Hemoglobin quantification in red blood cells via dry mass mapping based on UV absorption

Nischita Kaza,Ashkan Ojaghi,Francisco E. Robles

doi:10.1117/1.jbo.26.8.086501

Abstract

.Significance: The morphological properties and hemoglobin (Hb) content of red blood cells (RBCs) are essential biomarkers to diagnose or monitor various types of hematological disorders. Label-free mass mapping approaches enable accurate Hb quantification from individual cells, serving as promising alternatives to conventional hematology analyzers. Deep ultraviolet (UV) microscopy is one such technique that allows high-resolution, molecular imaging, and absorption-based mass mapping.Aim: To compare UV absorption-based mass mapping at four UV wavelengths and understand variations across wavelengths and any assumptions necessary for accurate Hb quantification.Approach: Whole blood smears are imaged with a multispectral UV microscopy system, and the RBCs’ dry masses are computed. This approach is compared to quantitative phase imaging-based mass mapping using data from an interferometric UV imaging system.Results: Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm.Conclusions: A full hematological analysis (including white blood cell identification and characterization, and Hb quantification) may be achieved using a single UV illumination wavelength, thereby improving the speed and cost-effectiveness.

Highlights

Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm
The dry mass of red blood cells (RBCs) is of clinical significance, and hematology analyzers routinely used to obtain a complete blood count (CBC) from peripheral blood samples measure indices such as the dry mass of Hb in the form of the mean corpuscular Hb (MCH), and the mean corpuscular Hb concentration.[7]
Deep-UV microscopy images have been used to successfully generate accurate nucleic acid and protein mass maps of live cells owing to their intrinsic molecular contrast, without the need for laborious fixing and staining procedures, 10,20,22,26 we recently demonstrated that deep-UV microscopy images acquired at 300 nm could accurately measure Hb mass.[24]

Summary

Introduction

The morphological and molecular properties of blood cells are important indicators of health and are diagnostic markers for several diseases.[1,2,3,4,5,6,7] The morphology, size, and hemoglobin (Hb) content of red blood cells (RBCs) serve as essential biomarkers to diagnose or monitor various types of anemias and other diseases, including malaria and thalassemia.[5,6,7] RBCs are enucleated, terminally differentiated cells that are composed of 95% to 98% Hb by dry mass (mass of all the constituents of a cell in the absence of water).[8,9,10] the dry mass of RBCs is of clinical significance, and hematology analyzers routinely used to obtain a complete blood count (CBC) from peripheral blood samples measure indices such as the dry mass of Hb in the form of the mean corpuscular Hb (MCH), and the mean corpuscular Hb concentration.[7] Along with mean values, single-cell measurements are invaluable to completely characterize a sample’s distribution.[11,12] In many clinical hematology analyzers, Hb concentration is obtained from the optical scattering of spherical cells.[13] Owing to the characteristic biconcave shape of RBCs, they have to be treated with a sphering agent, which lengthens the procedure and leaves cells in a Journal of Biomedical Optics

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