Poster Y6 - Blood cell agglomeration strategies for point-of-care assays in blood

Introduction: Today most diagnostic blood tests are conducted in certified central laboratories with trained technicians and expensive equipment. This paradigm results in significant delay time for results and is incompatible with the infrastructure in many low- and middle-income countries (LMICs). In contrast, point-of-care diagnostics (PoCDs) enable rapid results that lead to immediate treatment of the patient. Moreover, PoCDs fill the diagnostic gaps in LMICs. Diagnostic tests often require blood samples, but tests with an optical readout (e.g., colorimetric and fluorescence) cannot be performed on whole blood because of the optical absorbance of hemoglobin. Thus, for blood tests, the sample is centrifuged, and plasma is used for the test. However, PoCDs must be equipment-free, low-cost, and sample-to-answer. Thus, simple and automated methods for removing red blood cells (RBCs) from samples are needed. Here we investigate the use of RBC agglomerating agents (dextran [1] and hetastarch [2]) to clear blood samples for fluorescence measurements with a custom portable optical reader.

Materials and

Methods: For these experiments, we use double-stranded DNA (dsDNA) and intercalating fluorescent dye, based on our recently reported PoCD for the detection of circulating histones. [3] dsDNA (147 base pairs) was amplified from a bacterial genome in our lab and quantified with a Nanodrop. EvaGreen (Biotium, 20X), (27.6μL) was combined with 100nM dsDNA (123μL) and one of the varying dextran or hetastarch concentrations (16.3μL) and then incubated for 10 minutes. Whole blood (15 μL, collected via venous draw into collection tubes according to approved IRB protocols) was then added to the EvaGreen and dsDNA; blood samples were less than 48 hours old when tested. Dextran (Sigma-Aldrich) in deionized (DI) water was added in concentrations of 15 mg/mL, 25 mg/mL, and 75 mg/mL (final concentrations 1.3mg/mL, 2.2mg/mL, 6.7mg/mL). Hetastarch (6%) (STEMCELL Technologies, HetaSep) in DI water was added in concentrations of 1%, 3%, and 6% solutions to achieve final concentrations of 0.09%, 0.27%, and 0.54%. Fluorescence measurements were made in a standard 600 mL microtube placed in a custom-made fluorescence reader (Figure 1). The tube rests in an anodized aluminum heat block with an attached polymer heater. Slits are milled into the heat block (4.25 mm from the bottom of the sample tube) to guide excitation light from an LED (~ 470nm) and emission light to a photodiode. An excitation filter (483 nm) is located in front of the LED, while an emission filter (534 nm) is located in front of the photodiode.

Results, Conclusions, and Discussions: Fluorescence intensity was measured over time for each blood sample. The measurements for dextran are presented in Figure 2A (each line represents the average values for multiple samples; shaded areas represent the standard deviation). An increasing fluorescence signal indicates a clearing of the blood sample (i.e., settling of the RBCs below the optical slits in the portable reader). As Figure 2A shows, 15 mg/mL dextran did not clear the sample at all, while the higher concentrations resulted in easily detectable fluorescence signals. As expected, the addition of 75 mg/mL dextran resulted in the quickest and most significant clearing of the blood. The fluorescence measurements for hetastarch are presented in Figure 2B (each line represents the average values for multiple samples; shaded areas represent the standard deviation). All three resulted in some degree of fluorescence increase over time, indicating the settling of the RBCs below the optical slits. As expected, 6% resulted in the quickest and most significant clearing of the blood. We note that the 6% hetastarch (the highest concentration commercially available) results in a signal that is comparable to 25 mg/mL dextran. We conclude that both dextran and hetastarch can clear blood samples for optical measurements in portable readers. We note that 6% hetastarch clears blood, but not as well as dextran in concentrations higher than 25 mg/mL. Additionally, we note that in PoCD assays, these agglomerating agents must be added automatically to achieve sample-to-answer capability. In our assays, we utilize thermally responsive alkane partitions, which are higher order alkane layers that partition reagents when solid but breach when melted at moderate temperatures. [4]

References:
[1] Pribush A et al. Eur Biophys J. 2007, 85.
[2] Madkaikar M et al. British Journal of Biomedical Science. 2007, 157.
[3] Everitt ML et al. ACS Sens. 2021, 3006.
[4] Goertz JP et al. Analytical Chemistry. 2018, 3708.

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