Dear Collegues, I want to comment as we have done some work on retics and flow to validate the method. The standard flow method is to gate on red cells in a log log forward/sidescatter plot and to do a one dimensional histogram for green fluorecence (eg. as done by the BD reticount software). This is prone to many errors. The dyes in use are thiazol orange, auramin O, DEQTC or acridin orange, depending on the patent situation. The absorbing dyes (HeNe excitation) should not be used. The false positive cells (non reticulocytes) include: leukocytes (cave CLL and other leukemias) nucleated red cells (normoblasts, eg. thalassemia without retics) large platelets parasite infected red cells and inclusions like Howell Jolly bodies (as already pointed out) The dual parameter plot FSC vs fluorescence (as done in the Sysmex R3000) improves this situation a lot. Our solution we found optimal is to counterstain with the laser dye LDS751 (styryl 8), first introduced by Leon Teerstappen (published in 1988 in Cytometry 9, 548-556). This vital DNA stain emits in the red (FL3 channel in the FACScan etc., 488 nm ecitation) and can be plotted versus green (FL1). By this way it is possible to discriminate retics from the cells mentioned above. The method has beeen published: Bertsch T and Nebe CT, Flow-Cytometric Analysis of Reticulocytes, in: Aspects of the Flow-Cytometric Analysis of Red Blood Cells, K. Gutensohn, H.-H. Sonneborn and P. Kühnl (eds.), 69-80, Clin. Lab. Publications (1997) I fully agree that the analysis of reticulocytes improves the differential diagnosis of anemias a lot. It is therefore important not only to measure the increse but also the decrease of retics and to report absolute numbers with high precision. This requires dual fluorescence staining and multidimensional gating. Some new hematology analyzers present a good compromise and we include a review of a blood smear in problem cases (diagnosis). I appreciate the discussion on this subject. Regards Thomas Nebe Dr.med. C. Thomas Nebe Universitaetsklinikum Mannheim Zentrallabor Theodor-Kutzer-Ufer 1-3 D-68167 Mannheim * +49 621 383-3485 * +49 621 383-3819 e-mail: thomas.nebe@ikc.ma.uni-heidelberg.de http://www.ma.uni-heidelberg.de/inst/ikc/
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:51 EST