Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test

Abstract

Considerations for point-ofcare diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test," J. Biomed. Opt. 22(3), 035001 (2017), doi: 10.1117/1.JBO.22.3.035001. Abstract. There exists a broad range of techniques that can be used to classify and count white blood cells in a point-of-care (POC) three-part leukocyte differential test. Improvements in lenses, light sources, and cameras for image-based POC systems have renewed interest in acridine orange (AO) as a contrast agent, whereby subpopulations of leukocytes can be differentiated by colorimetric analysis of AO fluorescence emission. We evaluated the effect on test accuracy using different AO staining and postprocessing methods in the context of an image-based POC colorimetric cell classification scheme. Thirty blood specimens were measured for percent cell counts using our POC system and a conventional hematology analyzer for comparison. Controlling the AO concentration used during whole-blood staining, the incubation time with AO, and the colorimetric ratios among the three population of leukocytes yielded a percent deviation of 0.706%, −1.534%, and −0.645% for the lymphocytes, monocytes, and granulocytes, respectively. Overall, we demonstrated that a redshift in AO fluorescence was observed at elevated AO concentrations, which lead to reproducible inaccuracy of cell counts. This study demonstrates there is a need for a strict control of the AO staining and postprocessing methods to improve test accuracy in these POC systems. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 UnportedLicense. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Performance of a cost‐effective and automated blood counting system for resource‐limited settings operated by trained and untrained users

“…Users were not supplied with a timer or stopwatch and relied on their own judgment of the 3.5-minute incubation time of the blood with AO. While the fluctuation of the cell population fluorescence intensities within the 4-minute experiment time does not adversely affect our analysis, we speculate that when the incubation time becomes long, the granulocyte and monocyte populations become more challenging to distinguish, similarly to results recently reported by Powless et al [44] Further standardization of the incubation time through a more detailed protocol may eliminate even this minor effect.…”

Development of Blood‐Cell‐Selective Fluorescent Biodots for Lysis‐Free Leukocyte Imaging and Differential Counting in Whole Blood

“…In addition to RBC lysis, fluorescent labeling of a clinical blood sample is also required. [6,7] Typically, fluorescence staining is performed by applying a cocktail of fluorophore-labeled antibodies to detect specific types of leukocytes. The main limitations include the susceptibility of organic fluorophores to photobleaching which might lead to false results, while the use of expensive antibody reagents that lack long-term stability also add significant cost to Complete blood count with leukocyte (white blood cell, WBC) differential is one of the most frequently ordered clinical test for disease diagnosis.…”

“…To overcome this major challenge, a lysis‐free protocol for whole blood sample preparation is highly desired to enable faster and more reliable blood analysis. In addition to RBC lysis, fluorescent labeling of a clinical blood sample is also required . Typically, fluorescence staining is performed by applying a cocktail of fluorophore‐labeled antibodies to detect specific types of leukocytes.…”

“…Typically, fluorescence staining is performed by applying a cocktail of fluorophore‐labeled antibodies to detect specific types of leukocytes. The main limitations include the susceptibility of organic fluorophores to photobleaching which might lead to false results, while the use of expensive antibody reagents that lack long‐term stability also add significant cost to the diagnostic test . Thus, it remains a challenging and urgent need for developing multifunctional fluorescent reagents capable of lysis‐free counting and/or differentiating WBCs in whole blood directly toward faster, cheaper, and more reliable CBC for disease diagnostics.…”

“…[1,2] There are three major types the diagnostic test. [7] Thus, it remains a challenging and urgent need for developing multifunctional fluorescent reagents capable of lysis-free counting and/or differentiating WBCs in whole blood directly toward faster, cheaper, and more reliable CBC for disease diagnostics.…”