Fluorescence - Reticulocyte analysis

HORIBA Medical uses flow cytometry and impedance measure with a reagent named ABX Fluocyte which contains a fluorescent stain the thiazole orange (TO), a cyanine stain whose excitation occurs in the visible region at λ= 488nm. It is a nucleic-acid-specific dye that enters by permeability in the cells. When it binds to RNA, there is a 3000-fold increase of fluorescence emission. It is one of dyes recommended by the NCCLS-CLSI guidelines for reticulocytes count. TO has become the most common dye employed due to its counting accuracy and practicality for clinical laboratories with commercial clinical flow cytometers with relatively low-powered visible lasers.

The degree of fluorescence emission caused by the excitation bound dye is proportional to the amount of RNA in the erythrocyte. 

When the staining cell is achieved the suspension is then transferred to the laser optical bench to be measured. The laser optical bench simultaneously measures the fluorescence of the cells passing through the measuring point into the flowcell, and the volume by impedance. A cell passing through the flowcell gives 2 types of information: 

  • The size of the cell measured by resistivity CIS (Cell Impedance Signal) 
  • The Orthogonal Fluorescence Light (OFL): The fluorescence is collected using a lens focused on the optical flowcell and located at 90° from the light beam, an interferential filter specific to the TO stain selecting only the fluorescent wavelength and a photomultiplier tube. 
Yumizen H2500_Reticulocyte matrix_Fluorescence-Reticulocyte analysis_HORIBA_Medical

Yumizen H2500_Reticulocyte matrix_Fluorescence-Reticulocyte analysis_HORIBA_Medical

The Reticulocytes matrix is generated from 2 measurements: resistivity volume (CIS) and orthogonal fluorescence (OFL) of cells according on the X and Y axes respectively. Mature red blood cells without RNA show little or no fluorescent signal. They are located at the bottom of the matrix and horizontally distributed according to their MCV and RDW. Reticulocytes are separated from the red blood cells by their fluorescence which is proportional to the RNA content and their immaturity. The most fluorescent elements, which are saturated at the top of the matrix, are the most immature. Erythroblasts may also be found in this area.

The video shows you the entire Reticulocyte measurement process with HORIBA's high end hematology analyzer.

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