Abstract
Point-of-care (POC) biochemical assay is a highly important biochemical assay to estimate hemoglobin in the blood. High reagent volumes and complex-expensive optical setup requirements pose serious challenges when it comes to adopting conventional biochemical assays such as the Sodium Lauryl Sulfate (SLS) method into a POC device. Here, we report a modified SLS assay on a microfluidic platform, wherein the quantification is achieved using a simple microscopy-based imaging setup. Assay parameters, including SLS reagent-to-blood volume ratio, total reaction volume, the concentration of sodium dodecyl sulfate, and microfluidic chamber design, were optimized in order to achieve quantitation capability across a clinical range of hemoglobin using a path length suitable for the microfluidic platform. Besides quantitative correlation with a clinically accepted-validated standard method, the spectral absorption characteristics of the hemoglobin–SLS reagent mixture in the newly developed assay were compared with those of conventional SLS assays. The finalized chip design, including the reagent, cost 0.136 USD. The microfluidic chip in combination with an automated microscope was able to achieve a Pearson correlation of 0.99 in a validation study comparing the newly developed method and a commercially available hematology analyzer, with a turnaround time of 10 min, including incubation time. The clinical performance was ascertained, and the method achieved a sensitivity of 92.3% and a specificity of 53.8%. Overall, an automated microscopy-based biochemical assay was developed to estimate hemoglobin in whole-blood, using microfluidics technology, wherein the detector was a conventional camera associated with microscopy.
Highlights
Complete blood count (CBC) along with hemoglobin estimation is one of the most commonly performed preliminary medical diagnostic tests to ascertain the health of subjects
We present the results of a study involving microscopy imaging-based estimation of hemoglobin in blood using a microfluidic chip, wherein the photometric quantification was performed on an automated microscope designed to provide complete blood count and differential count
The assay format with a 1:5 volume ratio resulted in the maximum possible increase in absorbance value, and this was chosen for further detailed studies to compare the modified assay with conventional Sodium Lauryl Sulfate (SLS) assay
Summary
Complete blood count (CBC) along with hemoglobin estimation is one of the most commonly performed preliminary medical diagnostic tests to ascertain the health of subjects. The blood samples were lysed using a commercially available lysing reagent and subsequently loaded into the cuvette, which was placed in the holder for automated imaging followed by analysis for estimation of hemoglobin This was not performed on a microfluidic platform, and they used three separate add-on and three separate chambers for estimating hemoglobin, RBCs, and WBCs, which leads to a complicated setup with regard to usability. The imaging microscopy-based hemoglobin estimation required the development of a microfluidic chip along with optimization of the SLS reagent to enable quantitative estimation of hemoglobin across the required clinical range Photodetectors, such as CCDs and APDs, are widely used in POC medical devices associated with colorimetric assays, whereas cameras associated with microscopes are not as popular. The advantage is common illumination and a detection system between the cell imaging setup and the hemoglobin estimation setup
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