(G-246) A Microfluidic Device for Multiplexed Detection of 5 Blood Borne Pathogens at Plasma Donation Facilities

Introduction:: Blood plasma takes up ~55% of whole blood and is the light-yellow liquid that all blood parts flow. Plasma components can be industrially fractionated to isolate coagulation factors, immunoglobulins, albumin for treatment of hemophilia, immuno-deficiency disorders, severe burns, or shock, respectively [1].



65% of plasma worldwide is collected through apheresis, a 90-minute process where a donor’s plasma is separated from RBC's and returned to their body [2]. Collection and pooling of thousands of plasma donations for therapeutics production increases transmission risk of infectious diseases. WHO states that during human plasma fractionation, plasma collection facilities should conduct Nucleic Acid Tests and serological testing on donations for Hepatitis B and Parvovirus B19 (among 3 other viruses) along with HBsAg, HIV-1 Ab, and HCV Ab [3].



Presently, plasma collection facilities test for pathogens and their serological markers exclusively using Polymerase Chain Reaction (PCR) or Enzyme Linked Immunosorbent Assay (ELISA) at external labs. Creating diagnostic laboratories at collection sites is expensive. Collected plasma is held and stored until aliquots from samples are shipped and results are returned. Obstacles include the need for trained laboratory personnel, delayed sample-to-answer time, expensive instrumentation, reliance on cold-chain supply for reagents, and lengthy extraction and purification of the viruses [4].



There is a need for a point-of-collection accessible, rapid, sensitive, user-friendly-device that simultaneously detects the two target virus DNA and 3 target serological makers to determines eligibility of the donor’s plasma on site. Our team developed a microfluidic device combining chemical viral nucleic acid extraction, Fluorescence-linked Immunosorbent Assay (FLISA), Loop Mediated Isothermal Amplification (LAMP), and smart phone image capture. Fluorescence image analysis via our app gives a “yes/no” answer within 60 minutes, enabling rapid diagnostics at plasma donation facilities.

Materials and Methods:: Development and sensitivity determination of our LAMP and FLISA assays for two target viruses and three serological markers were first completed off chip, using a benchtop thermocycler instrument or 96 well plate + plate reader, respectively. We ran LAMP experiments with the target virus nucleic acid, inactive virus, or active virus serially diluted in NFW or plasma with literature sourced or in-house developed primers. Active/infectious viruses were lysed with 5% Tween-20 (in TE buffer) at a 1:1 ratio. LAMP protocol consists of combining buffer, primer, and reaction mixes, with sample serial dilutions, placed in a QuantStudio 3 Real-time PCR system (60 min., 65°C).



The 3D-printed microfluidic cartridge has the following elements: 1.) The sample tube for the sample, lysis buffer, and fluorescently tagged detection Ab's. Piercing at the sample inlet of the sample tube’s aluminum foil seal facilitates fluid entry into the chip. 2.) The suction pump, consisting of a bellow and twist-on cap, uses the cap’s counterclockwise turning motion to draw the sample from the sample vial through the chip. 3.) The FLISA 5-well (3 for target, 1 for positive control, 1 for negative control) detection region pre-coated with the capture antibody or antigen 4.) Thermal lysis reservoir to lyse viruses 95C 2 min 5.) 4-well LAMP detection region: 3 wells contain pre-dried-spotted LAMP primers for each virus target and the MS2 bacteriophage positive control, 1 well with no dried primers for the negative control. 5.) Wash buffer reservoir and pump to wash unbound detection antibodies after LAMP reaction is complete



This cartridge is placed into a reader, which contains a heater supplying a 65°C constant temperature for the LAMP reaction, lenses, and LEDs for image capture. On the reader, there is a slot for a smartphone app to capture (1/min) and analyze images for positive or negative fluorescence signals for both the LAMP and FLISA detection reservoirs (Fig. 1).


Results, Conclusions, and Discussions:: We have successfully developed 5 target virus off-chip LAMP assays. Our positive MS2 Bacteriophage control assay was finalized before this work. Our HAV, HBV, HCV, HIV-1, and B19V LAMP assay reached an LOD of E5 pfu/mL, E4 IU/mL, E6 IU/mL, E6 RNA copies/mL, and 5E3 IU/mL, respectively. All 5 virus targets, except HIV-1, were active, serially diluted in either plasma or NFW, and chemically lysed (3-4 replicates/concentration; Negative control=NFW or plasma with no virus spiked). In the case of HIV-1, inactive/active virus LAMP assay characterization will be completed once we receive the sample from the vendor. Representative LAMP threshold time and LOD’s results for each target are shown in Fig. 2a-e. Fig. 2f depicts a tabulated results summary of all off chip LAMP characterization completed for each target. Notably, all our LAMP assays can reach an LOD of less than or equal to 1,000 IU or copies/uL within 45 minutes, a sensitivity typical of microfluidic devices with less than 1 mL sample input volume and simplified chemical extraction. On chip experimental testing of clinically relevant viral concentrations spiked in plasma at different combinations/coinfections as well as ELISA sensitivity characterization of the different target antibody/antigen pairs is ongoing and results will be presented from spiked and clinical samples.



Overall, this multiplexed technology has the opportunity to improve the rapidness and efficiency of the viral plasma donor collection workflow. It can determine eligibility of plasma donors at the collection site as opposed to later, at an external laboratory facility. The ease of use of the device, combined with the quick chemical extraction, lack of cold chain reliance for LAMP reagents with our lyophilized pellets and dried primers, and facile results interpretation, provides a clear advancement in the lab on a chip literature. Future directions in this field would include one pot detection of proteins and nucleic acids.

Acknowledgements (Optional): : R.B., and E.V acknowledge the support of the Commonwealth Serum Laboratories (CSL) Behring at the University of Illinois at Urbana-Champaign. The following reagent was obtained through BEI Resources, NIAID, NIH: Hepatitis A Virus, HM175/18f, NR-137.


References (Optional): : [1] Mathew, J., Sankar, P., & Varacallo, M. (2018). Physiology, blood plasma.

[2] Burnouf, T. (2007). Modern plasma fractionation. Transfusion medicine reviews, 21(2), 101-117.

[3] WHO. (2005). Recommendations for the production, quality control and regulation of plasma for fractionation.

[4] Valera, E., Jankelow, A., Lim, J., Kindratenko, V., Ganguli, A., White, K., Kumar, J., & Bashir, R. (2021, May 13). COVID-19 Point-of-Care Diagnostics: Present and Future. ACSNano. 10.1021/acsnano.1c02981