocytes lose CD71 antigen expression at the stage of MFR. Moreover we were able to show that intracellular structures such as residual organelles disappear as the cells mature towards an RBC. This result was supported by immuno-electron microscopic analysis using CD71-gold colloid conjugate.

By electron microscopy we detected residual ribosomes in all of the immature cell fractions ( LFR, MFR and HFR ), albeit strongly decreasing towards the more mature stages. Since new methylene blue supravital staining, the standard method in reticulocyte analysis, targets residual ribosomes, we can conclude that reticulocytes identified by this staining corresponded to those sorted by FACS using simulated RET-channel. Moreover, this demonstrates that reticulocyte counting by the RET- channel is in accordance with that by new methylene blue supravital staining.

Many observations of the reticulocyte in vitro maturation process have been reported. This is one of the very few in vivo reports and we believe that our observations accurately reflect the in vivo maturation process.

KEY WORDS

RET Channel, Reticulocyte, Maturation, CD71 ( Transferrin Receptor ), Morphological