Quantitative Measurements of Red Blood Cell Indices Using Spectroscopic Differential Phase-Contrast Microscopy

Red blood cell (RBC) indices serve as clinically important parameters for diagnosing various blood-related diseases. Conventional hematology analyzers provide the highly accurate detection of RBC indices but require large blood volumes (>1 mL), and the results are bulk mean values averaged over a large number of RBCs. Moreover, they do not provide quantitative information related to the morphological and chemical alteration of RBCs at the single-cell level. Recently, quantitative phase imaging (QPI) methods have been introduced as viable detection platforms for RBC indices. However, coherent QPI methods are built on complex optical setups and suffer from coherent speckle noise, which limits their detection accuracy and precision. Here, we present spectroscopic differential phase-contrast (sDPC) microscopy as a platform for measuring RBC indices. sDPC is a computational microscope that produces color-dependent phase images with higher spatial resolution and reduced speckle noise compared to coherent QPIs. Using these spectroscopic phase images and computational algorithms, RBC indices can be extracted with high accuracy. We experimentally demonstrate that sDPC enables the high-accuracy measurement of the mean corpuscular hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, red cell distribution width, hematocrit, hemoglobin concentration, and RBC count with errors smaller than 7% as compared to a clinical hematology analyzer based on flow cytometry (XN-2000; Sysmex, Kobe, Japan). We further validate the clinical utility of the sDPC method by measuring and comparing the RBC indices of the control and anemic groups against those obtained using the clinical hematology analyzer.