Improved specificity of determination of immature erythrocytes (reticulocytes) by multiparameter flow-cytometry and thiazole orange using combined staining with monoclonal antibody (anti-glycophorin-A)

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Abstract
Applying thiazole orange stain and multiparameter flow-cytometry, immature erythrocytes, e.g., reticulocytes, can be discriminated from the mature erythrocytes by virtue of their higher RNA-content. One major problem of this method, however, consists in the inclusion of nucleated cells (leucocytes) and large RNA-containing platelets in the population defined by light scatter-patterns as erythrocytes. Due to the high intensity of staining with thiazole orange of all these cellular elements, all of them in the analysis are prone to be classified erroneously as reticulocytes. In order to classify elements in analysis properly as erythroid (reticulocytes+mature erythrocytes) or as non-erythroid (leucocytes+platelets), an alternative staining method is shown in this paper, consisting of thiazole orange combined to anti-glycophorin-A-PE-monoclonal antibody.