Qualification of a 48-Plex Cytokine Olink® Panel to Support Rare Disease and Pediatric Clinical Studies

Rare diseases present significant challenges in biomarker discovery and validation due to limited patient populations, complex disease mechanisms, and a lack of established or validated biomarkers. However, the ability to measure pathway-specific, physiologically relevant biomarkers is critical for the successful development, investigation, and approval of new therapeutics to address this unmet need. In this article, we explore how a the Olink Target 48 Cytokine panel can provide a powerful tool to support rare disease and pediatric clinical studies.

While individually uncommon, rare diseases collectively affect an estimated 30 million people in the US and 300 million people worldwide. Nearly 70% of these diseases manifest in childhood and approximately 80% percent are genetic in origin. To date, about 95% of rare diseases lack approved treatment options.[1]

There are many limitations in biomarker discovery for rare diseases, including:

• Limited assay sensitivity for low-abundance biomarkers
• Difficulty in obtaining high-quality, specific antibodies due to the limited availability of well-characterized antigens
• Low throughput assays, which is a significant limitation when comprehensive biomarker profiling is needed
• Inability to detect multiple biomarkers simultaneously
• Requirement for large sample volumes
• High variability between runs, operators, and laboratories

Thus, biomarker analysis for rare diseases—especially for use in pediatric clinical studies—can benefit from multiplexed platforms that can examine numerous analytes using minimal sample volume. In this poster summary, we describe the qualification of a multiplex Olink method that simultaneously measures a panel of 45 cytokine biomarkers in human serum using extremely low (<1 µl) sample volumes.

Purpose

The purpose of this project was to evaluate an off-the-shelf 48-plex cytokine panel as a biomarker panel for clinical studies. The Olink Target 48 Cytokine panel is based on the proximity extension assay (PEA^TM) technology, where two matched antibodies labeled with unique DNA oligonucleotides simultaneously bind to a target protein in solution during an overnight incubation. In close proximity, the DNA oligonucleotides hybridize and are extended by DNA polymerase, resulting in a unique DNA barcode for each protein. These barcodes are amplified by polymerase chain reaction (PCR) for readout by next-generation sequencing (NGS) or quantitative PCR (qPCR).[2]

Qualification criteria included:

• Inter-and intra-assay precision across operators and days
• Accuracy
• Dilutional linearity
• Specificity
• Stability of the intermediate PCR products

Method

Ten commercially obtained, healthy human serum donor samples were tested using 3-4 replicate samples per donor and 3-4 replicate samples of pooled serum from all 10 donors. Four runs were performed across two operators, on two different days to evaluate precision. Accuracy was assessed by mixing the human serum with Olink-provided sample control. Dilutional linearity was tested by mixing the human serum with Olink-provided sample control and then diluting with Olink-provided diluent. A second assessment of dilutional linearity was completed using samples spiked with Olink-provided sample control and diluted in non-human matrix, specifically rat plasma. Stability of the intermediate PCR products stored at 4º C was tested fresh, at one week, and at one month.

Results

Table 1. Dilutional linearity, accuracy, and precision

As shown in Table 1:

• For the precision evaluation, assay results from two operators on two different days demonstrated mean coefficients of variance (%CV) were 2.0-29% across the 45 cytokines.
• For accuracy assessment, normal human serum mixed 1:1 with sample control showed ≥80% recovery across all 45 cytokines.
• For dilutional linearity/lower limit of quantification (LLOQ) tests performed using normal human serum mixed 1:1 with sample control diluted serially in rat plasma, passing was defined as 70-130% recovery of neat signal. With the exception of CCL19, this evaluation was within the acceptance criteria.

Stability of the intermediate PCR products remained within 0.5 deviations from the median for up to one week stored at 4ºC (see Figure 1).

Figure 1. Stability of intermediate PCR products stored at 4ºC

Conclusion

While commercially available panels can be used off-the-shelf, independent qualification adds scientific rigor. This independent qualification of the Olink Target 48 Cytokine panel demonstrated excellent precision across multiple operators and multiple days and excellent accuracy when spiking with Olink-provided sample control. Dilutional linearity was acceptable and varied depending on the analyte, and the large detection range suggests that diluting into the range of the assay is likely not necessary. To avoid using precious sample volume on re-tests, it is possible to store intermediate PCR products short-term at 4ºC.  Overall, this independent qualification shows that the Olink Target 48 Cytokine panel is appropriate for highly multiplexed analysis and can detect low levels of target analyte from low volumes of sample, making it suitable for biomarker analysis to support rare disease and pediatric studies.

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[1] The Lancet Global Health. The landscape for rare diseases in 2024. Lancet Glob Health. 2024;12(3):e341.

[2] Olink. What is PEA? Available at https://olink.com/technology/what-is-pea. Accessed August 22, 2024.

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