Assays and Performance

Point-of-care immunoassays with unmatched sensitivity and speed

Novilux products are not approved for diagnostic use

Multiplexed Cardiac Panel
(UHS-Cardiac Troponin I, NT-proBNP, D-dimer)

Novilux SMC™ technology can multiplex assays both spatially and optically. The lynchpin of the Novilux 3-Plex Cardiac Panel is the Ultra-High Sensitivity Cardiac Troponin I assay, a biomarker which has become the definitive blood test for diagnosis of myocardial infarction. Multiplexed panels are used to quickly rule-in or rule-out various conditions that often present with the same symptoms. For example, Acute Coronary Syndrome (ACS), Heart Failure (HF), and Pulmonary Embolism (PE) all share shortness-of-breath as a symptom. Cardiac Troponin I (cTnI) is critical to aid in the diagnosis of ACS (myocardial infarction in particular), NT-proBNP aids in the diagnosis HF and D-Dimer is used to help diagnose PE. Providing all three markers simultaneously can be of enormous benefit to Acute Care Physicians seeking to differentiate diagnosis of cardiac or pulmonary conditions with different etiologies when their presenting symptoms overlap. The Novilux 3-Plex Cardiac Panel can provide results for all three markers in less than 13 minutes from whole blood specimens.

Results from CLSI test protocols for the Novilux 3-Plex Cardiac Panel performed on the Phoenix PoC platform are shown below.

Ultra-High Sensitivity cTnI

The table below shows LoBDQ results from CLSI EP17-A2: Detection Capability study:

o Blanks made from cTnI depleted plasma

o Other samples are endogenous plasma and spikes

The Detection Capability study was run post calibration. Calibration of Cardiac Troponin I material was value assigned using NIST Standard Reference Material (SRM) 2921: Human Cardiac Troponin Complex. The instruments were calibrated between 0 ng/L and 160,000 ng/L.

NT-proBNP

Results for NT-proBNP LoBDQ Detection Capability shown in table below and Measurement Range with test concentrations between 0 ng/L - 36,000 ng/L shown in plot to the right

D-Dimer

Results for D-Dimer LoBDQ Detection Capability shown in table below and Measurement Range with test concentrations between 0 ng/L - 13,000 ng/L shown in plot to the right

The graph below shows four samples in the Detection Capability study between 0 ng/L and 7.90 ng/L used to compute LoBDQ results shown in the table on the left.

Measurement points are shown as multi-colored circles and the three lowest calibration points are shown as black diamonds in the plot below.

CLSI EP06-A Linearity of Quantitative Measurement was also run. The Phoenix demonstrates excellent linearity and no hooking out to 190,000 ng/L (a larger measurement range than Central Lab and Point-of-Care instruments).

NT-proBNP run in multiplex with cTnI and D-Dimer, spatially and spectrally multiplexed to assess performance of all three biomarkers.

D-Dimer run in multiplex with cTnI and NT-proBNP, spatially and spectrally multiplexed to assess performance of all three biomarkers.

Cardiac Troponin I Flexibility
(Turnaround Time | Sensitivity | Multiplexing)

Cardiac Troponin I (cTnI) is critical to aid in the diagnosis of Acute Coronary Syndrome, myocardial infarction in particular. In the acute setting where cTnI is utilized, medical practitioners often cite that "time is muscle" when emphasizing the need for timely intervention to minimize potential ischemic damage to the heart.

The Phoenix PoC system is designed to deliver high sensitivity cTnI results with a 9 minute time-to-result (TTR) at the point-of-care from whole blood. Ultra-high sensitivity cTnI performance can be achieved within 12 minutes. Ultra-high sensitivity may enable rule - out of MI at the point-of-care. UHS cTnI might also enable routine cardiac health monitoring.

o hs-cTnI results in 9 minutes o ultra-hs-cTnI results in 12 minutes o multiplexed panels

Fast Turnaround hs-cTnI (9 min)

Results summarized in the table below are from a single-plex test protocol where assay parameters were adjusted to optimize time-to-result over sensitivity. As shown even with a 9 minute TTR the Phoenix platform generates sensitivity results superior to large diagnostic instruments in the reference lab.

All samples

Graph below shows LoBDQ data for cTnI run as a single-plex with assay parameters adjusted for speed vs. sensitivity. With these settings, the lowest concentration sample at 0.35 ng/L is differentiated from the blank by roughly 3 standard deviations vs. more than 10 standard deviations for the Ultra-high sensitivity settings.

SARS-CoV-2 Antigen (demonstration)

Novilux SMC™ technology is well suited to just about any immunoassay including the SARS-CoV-2 antigen. The SARS-CoV-2 assay is complementary to the cTnI assay owing to the established connection between the severity of COVID-19 infection and cardiomyocyte damage. Most SARS-CoV-2 antigen tests used today are lateral flow immunoassays with Emergency Use Authorization (EUA) by the US FDA. The SMC™ SARS-CoV-2 assay developed at Novilux for demonstration purposes is 100-fold more sensitive than these tests.

SARS-CoV-2 Antigen LoD

The graph on the right shows the relative sensitivity of a Novilux prototype of the SARS-CoV-2 antigen SMC™ assay (in blue at 0.6 pg/ml) compared to many of the commercially available tests. These assays have a median LoD of 100pg/ml and with only one exception all have LoD's higher than 10 pg/mL. Other than Novilux, only the Quanterix Simoa has an LoD of less than 1 pg/ml. However, the Simoa assay cannot be done at Point-of-Care, and results take more than an hour after sending to a Centralized Lab running an LDT.

Note: These results are for demonstration purposes. The results were generated using the Novilux RDP assay development platform. The assay has not been ported to the Phoenix Point-of-Care system.

Hemolysis & Hematocrit Measurement

The same technological innovations that facilitate rapid SMC™ immunoassays at the point-of-care from anti-coagulated whole blood samples also enable the characterization of critical hematological properties of the applied blood specimens. The amount of free hemoglobin remaining in the blood sample following initial centrifugation can be easily measured, and therefore the degree of hemolysis can be rapidly reported as well. Hematocrit is the fraction of the sample that is attributed to red blood cells (RBCs). Initial centrifugal processing of applied blood specimen to the disc allows for the accurate and rapid determination of percent hematocrit. While important biomarkers in their own rights, hemolysis and hematocrit determinations are important elements of our SAFE realtime run QC that can flag samples with anomylous levels of either or both of these hematological parameters.

Free Hemoglobin / Hemolysis

Clinical conditions and/or preanalytical sample handling can cause hemolysis of a blood specimen. The Phoenix SAFE QC system measures the free hemoglobin (and hematocrit) during initial centrifugation to determine the percent hemolysis in the sample. SAFE QC flags samples above thresholds for assay interference.

Panels A. and B. (top right) show examples of non-hemolyzed and hemolyzed blood. SAFE measured free hemoglobin for these samples at 0.03 g/L and 2.68 g/L respectively. (Note: pinkish color of "B" is easily quantitated in RGB spectra at runtime.)

Panel C. shows the correlation of the measured and known average plasma free hemoglobin concentration (8 replicates across 2 instruments) based on gravimetric dilution series. Linear regression is shown with ± 1-SD error bars. Hemolysate was produced in accordance with CLSI EP7-A2 Appendix G and quantified with the Alere HemoPoint H2 system.

Hematocrit Determination

Hematocrit is an important hematological marker that can also have an impact on immunoassay performance. Similar to plasma free hemoglobin, the SAFE imaging system accurately measures the volumes of the plasma and RBCs during centrifugation to compute hematocrit levels.

Panels A, and B. (right) show SAFE imagery immediately after centrifugation for a low hematocrit sample (9.3%) and for a high hematocrit sample (66%) respectively. Panel C. shows the correlation of the Phoenix determined hematocrit level (8 replicates across 2 instruments) compared to hematocrit assignment (in triplicate) using the microhematocrit standard method. Linear regression is shown and error bars represent ± 1-SD about each mean.

SAFE measurement of plasma free hemoglobin and hematocrit enables the calculation of hemolysis on every sample.

Blood Chemistries

Many of the technological innovations that enable ultra-high sensitivity SMC™ immunoassays at the point-of-care are also useful for the development of assays to measure electrolytes, metabolites, and blood gasses. There is also a need for these same markers to be concomitantly delivered along with acute care markers like those in the Novilux 3-Plex Cardiac Panel as electrolyte dysregulation and/or renal function are important factors for health care professionals to consider as part of a comprehensive diagnostic workup. The Basic Metabolic Panel (BMP) is one of the most ordered blood tests, tied in frequency with the Complete Blood Count (CBC). Beyond acute care, the ability to measure a plurality of these blood chemistries at the point-of-care allows for utility to be expanded into non-critical setting such as physican office labs and retail clinics.

Basic Metabolic Panel

Novilux is currently developing a Basic Metabolic Panel as the initial proof-of-concept for the demonstration of blood chemistries on the Phoenix platfom. The eight markers that make up the Novilux initial BMP are:

Glucose (RDP prototype assay results in Panel A.)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca2+)
Creatinine
Blood Urea Nitrogen (BUN)
Total CO2 (tCO2)

The development of this panel of assays follows a similar approach that Novilux follows for the development of immunoassays. Prototype assays are first configured on the Rapid Development Platform using liquid reagents. These assays are then subsequently ported to lyophilized formulations for ultimate Phoenix platform implementation. Like with Novilux's immunoassays and hematological assays, the SAFE realtime QC system is integral to the devlopment of this expanding content.

Growing Ecosytem for Biomarker Development

A Novilux out-license to MilliporeSigma (Merck KGaA) has enabled the development of a thriving research market using SMC™ technology. A large and growing installed base of SMCxPRO™ and FemtoQuest™ instruments, along with MilliporeSigma assay kits, has created an ecosystem of new biomarker development based on SMC™ discoveries.

As groundbreaking new clinical research validates important new biomarkers using SMC™ technology, Novilux is poised to collaborate and implement those biomarkers on its Phoenix Point-of-Care platform.

> 130 SMC™ Immunoassays

The graphic to the right shows a collection of 54 of the more than 130 ultrasensitive immunoassays developed to date on SMC™ platforms. The biomarkers enumerated here are important for monitoring a broad array of health conditions, many of which could be developed into diagnostic tests or panels.

An ecosystem for ultrasensitive assays is now taking shape where commercial Life Science discoveries can become validated diagnostic tests/panels via porting to Novilux ultra-high sensitivity, low-cost diagnostic platforms.

Infographic Footnote Key. Assays developed or sold by:

1. Novilux

2. Singulex

3. MilliporeSigma (SMCxPRO™ & FemtoQuest™)

4. External, SMCxPRO™ user developed

ultrasensitive detection of biomarkers