key: cord-1023879-r8qxve63
authors: Song, Y.; Ye, Y.; Su, S.-H.; Stephens, A.; Cai, T.; Chung, M.-T.; Han, M.; Newstead, M. W.; Frame, D.; Singer, B. H.; Kurabayashi, K.
title: A Digital Protein Microarray for COVID-19 Cytokine Storm Monitoring
date: 2020-06-17
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.06.15.20131870
sha: fb4be32fe61238f6a0983edac90b3f44830f1810
doc_id: 1023879
cord_uid: r8qxve63

Despite widespread concern for cytokine storms leading to severe morbidity in COVID-19, rapid cytokine assays are not routinely available for monitoring critically ill patients. We report the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit (ICU) at the University of Michigan Hospital. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing/mixing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a 12-12-15 inch compact fluorescence optical scanner for the potential near-bedside readout. The platform enabled daily cytokine analysis in clinical practice with high sensitivity (<0.4pg/mL), inter-assay repeatability (~10% CV), and near-real-time operation with a 10 min assay incubation. A cytokine profiling test with the platform allowed us to observe clear interleukin-6 (IL-6) elevations after receiving tocilizumab (IL-6 inhibitor) while significant cytokine profile variability exists across all critically ill COVID-19 patients and to discover a weak correlation between IL-6 to clinical biomarkers, such as Ferritin and CRP. Our data revealed large subject-to-subject variability in a patient's response to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays.

With the global outbreak of the novel coronavirus pneumonia , accumulating evidence 1-3 indicates that cytokine storm or cytokine release syndrome (CRS) is associated with severe illness. CRS is observed in several disease states associated with dysregulated immunity, including as a consequence of CAR-T cell immunotherapy 4 , a manifestation of hemophagocytic lymphohistiocytosis (HLH) in malignancy, macrophage activation syndrome in autoimmune disease 5 , or severe sepsis 6 . Selective cytokine blockade is a mainstay of care for CRS related cancer immunotherapy 4, 7, 8 , and macrophage activation syndrome 9 . In COVID-19, early translational studies suggest that high serum cytokines are a result of a complex interplay between lymphocytes and myeloid cells 10 . Modulation of cytokine signaling pathways is currently the subject of over 50 clinical trials worldwide 11 . However, most studies enroll based on clinical criteria without rapid assessment of specific cytokine levels, despite delivering therapies that are targeted to specific cytokines, such as interleukin (IL)-6. In our center, the current clinical practice is to use a variety of less specific surrogate markers, such as ferritin and CRP, to gauge a patient's overall level of inflammation. While cytokine levels are being checked in patients with severe COVID-19, in practice, the results of these tests return in days, not hours. Ideally, treating physicians would understand the "real-time" level of a variety of cytokines in a particular patient before administering specific medications to blunt cytokine storm in critical illness, which urgently requires a low-cost near-bedside multiplex cytokine profiling assay with a rapid assay turnaround.

Digital immunoassay 12, 13 has been considered as the next generation protein detection method which provides single-molecular sensitivity (aM-fM) detection by digitizing and amplifying enzymatic reaction in extremely confined volumes (fL-nL). Several groups invented microfluidic platforms for lab-on-a-chip operation of digital assays [14] [15] [16] [17] and notably, Yelleswarapu et al 18 demonstrated a mobile-phone-based, droplet microfluidic digital immunoassay for pointof-care (POC) settings. However, few studies have implemented a digital assay platform applicable to the clinical treatment of a COVID-19-induced cytokine storm. If continuous monitoring of the cytokine profiles of a COVID-19 patient is needed, the assay requires more than speed, sensitivity, and multiplex capacity. Other important but often overlooked requirements include (1) flexibility of running a small number of samples based on the demand of the physician with minimum preparation; (2) great inter-assay precision between multi-time point measurements, which is not an issue in conventional large batch-based retrospective tests; (3) a low-cost, compact, automated fluidic handling and readout instrumentation that can be operated inside the bio-safety cabinet with minimum user exposure to virus-contaminated blood samples.

Here, we report the development and application of an automated digital assay platform using a method termed the "pre-equilibrium digital enzyme-linked immunosorbent assay (PEdELISA) microarray" for rapid multiplex monitoring of cytokine: IL-6, TNF-α, IL-1β and IL-10 from COVID-19 patients admitted to the ICU in the University of Michigan hospital. The PEdELISA microarray analysis employs magnetic beads trapped into spatially registered microwell patterns on a microfluidic chip. The locations of the microwell patterns on the chip indicate which target analytes are detected. Unlike the existing digital assays, our method employs an approach of quenching the assay reaction entirely on-chip at an early pre-equilibrium state. This approach achieves near-real-time assay speed (<10 min incubation) with a clinically relevant fM-nM dynamic range without losing assay linearity. Furthermore, using a simple microfluidic spatial encoding technique and machine learning-based image processing algorithm, we achieved multiplex detection with high-accuracy counting and eliminated significant bead loss faced by the commercial state of the art platform 19 . The advancements of our digital assay demonstrated here enable it as a great candidate for near-bedside cytokine profiling with the combination of speed and sensitivity, both greater than those of current analog 20-23 and label-free POC diagnostic systems [24] [25] [26] [27] .

The PEdELISA microarray assay platform comprises a cartridge holding a disposable microfluidic chip with capture antibody (CapAb)-conjugated magnetic beads pre-settled in the designated microarray locations according to the antibody type, a parallel pipetting module controlled by Arduino for on-chip fluidic dispensing and mixing, and a 2-axis cartridge scanning and fluorescence imaging module (Fig. 1A , see Supporting Information for system details). In this setup, the disposable microfluidic cartridge (Fig. 1A , inset) was designed to handle 16 samples per chip with up to 16-plex maximum capacity. The chip contains two polymethyl methacrylate (PMMA) layers top (venting) and bottom layer (substrate) with countersink connectors that are seamlessly interfaced with fluidic dispensing tips, a thin polydimethylsiloxane (PDMS) layer (200 µm) which contains fL-sized microwell arrays for digital assay, and a polyethylene terephthalate (PET) thin (120 µm) film with microfluidic channels fabricated by laser cutting (see Fig. S1 for cartridge fabrication). The use of the materials and processing methods significantly reduced the chip manufacturing cost (< $0.5/chip).

The PEdELISA assay was carried out by the programmed pipetting module that allowed for microfluidic loading and handling in a consistent and repeatable manner (Fig. S2A) . The module first mixed patient samples or assay standards with a detection antibody (DeAb) solution and then loaded them into the cartridge in parallel, followed by 50 automated cycles of on-chip mixing during incubation (8 min), washing (2 min) and enzyme labeling (1 min), washing (5 min), substrate loading, and oil sealing (Fig. 1B , see Supporting Information for assay details). The chip was subsequently scanned and imaged by the compact and low-cost (<$5000) fluorescence imaging module using a consumer-grade CMOS camera (Fig. S2B) , and the data was analyzed by a high-throughput in-house image processing algorithm based on convolution neural network and parallel computing (Fig. 1C ). This algorithm performed autonomous classification and segmentation of image features such as microwells, beads, defects, and backgrounds, so that the digital assay counting results were generated without human supervision. The assay involved some minor manual work for assay reagent preparation and serial dilution, fluid waste collection, z-axis focusing, and origin/endpoint positioning to trigger the optical scanning.

We ensured the x-y optical scanning motion control accuracy each time by repetitively scanning and imaging the microarray structures on the cartridge. Post-image processing was used to calculate the x, y offset, which may be induced by the imperfection of system alignment, lead screw backlash, or motor step missing. We developed a mathematical algorithm to correct these offsets, and the scanning module was able to achieve less than 5 µm bidirectional repeatability and 0.31 µm minimum incremental movement (Fig. S3 ). Using the programmed fluidic dispensing system, we optimized the assay reaction parameters (incubation time and reagent concentration) and achieved a limit of detection (LOD) less than 0.4 pg/mL with both assay reaction and labeling incubation time in 9 min (Table 1) . We also assessed the 4-plex assay's specificity and signal to noise ratio (SNR) by spiking-in each cytokine analyte in 100% fetal bovine serum buffer (FBS) to mimic the patient serum detection. Fig 2A shows the assay results of "all-spike-in," "single-spikein," and "no-spike-in" using 200 pg/mL recombinant cytokine standards (a typical clinical threshold for cytokine storm). Negligible antibody cross-reactivity was observed between each All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 17, 2020. . cytokine analyte and SNR=488.0 was calculated on average (averaged assay signal over background signal).

In order to facilitate the care of patients with COVID-19 at the University of Michigan Hospital, we undertook a pragmatic study to rapidly return same-day cytokine levels to the clinical teams treating critically ill COVID-19 patients in ICU at the physicians' request from April 9 th to May 29 th in 2020. Given the investigational nature of the assay, patients or their representatives provided informed consent for cytokine measurements to be provided for clinical use (UM IRB HUM00179668). To ensure the accuracy of our data, the COVID-19 patient samples were run in quadruplicate with an assay standard curve calibrated every day. Fig. 2B shows assay standard curves that were accumulated in 10 different workdays of the patient cytokine monitoring period.

The multiple assay standard curves yielded excellent repeatability with the inter-assay coefficient of variation (CV) of ~ 10% due to the programmed fluidic handling and reaction (Table 1) . We also characterized the intra-assay CV for five representative COVID-19 patient serum samples with cytokines at concentrations ranging from 6-600 pg/mL, each tested in quadruplicate measurements (Table 1) . We compared assay data for these five patients resulting from near-realtime measurements of fresh samples drawn daily and retrospective measurements of stored samples after one freeze-thaw cycle. We observed a good linear correlation (R 2 =0.99) between the two measurement modes except for TNF-α. This suggests that TNF-α in the stored serum could degrade by 20-40% after the freeze-and-thaw banking at -80 °C (Fig. 2C) . Additionally, to validate our PEdELISA microarray assay, we compared the assay results with those of a conventional single-plex ELISA method that retrospectively measured 15 banked samples from identical patients. Because conventional ELISA requires a much larger sample volume (>200 L for each measurement, in duplicate per analyte) relative to PEdELISA, it was practically difficult for us to manage the acquisition of a sufficiently large blood sample volume from critically ill COVID-19

patients. Therefore, we only validated our assay against IL-6 detection results (Fig. 2D ). The data between these two methods overall matched linearly (R 2 =0.95, P<0.0001). Some discrepancy was observed at concentrations below 50 pg/mL and may be potentially due to the limited sensitivity and linearity of the ELISA assay ( Fig. 2D inset) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 17, 2020. . practical operation of our test, a larger amount of time was spent on sample processing, transport, and team coordination, as well as biosafety and disinfection protocols in handling COVID-19 samples. Nevertheless, the <4-hour blood draw to result turnaround is still rapid as compared to typical clinically deployed tests. Our rapid cytokine measurement in patients with respiratory failure due to COVID-19 revealed significant subject-to-subject heterogeneity despite all patients being critically ill. As expected, interruption of IL-6/IL-6R signaling in patients who received tocilizumab resulted in marked elevation of IL-6 levels in the setting of ongoing illness (p<0.0001, Fig. 3B ). 28 Among patients who did not receive tocilizumab, we observed a large degree of variability in IL-6 levels, with a quarter of subjects having IL-6 <15 pg/mL, the median value of 106 pg/m, and the CV of 114%. Variability of TNF-α (CV 164%) and IL-1β (CV 193%) was driven by a small number of subjects with elevated levels. However, like IL-6, levels of IL-10 were also broadly distributed in patients who had not received tocilizumab (CV 93%).

Given the heterogeneity of cytokine levels in critically ill patients with COVID-19, we asked whether IL-6 levels were reflected in surrogate biomarkers. In current, rapidly evolving clinical practice, the presence of cytokine storm and risk of clinical deterioration is frequently judged by inflammatory markers such as CRP and ferritin in the absence of direct cytokine measurement. Ferritin did not predict IL-6 levels (Fig. 3C , R 2 = 0.01, P=0.71). CRP was significantly associated with IL-6 ( Fig. 3D , R 2 =0.41, P=0.018). However, this association was driven by low IL-6 in subjects with low levels of CRP, while IL-6 values in subjects with high CRP were widely distributed. Neither CRP nor ferritin is a reliable predictor of IL-6. Note that our pragmatic study of rapid cytokine measurements in patients with COVID-19 was designed to provide information to clinicians, rather than systematically study the biology of COVID-19. We therefore, enrolled subjects without regard to time from the onset of infection. Furthermore, due to these subjects' critical illness, many received empiric antibiotic therapy, limiting our ability to determine bacterial co-infection. These factors, as well as our small sample size, may have contributed to the heterogeneity of the cytokine response. Nevertheless, all subjects in this study were critically ill and had respiratory failure, underscoring the diversity of biological mechanisms that may lead to critical illness in COVID-19 and the importance of measuring, rather than inferring, cytokine storm.

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We purchased human IL-6, TNF-α capture, and biotinylated detection antibody pairs from Invitrogen™, and IL-1β, IL-10 from BioLegend. We purchased the corresponding ELISA kits from R&D Systems (DuoSet®). We obtained Dynabeads, 2.7μm-diameter epoxy-linked superparamagnetic beads, avidin-HRP, QuantaRed™ enhanced chemifluorescent HRP substrate, bovine serum albumin (BSA), TBS StartingBlock T20 blocking buffer, and PBS SuperBlock blocking buffer from Thermo Fisher Scientific. We obtained Phosphate buffered saline (PBS) from 

We conjugated human IL-6, TNF-α, IL-1β, IL-10 capture antibodies using the epoxylinked Dynabeads (2.7 μm) with the capture antibody molecules at a mass ratio of 6 μg (antibody):

1 mg (bead) following the protocols provided by Invitrogen™ (Catalog number: 14311D). The beads were then quenched (for unreacted epoxy groups) and blocked with TBS StartingBlock T20 blocking buffer. We stored the antibody-conjugated magnetic beads at 10 mg beads/mL in PBS (0.05% T20 + 0.1% BSA + 0.01% Sodium Azide) buffer sealed with Parafilm at 4 °C. No significant degradation of the beads was observed within the 3-month usage.

The disposable microfluidic cartridge used for PEdELISA assay is plastic-based and fabricated by laser cutting and PDMS molding. It has a transparent sandwich structure for optical imaging as shown in Fig. S1 . The top and bottom layers are laser-cut using 3.175 mm (1/8 inch) All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 17, 2020. . and 1 mm thin clear polymethyl methacrylate (PMMA) boards which contain through-holes for venting and screw assembly purposes. The microfluidic channels (designed with AutoCAD software) are laser-cut through a 120 µm high definition transparency polyethylene terephthalate (PET) thin film (adopted from standard screen protector) which has a silicone gel layer to create a vacuum for securely sealing to the top acrylic layer without adhesives. The power and speed of the laser cutter are optimized to ensure a high-resolution smooth cut so that resistance difference or bubbles generation can be minimized during the fluidic handling process. The femtoliter-sized microwell (d=3.4 μm) array layer (~300 μm) was made by polydimethylsiloxane (PDMS) through a standard SU-8 molding. First, we constructed SU-8 molds on oxygen plasma treated silicon wafers by standard photolithography which involved depositing negative photoresist (SU-8 2005 MicroChem) layers at 5000 rpm to form the desired thicknesses 3.8±0.1 µm. Subsequently, a precursor of PDMS was prepared at a 10:1 base-to-curing-agent ratio and deposited onto the SU-8 mold by spin coating (300 rpm) and baking overnight at 60 °C. We then transferred the fully cured PDMS thin film onto the bottom acrylic layer using a modified surface silanization bonding method based on a previous publication 30 . We also drilled 2 mm countersink holes (60°) using a benchtop mini drill press (MicroLux®) on the top venting layer for guiding the multi-pin fluidic dispensing connector. Each layer was thoroughly cleaned through water bath sonication and the PET microchannel layer was carefully attached to the top venting layer for the later bead patterning process.

The PEdELISA bead patterning process involves first attaching the bead settling layer (containing long straight PDMS channels perpendicular to the PET microchannel layer) to the PDMS microwell array layer on the bottom PMMA substrate. Then, we prepared 4 sets of a 25 µL bead solution at the concentration of 1 mg/mL for IL-1β, TNF-α, IL-10, and IL-6 bead respectively.

The bead solution was loaded into four different physically separate patterning channels in the bead settling layer. After waiting 5 min for beads settling inside the microwells, we washed the patterning channels with 200 uL PBS-T (0.1% Tween20) to remove the unstrapped beads. At this step, we imaged the microarray under the microscope to ensure that the microwells were filled with the beads at a sufficient rate (typically above 50%). If not, the bead mixture solution was reloaded and washed again. Finally, the bead settling layer was peeled off and replaced with PET microchannel and top venting layer. Four layers of the cartridge were sandwiched together using M2 bolt screws. Note that the bonding between the PET layer and the PDMS layer was not All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 17, 2020. . https://doi.org/10.1101/2020.06.15.20131870 doi: medRxiv preprint permanent but through pressure-based self-sealing, which can be later easily peel off and replaced.

We then slowly primed each sample detection channel with Superblock buffer to passivate the cartridge surface and incubated the whole chip for at least 1 hour before the assay to avoid nonspecific protein adsorption. The cartridge was typically prepared in batch and sealed in a moisturecontrolled petri-dish at room temperature for up to a week with no significant degradation.

The for the digital counting process.

The scanning system was used to scan the image of the bead-filled microwell arrays on the PEdELISA cartridge right after the oil sealing step to detect the enzyme-substrate reaction activity.

The imaging stage was pre-programmed to follow the designated path to scan the entire chip (64 microarrays) twice: 1. Scan the QuantaRed channel (545nm/605nm, excitation/emission) 2. Scan the brightfield with the transmission light source on. It typically took around 6 min to scan the entire chip for 16 samples in 4-plex detection.

This study was approved by the University of Michigan Institutional Review Board (HUM00179668) and patients or their surrogates provided informed consent for the investigational use of this test. Patients with positive SARS-CoV-2 test via PCR and respiratory failure requiring All rights reserved. No reuse allowed without permission.

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Experiments with synthetic recombinant proteins were performed daily with 2 on-chip repeats averaged to calculate the patient serum cytokine levels. 10-day standard curves using 10 microfluidic cartridges were accumulated to calculate the inter-assay coefficient of variance. The COVID-19 patient serum samples were performed in quadruplicate and averaged for the near-realtime daily cytokine profile monitoring test. Conventional ELISA test was conducted retrospectively for IL-6 in duplicate for selected banked patient samples. Here, Pearson's R-value was used to quantify the PEdELISA to ELISA correlations and the t-test was used for group analysis of the Tocilizumab treatment. A p-value of < 0.05 was considered to be statistically significant.

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There are no conflicts to declare. The 4-step assay procedure includes (i) automated injection and subsequent on-chip mixing of serum and a detection antibody solution with capture antibody-coated magnetic beads pre-deposited in microwell arrays, which is accompanied by a short incubation (8-min) and followed by washing (2-min), (ii) HRP enzyme labeling (1-min), followed by washing (5-min), (iii) fluorescence substrate loading and oil sealing (2-min), and (iv) x-y optical scanning and imaging (12-min). (C) Data analysis using a convolutional neural network-guided image processing algorithm for high throughput and accurate single-molecule counting that corrects image defects and accounts for signal intensity variations. Both the fluorescence substrate channel (Qred CH) and brightfield channel (BF CH) are analyzed to calculate the average number of immune-complexes formed on each bead surface. The unlabeled scale bars are 25 μm. The data points were fitted with four-parameter logistic (4PL) curves. The black dotted line represents the signal level from a blank solution. The blue dotted line shows 3σ above the blank signal, which is used to estimate the limit of detection (LOD) for each cytokine. (C) Linear correlation (R 2 =0.99, P<0.0001) between rapid measurements of fresh samples and retrospective measurements of stored samples (1 freeze-and-thaw at -80 °C) in quadruplicate for 5 representative COVID-19 patients. (D) Good agreement (R 2 =0.95, P<0.0001) observed between single-plex IL-6 ELISA and multiplex PEdELISA measurements for 16 COVID-19 patients. The inset shows the circled region.

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The copyright holder for this preprint this version posted June 17, 2020. . Ferritin does not correlate well with IL-6 (R 2 = 0.01, P=0.71). CRP correlates with IL-6 (R 2 =0.41, P=0.018) better, but the IL-6 levels were widely distributed for patients with high levels of CRP. Table 1 . Limit of detection (LOD), limit of Quantification (LOQ), and coefficient of variation (CV) of PEdELISA for a panel of 4 cytokines. Here, the LOD and LOQ values were determined from the blank signal + 3σ and the blank signal + 10σ, respectively. The intra-assay CV was determined by quadruplicate measurements of five COVID-19 patient samples at the range of 6-600 pg/mL in both near-real-time and retrospective assay modes. The inter-assay CV was determined by taking the root-mean-square average of signals from 40, 200, and 1000 pg/mL assay standard in 10-day continuous measurements of COVID-19 patients. All rights reserved. No reuse allowed without permission.

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PHASE 2/3 ADAPTIVE-DESIGNED TRIAL OF KEVZARA® (SARILUMAB) IN HOSPITALIZED COVID-19 PATIENTS

We thank the College of Engineering, Department of Internal Medicine at the University of Michigan for their emergency approval of this COVID-19 related research. We specifically thank the UM Mechanical Engineering machine shop, Mr. Charles Bradley, Donald Wirkner, and Kent Pruss for the great help offered in designing and machining the automated system during the COVID-19 pandemic period. We acknowledge the funding support from the National Institute of