A-143 Imaging Flow Cytometry: A Novel Method Using ADVIA 2120i System Reagents and Microscopic Image Analysis

Abstract

Abstract Background Flow cytometry based ADVIA 2120i Hematology System reagents are used to modify blood cells. Laser-scattering flow cytometry replaced with digital microscopic image analysis significantly simplifies the complex system to a compact, low-cost microscope. A novel method for imaging flow cytometry is proposed here that simultaneously generates complete blood count (CBC) and high-resolution images of blood cells. Methods Image processing algorithms are implemented to process the pixels containing blood cells. RBCs are made isovolumetrically spherical using the RBC reagent (Fig. 1A) and imaged. RBC volumes are easily determined from the diameter of the isovolumetrically spherical RBCs. By illuminating the spherical RBCs with approximately400 nm light, the increasing grayscale values of the pixels corresponds to increasing haemoglobin concentration due to absorption. WBC nuclei are stained using the Perox reagent (Fig. 1B). The grayscale values of the nuclei pixels can be used to determine the peroxidase stain component, thereby obtaining cell size-to-peroxidase activity scatter plots similar to those on the ADVIA 2120i system. Using the Baso reagent, the cytoplasm of all WBCs are stripped off except for basophils, as shown in Fig. 1C. All the microscopic images have good contrast and high resolution. Image processing techniques can thus provide a 5-part differential using the nuclei/lobular density of WBCs. Results Images were obtained under a 20×x Nikon microscope with a Thorlabs DCC1545M monochrome camera. ADVIA reagents were mixed with venous blood and <5 µL of reagent treated sample was spotted between a microscopic slide and coverslip. Image analysis was performed using ImageJ particle analysis tools. Conclusion Analysis of digital microscopic images of blood cells acted upon by ADVIA 2120i system reagents can offer a novel, simplified, cost-effective method of hematology analysis. Simultaneous CBC scatter plots and high-resolution images of the blood cells can be obtained using this new approach.Fig. 1.(a) Isovolumetricallly spherical RBCs, (b) peroxidase stain of WBC nuclei, and (c) cytoplasm of WBCs stripped off (except for basophils). Images taken under a 20× Nikon microscope. *Under development and not available for commercial sale.