Morphological definition of CD71 positive reticulocytes by various staining techniques and electron microscopy compared to reticulocytes detected by an automated hematology analyzer.

Background: The enumeration of peripheral blood reticulocytes plays an important part in clinical hematology. Although reticulocyte enumeration is currently performed with visible dyes such as New Methylene Blue (NMB), fluorescent dyes, or anti-CD71 (transferrin receptor) antibody, it has not been demonstrated whether the reticulocytes detected in each method are the same or not.

Methods: We prepared the reticulocyte rich fraction with density gradient centrifugation, stained with both anti-CD71 and Sysmex's fluorescent stain RET SEARCH (II), and detected the cells by both confocal laser scanning microscopy and flow cytometry. We also stained the reticulocyte rich fraction and the CD71+ reticulocytes with NMB and compared them by microscopy. We also observed the CD71+ reticulocytes by electron microscopy.

Results: Almost all CD71+ reticulocytes were intensely stained with both NMB and RET SEARCH (II). These cells were therefore classified as highly immature reticulocytes. During the stages of reticulocyte maturation, the expression of CD71 antigen decreased prior to the reduction of the reticular structures. The electron microscopic observation showed that CD71+ reticulocytes had some typical morphological characteristics found in highly immature reticulocytes.

Conclusions: The CD71+ reticulocytes consisted of highly immature reticulocytes and were not equal to the reticulocytes defined with NMB or RET SEARCH (II).