Evaluation of a research use only (RUO) TIMP-1 assay for use with the automated Alinity i platform

Abstract

Tissue inhibitor of metalloproteinase-1 (TIMP-1) binds matrix metalloproteinases to regulate extracellular matrix turnover; elevated TIMP-1 has been associated with liver fibrosis, tumor progression, and fibrotic heart disease. We previously developed a quantitative, research use only (RUO) automated TIMP-1 immunoassay for use with the Abbott ARCHITECT i System; here, we describe the modification of the assay for use with the Abbott Alinity i system. The observed limit of detection was 1.42 ng/mL, and the calculated limit of quantitation was 2.44 ng/mL. In 100 normal plasma samples, TIMP-1 levels ranged from 106.23 ng/mL to 329.68 ng/mL. Deviation from linearity was ≤10% and precision was ≤10%CV across the analytical measuring interval from 2.44 to 500 ng/mL. No significant interference was observed for both conjugated and unconjugated bilirubin and hemoglobin at industry recommended test concentrations. Slight interference for protein and triglyceride resolved with titration to 12.15 g/dL and 750 mg/dL, respectively. Assay performance was optimal for plasma specimens in LiHep or EDTA. TIMP-1 in plasma samples was stable after multiple freeze-thaw cycles, up to 7 days at 2‒8 °C, and for up to 3 h on-board the Alinity i instrument. In cardiac plasma specimens, the assay consistently detected higher TIMP-1 in specimens with elevated troponin-I. The TIMP-1 immunoassay, modified for the Alinityi platform, detected TIMP-1 at physiologically relevant ranges. Future clinical development of the assay will focus on its clinical utility in various cardiac cohorts as a potential biomarker of fibrotic heart disease.

Highlights

• •A quantitative immunoassay for TIMP-1 was modified for use on the Alinity i platform.
• •The immunoassay detected TIMP-1 within physiologically relevant ranges on Alinity i.
• •The preferred sample type is plasma collected in Lithium Heparin and EDTA Tripotassium tubes.
• •Elevated TIMP-1 was observed in cardiac plasma along with elevated troponin-I.

1. Introduction

Matrix metalloproteinases (MMPs) are inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1), which binds MMPs with high affinity and forms stable complexes to regulate extracellular matrix turnover [1]. Elevated concentrations of TIMP-1 have been associated with fibrosis [2] and tumor progression as well as worse overall survival [3]. Variations in TIMP-1 levels have been linked to colorectal cancer [4], liver fibrosis [2], and heart disease [5,6]. In fibrotic liver disease, TIMP-1 concentrations have been shown to increase with disease progression [7].

These studies suggest that TIMP-1 may be a clinically useful biomarker for monitoring response to therapy as well as to aid in the initial diagnosis of disease in high-risk populations. We previously developed a research use only (RUO) double monoclonal antibody sandwich TIMP-1 immunoassay to run on the automated ARCHITECT i system (Abbott Laboratories) [8]. This quantitative assay was initially developed for the detection of TIMP-1 as a biomarker of colorectal cancer with potential applications in other disease states associated with TIMP-1 elevation. The assay demonstrated high sensitivity with a limit of detection (LoD) of less than 0.13 ng/mL and strong precision performance (≤5.25% coefficient of variance [CV]) [9].

Here, we describe reformulation of the RUO TIMP-1 immunoassay for migration to the high-throughput automated Alinity i platform (Abbott Laboratories) and assessment of its analytical performance. The clinical utility of TIMP-1 as a potential cardiac biomarker was also evaluated.

2. Materials and methods

2.1. Assay description

The TIMP-1 immunoassay is a two-step dual monoclonal sandwich assay. The TIMP-1 analyte in the sample is captured by the first monoclonal antibody (capture mAb), which is coated on the surface of paramagnetic microparticles (Supplemental Fig. 1). The captured analyte is then detected by a second monoclonal antibody labeled with acridinium (detection mAb). Addition of pre-trigger and trigger reagents results in chemiluminescent reaction measured as relative light units (RLU) that is proportional to the amount of TIMP-1 analyte captured.

2.2. Assay modification

To develop the TIMP-1 immunoassay for the Alinity i system, we used the same two-step process with the same capture and detection antibodies as described previously [8,9]. A new lot of the capture and detection antibodies were prepared and purified. In addition, the assay was modified to improve signal intensity, including lowering of the pH of the particle coating buffer (2-(N-morpholino) ethanesulfonic acid [MES]) from 6.2 to 5.5 and increasing the conjugate bulk concentration from 37.5 to 60 ng/mL. Additional modifications were made to the diluents as follows: the surfactant Triton X-100 was replaced with Tergitol 15-S-9 in the conjugate diluent and Tergitol 15-S-40 in the microparticle and assay specific diluents; BSA was replaced with a protease-free version in the conjugate, microparticle, and calibrator diluents; and the defoaming agent was replaced with Korasilon.

The calibrators and controls were prepared with an externally sourced TIMP-1 antigen (R&D Systems, Minneapolis, MN). The same reaction volumes developed for the ARCHITECT system (25 μL assay sample mixed with 50 μL of the capture antibody-coated microparticles, washed, and then mixed with 50 μL of the acridinium-labeled detection antibodies) [9] were used for the Alinity i assay. A 1:5 auto-dilution protocol using line diluent was included in the assay file and the internal calibrator range was 0 to 500.00 ng/mL. The effect of each change made to the TIMP-1 assay reagents on the RLU signal is shown in Supplemental Table 1.

2.3. Tube type evaluation

Clinical specimens for tube type testing were collected at Abbott Laboratories using a protocol approved by an external Institutional Review Board (WCG 202238893). Participants provided informed consent prior to specimen collection. Specimens were collected from 12 donors in “No Additive” tubes, pooled, and aliquoted into Lithium Heparin (LiHep) (control), Tripotassium (K3) EDTA, Serum, Serum Separator, and Sodium Citrate collection tubes and processed according to blood tube manufacturer's instructions. Acceptable performance was defined as ≤10% bias in concentration compared to the control tube.

2.4. Detection limits

A three-day study was performed on two Alinity i instruments using one lot of reagents and a six-member low-level dilution panel. 3% BSA in SeraSub was used as the zero-analyte sample for determination of the limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ). In accordance with Clinical and Laboratory Standards Institute (CLSI) EP17-A2 section 5.3.3.1, the limit of blank was estimated using the non-parametric approach by determining the 95th percentile of the zero-analyte sample concentration of 60 replicates on 2 instruments across 3 days. The limit of detection was estimated using the parametric approach per CLSI EP17-A2 section 5.3.3.2. The limit of quantitation was estimated per CLSI EP17-A2 section 6.5 and defined as the lowest concentration at which the assay demonstrates a ≤20% CV.

2.5. Linearity

A linearity study was performed on one Alinity i instrument using a high specimen pool targeted to ≥500 ng/mL and 3% BSA SeraSub as the zero-analyte diluent to generate nine intermediate levels by gravimetric preparation. The panels were equilibrated at 2-8°C overnight prior to testing on the TIMP-1 immunoassay. The linear range was defined as the concentration range for which the assay was linear or had a deviation from linearity of ≤10%.

2.6. Precision and reproducibility

A five-day precision study was performed on one Alinity i instrument using a human plasma panel with two runs per day. A five-day reproducibility study was performed on three Alinity i instruments with two runs per day. Acceptable precision and reproducibility values was defined by ≤ 10% CV.

2.7. Cardiac sample acquisition

Eighty-eight cardiac samples with known troponin-I levels were acquired from Precision Biospecimens (Frederick, MD).

2.8. Normal range/expected values

A total of 100 human plasma samples from healthy donors (Gulf Coast, received Dec 2023) were tested with the Alinity i TIMP-1 immunoassay in singlicate to establish preliminary expected values on the Alinity i system.

2.9. Manual versus auto dilution

The auto dilution capability of the assay was evaluated by testing cardiac samples with elevated TIMP-1 and comparing the assay result of samples diluted 1:5 on-board the instrument versus samples manually diluted with the same diluent. Acceptable performance was defined as within or equal to ±10% difference for automated versus manual dilutions.

2.10. Endogenous interference

Two lithium heparin plasma pools at an approximate TIMP-1 concentration of 120 and 175 ng/mL were used for endogenous interference testing. Pools were spiked with conjugated or unconjugated bilirubin, hemolysate (containing hemoglobin), a high protein solution consisting of bovine gamma globulin (BGG) and human serum albumin (HSA), or human triglycerides and tested with the Alinity i TIMP-1 immunoassay to assess interference. Testing for endogenous interference followed CLSI EP07 Ed. 3, which recommends testing interferents at three times the highest concentration observed clinically. For rheumatoid factor (RF) interference, plasma samples were spiked with RF stock at either 200 IU/mL or 1200 IU/mL and tested with the TIMP-1 immunoassay. To assess human anti-mouse antibody (HAMA) interference, plasma samples were spiked with HAMA stock at 300 ng/mL, 600 ng/mL, and 900 ng/mL and tested with the TIMP-1 immunoassay. The test (with interferent) and control (without interferent) samples were considered equivalent if the two-sided 95% confidence interval (CI) around the % difference was within or equal to ±10%.

2.11. Specimen stability

Specimen stability was assessed by storing specimens in LiHep or K3 EDTA collection tubes from the Tube Type study either on the cells/clot in LiHep or K3 EDTA collection tubes at room temperature (RT) or 2‒8 °C for 24 h and 7 days or off the cells/clot and subjected to 3 freeze-thaw cycles then tested on the Alinity i TIMP-1 immunoassay. The difference and % difference in mean measurand values between each test (storage condition) and control (baseline test time point) condition was calculated. The test (each storage condition) and control (baseline test time point) samples were considered equivalent if the average % difference (across donors) was within or equal to ±10%.

2.12. Sample on-board stability

Sample on-board stability was evaluated by storing specimens on-board the Alinity i instrument for up to 3 h prior to testing. The test (each storage condition) and control (initial test time point) samples were considered equivalent if the two-sided 95% CI around the % difference (across donors) was within or equal to ±10%.

2.13. Reagent on-board stability

The previous TIMP-1 immunoassay, developed for use with the ARCHITECT system, claimed a 14-day reagent on-board stability and calibration frequency. We examined the stability of the assay reagents on the Alinity i instrument in a 16-day study performed using controls and the low-analyte panel. Samples were tested at 10 time points over 16 days. Samples were regressed and acceptable performance was defined as a shift within or equal to ±10%.

3. Results

3.1. Specimen collection

Because TIMP-1 is expressed in platelets and white blood cells during coagulation, collection and handling of blood specimens can affect TIMP-1 assay results, and plasma is preferred over serum as the test matrix for the TIMP-1 immunoassay [10]. Therefore, as part of development of the TIMP-1 assay for use with the Alinity i system, we assessed the effect of the collection tube on assay performance. Similar to previous results [8], collection in serum tubes led to a significant difference in TIMP-1 concentration compared to the control LiHep plasma tubes. The presence of gel in the serum separator tubes did not improve assay performance. Finally, collection in Sodium Citrate tubes, typically used for coagulation assays, also showed a significant difference in TIMP-1 concentration compared to the control LiHep plasma tubes. Thus, the use of plasma specimens using LiHep or EDTA anticoagulants are recommended for the TIMP-1 immunoassay on the Alinity i system (Supplemental Table 2).

3.2. Analytical performance

3.2.1. Detection limits

The observed LoD was 1.42 ng/mL (Table 1). The highest observed LoQ across two reagent lots and two instruments was 2.44 ng/mL (Table 2). After removing outliers, the calculated LoQ decreased to 2.39 ng/mL; however, it is important to note that the pooled %CV for the low-analyte Level 1 panel, which had an observed concentration 0.95 ng/mL, was near the 20% CV criterion (22.4%) on the second instrument.

Table 1.

Detection Limits of the TIMP-1 assay on two Alinity i Instruments.

Instrument No.	Zero Level Analyte		Low-Level Analyte
	N	Limit of Blank (ng/mL)	N	Pooled Mean (ng/mL)	Pooled SD	Limit of Detection (ng/mL)
Ai01004	30	1.06	30	2.30	0.142	1.30
Ai01008	30	0.90	30	2.44	0.311	1.42

Table 2.

Limit of Quantitation of the TIMP-1 assay on two Alinity i Instruments.

Low-level Analyte Level	N	Pooled Mean (ng/mL)	Pooled SD	Pooled %CVa	LoQ (ng/mL)
Instrument No: Ai01004
1	30	1.11	0.198	17.8	1.30
2	30	2.30	0.142	6.2	NA
3	30	4.93	0.223	4.5	NA
4	30	7.09	0.196	2.8	NA
5	30	9.73	0.243	2.5	NA
6	30	19.93	0.245	1.2	NA
Instrument No: Ai01008
1	30	1.05	0.602	57.4	NA
2	30	2.44	0.311	12.8	2.44
3	30	5.12	0.196	3.8	NA
4	30	7.39	0.211	2.9	NA
5	30	10.07	0.272	2.7	NA
6	30	20.43	0.344	1.7	NA

^a%CV = Pooled SD x 100/Pooled mean.

3.2.2. Linearity

All samples across the measurand range evaluated had a deviation from linearity within or equal to ±10% (Fig. 1, Supplemental Table 3).

Fig. 1.

Linearity performance of the Alinity i TIMP-1 immunoassay across the analytical measuring interval

Observed concentrations for gravimetrically prepared dilution levels are plotted against expected values. All levels demonstrated a deviation from linearity within ±10%, confirming linear response throughout the validated range.

3.2.3. Automated versus manual dilution

The results demonstrate that manual dilution using the same diluent is consistent with the Alinity i automated dilution protocol (Supplemental Table 6).

3.2.4. Precision

In both the precision and reproducibility studies, all samples met the precision criteria of ≤10% CV (Supplemental Tables 4 and 5).

3.2.5. Reportable Interval

The Analytical Measuring Interval (AMI), Extended Measuring Interval (EMI), and Reportable Interval (RI) were established in accordance with CLSI EP34 Ed. 1. The AMI is determined by the range of values that demonstrated acceptable performance for linearity, imprecision, and bias and extends from the LoQ to the upper limit of quantitation (ULoQ). The AMI for the Alinity i TIMP-1 assay was determined to be 2.44 to 500 ng/mL. The EMI extends from the ULoQ to the ULoQ × dilution factor. The EMI for the Alinity i TIMP-1 assay was determined to be 500-2500 ng/mL. Finally, the reportable interval extends from the LoD to the upper limit of the EMI and is 1.42 to 2500 ng/mL.

3.2.6. Normal range/expected values

Testing was conducted on plasma specimens from 100 apparently healthy individuals. The mean concentration was 170.98 ng/mL (Fig. 2). TIMP-1 values ranged from a minimum of 106.23 ng/mL to a maximum of 329.68 ng/mL, with a median of 163.82 ng/mL.

Fig. 2.

Distribution of TIMP-1 concentrations in normal plasma samples (n = 100) and cardiac specimens with elevated troponin-I (n = 88)

The normal donor cohort establishes the physiological concentration range, while cardiac specimens exhibit higher TIMP-1 levels.

3.2.7. Cardiac samples

The TIMP-1 immunoassay results for 88 cardiac specimens with known elevated troponin-I results are also shown in Fig. 2. Higher TIMP-1 concentrations were detected in specimens with elevated troponin-I.

3.2.8. Endogenous interference

No significant interference was observed for both conjugated and unconjugated bilirubin and hemoglobin at the recommended CLSI test concentrations (40 mg/dL and 1000 mg/dL, respectively; Supplemental Table 7). Interference by hemoglobin was evaluated at both 500 mg/dL and 1000 mg/dL based on previous interference observed at 750 mg/dL in the ARCHITECT TIMP-1 immunoassay; similar interference was not seen for the assay on the Alinity i platform. Slight interference with protein was observed at the recommended test concentration of 15 g/dL. Interference was not observed at a total protein concentration of 12.15 g/dL. Similarly, triglyceride was titrated from the CLSI recommended test concentration of 1500 mg/dL to 750 mg/dL to reduce interference to within ±10%. No assay interference was observed for RF up to 200 IU/mL (Supplemental Table 8), and no interference was observed with HAMA up to 900 ng/mL except in a single sample (Supplemental Table 9).

3.2.9. Specimen stability

Under all storage conditions and time, average shifts in RLU compared to baseline were within ±10% except for K3 EDTA at 7 days at room temperature, which had a mean % difference compared to baseline of 14% (Supplemental Table 10a and Supplemental Table 10b). However, removal of one sample that consistently showed an elevated RLU shift regardless of collection tube type, storage temperature, or time resulted in a mean % difference of 10%. Data supports storage of up to 7 days at either 2-8 °C or room temperature; however, samples should be storage at 2-8 °C when possible. Samples were stable up to 3 freeze-thaw cycles (Supplemental Table 10c).

3.2.10. Sample on-board stability

Normal plasma and cardiac samples were tested after 3 h on-board the Alinity i system. The largest observed shift in RLU was 4.7%, below the ≤10% target (Supplemental Table 11).

3.2.11. Reagent on-board stability

The assay reagents and calibration were demonstrated to be stable on-board the Alinity i instrument for 16 days (Supplemental Table 12).

4. Discussion

The reformulated TIMP-1 immunoassay for use with the Alinity i system, in accordance with current best practices for microparticle coating and bioconjugation of mAbs, showed high accuracy, precision, and reproducibility.

The observed LoD at 1.42 ng/mL on Alinity i was higher than the LoD observed on ARCHITECT (0.13 ng/mL) as the analytical methodologies differed. While both methods calculated the LoD across two instruments, a more robust study design in accordance with modernized guidance was used to calculate the LoD on the Alinity i with an increased number of replicates and incorporation of the standard deviation of all low analyte-level samples. If the LoD for Alinity i were to have been calculated following the ARCHITECT calculation, LoD = Calibrator A Standard Deviation ∗ 2 ∗ (Calibrator B Target 20 ng/mL)/[Calibrator B Average RLU- Calibrator A Average RLU]), it would yield a similar LoD of 0.19 ng/mL.

The calculated LoQ ranged from 1.30 to 2.39 ng/mL across two instruments. In plasma samples from healthy donors, TIMP-1 levels ranged from 106.23 to 329.68 ng/mL. Previous studies of TIMP-1 levels in specimens from individuals at high risk of colorectal cancer reported values between 50 and 250 ng/mL [11]. Thus, the TIMP-1 assay on Alinity i is able to detect TIMP-1 within physiologically and clinically relevant ranges.

The assay reagents demonstrated on-board stability, accurate automated dilution, and linearity, and no significant interference. The tube type studies were consistent with those performed with the ARCHITECT system, with plasma collected in LiHep and K3 EDTA tubes as the preferred sample type. Release of TIMP-1 from platelets in serum samples and suppression of TIMP-1 in sodium citrate samples may interfere with assay accuracy [10,12].

The TIMP-1 analyte in plasma samples was stable for up to 7 days at 4‒6 °C and after multiple freeze thaw cycles. In addition, the specimens were stable up to 3 h on-board the Alinity i instrument. The single specimen with an elevated shift may have been due to abnormal baseline test results, as all test results after storage were ≥100 ng/mL.

TIMP-1 elevation has been reported in a number of fibrotic disease states, including liver fibrosis leading to cirrhosis and hepatic cancer, cardiac fibrosis preceding heart failure and acute myocardial events, and gastrointestinal cancers, such as colorectal cancer. TIMP-1 is one of three biomarkers included in the Enhanced Liver Fibrosis (ELF) test, which has been shown to predict liver disease progression and outcomes in various populations [2,13]. TIMP-1 has also been evaluated extensively as a biomarker of colorectal cancer risk [3,[14], [15], [16]], for diagnosis [17], and to predict outcomes [4], as well as to evaluate treatment response [18,19]. With the TIMP-1 immunoassay, we observed higher TIMP-1 concentrations in cardiac samples with elevated troponin-I, an established marker of cardiovascular disease, compared to plasma samples from healthy donors. These preliminary findings are consistent with the literature reporting elevated in TIMP-1 in patients with heart failure [20,21] and diabetes [22] and its use as a predictor of treatment response after aortic valve replacement [23] and cardiac resynchronization therapy [24].

5. Conclusion

This study describes the reformulation and analytical evaluation of an RUO TIMP-1 immunoassay for migration from the Abbott ARCHITECT system to the high-throughput Alinity i platform. TIMP-1, a key regulator of extracellular matrix turnover, is clinically associated with fibrotic and oncologic disease states, including liver fibrosis, colorectal cancer, and cardiac fibrosis. The modified assay retained the dual monoclonal antibody sandwich format and incorporated reagent and buffer optimizations to enhance signal performance.

Analytical evaluation demonstrated robust performance: an LoD of 1.42 ng/mL, an LoQ of 2.44 ng/mL, and within-laboratory imprecision ≤10% CV across the analytical measuring interval (2.44–500 ng/mL). Linearity, automated dilution accuracy, and reagent stability were confirmed, with no significant interference from bilirubin, hemoglobin, rheumatoid factor, or human anti-mouse antibodies. Plasma collected in Lithium Heparin or EDTA tubes was identified as the preferred specimen type. TIMP-1 concentrations in healthy donors ranged from 106.23 to 329.68 ng/mL, while elevated levels were observed in cardiac samples with increased troponin-I, supporting its potential role as a biomarker of fibrotic heart disease.

The Alinity i TIMP-1 immunoassay provides sensitive and reproducible detection of TIMP-1 within physiologically relevant ranges. Future clinical development will focus on validating its utility in cardiac cohorts and other disease states associated with fibrosis and tumor progression.

6. Limitations and prospects

With respect to cross-platform validation, the assay evaluated in this study is specifically intended for use on the Alinity i system; therefore, cross-platform comparison studies were not performed. This represents a limitation in terms of generalizability to other analytical platforms, and the performance conclusions of this study should be interpreted within the context of the intended system only. Although a high-dose hook effect was not directly evaluated for the TIMP-1 assay on the Alinity i system, this risk is mitigated by the two-step sandwich assay design, consistent with CLSI EP34 Ed.1 guidance. Furthermore, no modifications were made to the assay biologics or format for implementation on the Alinity i system, and both the Alinity i and ARCHITECT TIMP-1 assays utilize identical reaction ratios and timing. Importantly, no hook effect was observed during prior evaluation of the TIMP-1 assay on the ARCHITECT system [24].

Assay performance testing was conducted primarily using specimens from healthy donors, with the exception of a limited cohort of cardiac specimens exhibiting elevated troponin-I concentrations. As such, additional studies incorporating specimens from diverse disease states with endogenous TIMP-1 concentrations spanning the analytical measuring interval would be valuable to further characterize assay performance and clinical utility across broader patient populations.

Specimen stability was evaluated at baseline, 24 h, and 1 week under room temperature and refrigerated storage conditions, as well as following multiple freeze–thaw cycles, using samples from 12 individual donors. This study design is consistent with current clinical laboratory guidelines and reflects routine clinical laboratory handling and storage workflows. However, this assessment does not encompass long-term storage conditions or non-standard preanalytical handling scenarios. Accordingly, the stability conclusions presented here are limited to the conditions evaluated and should be interpreted in the context of typical clinical laboratory practice.

Future clinical development of the Alinity i TIMP-1 immunoassay will focus on addressing the current study limitations through targeted, hypothesis-driven investigations.

Large-scale clinical validation should include well-pedigreed cardiac cohorts spanning early-stage to advanced fibrotic heart disease, with appropriate inclusion of longitudinal sampling. These studies should evaluate the association of circulating TIMP-1 concentrations with clinically relevant endpoints such as disease progression, risk stratification for adverse cardiovascular events, and response to antifibrotic or cardioprotective therapies. Inclusion of diverse patient populations and disease severities will be critical to defining the prognostic value of TIMP-1 across the analytical measuring interval and beyond healthy reference ranges.

Expanded sample matrix applicability represents an additional area for future investigation. While the current work focused on serum and plasma under routine clinical laboratory handling conditions, further studies evaluating alternative specimen matrices, extended storage durations, and nonstandard preanalytical conditions will help define assay robustness and inform broader clinical implementation strategies.

Multiplex panel development is another prospective direction, particularly the integration of TIMP-1 with complementary biomarkers of myocardial injury, inflammation, and extracellular matrix turnover. Such panels may improve diagnostic and prognostic performance relative to single-analyte testing and enable more comprehensive assessment of fibrotic remodeling within heterogeneous cardiac disease populations.

Finally, mechanistic exploration of TIMP-1 biology will be essential to refining its clinical interpretation. Future studies should investigate the role of TIMP-1 in key extracellular matrix remodeling pathways, its interaction with matrix metalloproteinases, and its crosstalk with established cardiac biomarkers. Elucidating these mechanisms across normal and pathological states may clarify whether TIMP-1 functions predominantly as a marker of fibrosis burden, disease activity, or therapeutic response, thereby informing both assay positioning and the design of subsequent clinical validation studies.

Author contributions

P Hemken and M Anderson designed the study and drafted the manuscript. J Grieshaber, J Ortiz, M Baclig, G Pacenti, J Munoz, C Rudolph, K Otis designed and performed experiments, analyzed the data, and revised the manuscript. J Corby and J Prostko analyzed the data and revised the manuscript. All authors reviewed the manuscript, accepted responsibility for the entire content of this manuscript, and approved its submission.

Funding

This study was supported by Abbott Laboratories.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Philip Hemken reports financial support, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by Abbott Laboratories. All authors report a relationship with Abbott Laboratories that includes employment and equity or stocks.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

The data that has been used is confidential.