Quicker and digital: the way on protein biomarkers?

In this issue of Blood, Song et al¹  report the development of a new biomarker assay for the continuous monitoring of systemic immune disorders based on single-molecule digital detection of proteins. The technique is very rapid, multiplexed, and easy to handle.

For most chronic diseases, biomarker assays should be highly reproducible, with high sensitivity and specificity. The tradeoff for these advantages is usually the time of execution of the assays (blood draw-to-answer time), which can be hours or days.²  In the case of acute, lethal conditions, such as septic shock or various types of cytokine release syndrome (CRS), including postchimeric antigen receptor T-cell (post–CAR-T) therapy³  or COVID-19–related cytokine storm,^4,5  the quickness of diagnosis is critical for patient survival. Therefore, for such conditions, rapidity is crucial.

In line with this, Song et al report a very interesting advance of the classics assay of enzyme-linked immunosorbent assay (ELISA) that they named, suggestively, PEdELISA (pre-equilibrium digital ELISA). This assay is based on 2 principles: the instantaneous single-molecule binary counting of nonequilibrium protein-binding events (the “digital” part, to achieve simultaneously speed and sensitivity) and the intentional stopping of the immunologic reaction with a washing buffer in its pre-equilibrium state (the “pre-equilibrium” part, to achieve the single-molecule counting condition). This produces a result in minutes (approximately half an hour) from the sample collection (and the authors have a clear plan to further reduce this interval) vs hours to days for the classic ELISA test or mass spectroscopy assays, respectively. The disease state used for testing was profiling of multiple plasma cytokines in patients manifesting post–CAR-T therapy CRS. This is a deadly complication for one of the most expensive nonsurgical therapies (yet rapidly expanding in applications) in the entire medical field.

The research behind is solid, wise, and simple. First, the authors developed the PEdELISA platform by combining the micro‐array biosensor design with the microfluidic chip for multiplex analysis. Then, they developed the theoretical basis of the method by prediction of “quench-and-snapshot” measurement, which includes accounting for mass transport and surface reaction for a theoretical “reaction volume” with a bead placed in its center, followed by the kinetics of the antibody-antigen antibody immune complex formation. They continued with the analytical validation of the assay based on 10 representative cytokine biomarkers and identified the signal-to-noise ratio. The linearity of the assay was confirmed over a three-order-of magnitude concentration range regardless of the cytokine and was well maintained even for the ultrafast (15 seconds only) PEdELISA interleukin-6 assay. Importantly, the authors demonstrated close concordance between the results of the conventional 3-step sandwich ELISA and PEdELISA. Finally, they tested the assay with a 300-second incubation time to monitor the cytokine profiles of a set of hematological can‐cer patients with various levels of CRS symptoms after CAR T cell therapy. The analyses included a near-real-time cytokine profile analysis for the patient with the most severe condition, as well as a side-by-side comparison in 3 different patients with unknown concentrations of cytokines using both PEdELISA and a commercial multiplex assay LEGENDplex (BioLegend) that showed a high linear correlation between the 2 assays.

Why is this study important for the readers of a hematology journal? First, the assay can be used for multiple disorders where no such assays are available. Cytokine storm is a devastating condition where therapies based on a mechanistic understanding of the pathogeny are needed.⁵  But the disorder often occurs in medically complex patients for whom the initial findings can be due to many etiologies. Therefore, one way to decrease mortality is quick diagnosis. Certainly, having a cytokine biomarker tool with reliable data in minutes will be an excellent news for intensive care unit doctors and any physician involved in the treatment of this condition. Second, this can be applied to medical conditions affecting other disorders, such as COVID-19, which can be deadly in individuals with secondary conditions,⁶  or to acute posttraumatic injuries syndrome, which can affect young individuals without secondary conditions.⁷  Many lives depend on how quick an assay can be run directly at the place where the individual is located. Third, the research behind the assay’s development is strong, and the authors already plan the development of a more automated and precisely volume/time-controlled sample/reagent handling system into the PEdELISA platform for near-bedside operation. This would allow the whole test to be completed with a blood draw-to-answer time of <30 minutes and truly enable “real-time” biomarker detection.

As the authors point out, one future essential requirement is validation using large multi-institutional sets of patients with acute diseases where cytokines play an essential pathogenic and biomarker roles. The development of assays for additional types of biomarkers is also important. This could include detection of RNAs and lipids, as well as novel microparticles such as the elongated neutrophil-derived structures formed by rolling neutrophils in sepsis.⁸  Also, the development of mobile units of manageable size and weight is essential to be able to also use this new and quick assay in nonhospital settings.⁹  It is fair to say, we are waiting with curiosity and hope to see if this rapid single-molecule digital detection of protein biomarkers will live up to its potential.