The CMI-PB team initially measured cytokine concentrations in plasma using the Olink assay. However, after the targeted cytokine panel from Olink was discontinued in 2024, we transitioned to using the Legendplex assay for measuring cytokine concentrations in plasma. Cytokine concentrations were measured using Olink for the complete cohorts of 2020, 2021, and 2022, as well as for the partial cohort of 2023. In contrast, cytokine concentrations were measured using Legendplex for the complete cohorts of 2021, 2022, and 2023, with the exception of the 2020 cohort. Although predicting cytokine measurements is not a specific task in the CMI-PB public challenge, these measurements may play a significant role in predicting outcomes involving different assays in other tasks.
The CMI-PB team recommends two options:
- Option 1: Utilize cytokine measurements from both methods for building prediction models unless there is a specific preference for one assay over the other.
- Option 2: LegendPlex (n=14) and Olink (n=45 from 2021 and 2022 cohorts) have 14 overlapping cytokine measurements: CXCL8, CXCL10, IFN-γ, IFN-α2, IL-10, CCL3, G-CSF, IL-1RA, CCL2, CCL5, IL-2, IL-6, TNF, and IL-7. Utilize these overlapping cytokine measurements from the LegendPlex assay and incorporate the unique measurements from the Olink assay.
When comparing Legendplex and Olink for measuring cytokine concentrations in blood plasma to capture immune responses, several key differences emerge in technology, sensitivity, specificity, and overall application. The following are key differences between LegendPlex and Olink assays:
- Legendplex utilizes bead-based multiplex immunoassay technology, where each bead is coated with a specific antibody targeting a cytokine. The assay involves binding cytokines from the sample to these beads and adding a detection antibody and a fluorescent reporter, with the fluorescence intensity measured by flow cytometry. This intensity correlates with cytokine concentration, offering a moderately high sensitivity and good specificity. However, potential cross-reactivity between closely related cytokines can occur. On the other hand, Olink uses Proximity Extension Assay (PEA) technology, which employs pairs of antibodies to bind to the target cytokine, bringing their attached DNA oligonucleotides into proximity. This leads to DNA extension and amplification via PCR, quantified by next-generation sequencing (NGS) or qPCR. Olink’s technology provides very high sensitivity and specificity, reducing the likelihood of cross-reactivity making it particularly advantageous for detecting low-abundance cytokines in complex samples like blood plasma.
- Regarding multiplexing capability, Legendplex can measure up to ~ 50 cytokines simultaneously in a single assay, which is generally sufficient for many studies. However, the number of cytokines that can be measured is limited by the availability of compatible bead sets and potential cross-reactivity. Olink, however, is highly scalable, allowing for the measurement of up to ~92 cytokines per panel and the possibility of combining multiple panels for larger-scale studies. While this scalability is a significant advantage, it also increases the cost per sample as more panels are used.
- Sample volume requirements also differ between the two platforms. Legendplex typically requires around 50-100 µL of sample per assay, which is relatively low and suitable for studies with limited availability, such as pediatric studies or rare sample collections. In contrast, Olink requires only about 1 µL per panel, making it highly advantageous when sample quantity is extremely limited. It is ideal for large-scale studies or situations where minimal sample use is crucial.
- Data analysis and throughput are other areas where the two platforms differ. Legendplex requires manual data analysis through software provided by the manufacturer, often involving manual gating and interpretation of flow cytometry data, leading to medium throughput. Flow cytometry allows for the processing of multiple samples but may be slower compared to automated platforms. In contrast, Olink provides an automated and standardized data analysis pipeline, offering results as normalized protein expression (NPX) values and simplifying data interpretation. Olink is also designed for high-throughput screening, with the capability to process hundreds to thousands of samples in parallel.
- Cost and accessibility are critical considerations when choosing between these two platforms. Legendplex is generally more cost-effective per assay, making it suitable for smaller studies or research with budget constraints. Additionally, it only requires a standard flow cytometer, which is common in many labs, enhancing its accessibility. Olink, while offering superior sensitivity and specificity, comes at a higher cost per sample due to the sophisticated technology involved. It also requires access to specialized equipment for PEA and NGS or qPCR, which may not be as widely available as the equipment needed for Legendplex.
In summary, Legendplex is a robust, cost-effective option for multiplex cytokine measurement with moderate sensitivity. It is well-suited for many immune response studies, particularly when resources or sample volume are limited. Olink provides superior sensitivity and specificity with a high-throughput and low sample volume requirement. It is ideal for comprehensive, large-scale immune response studies, especially where detecting low-abundance cytokines is critical. However, this comes at a higher cost and requires access to specialized equipment, which may limit its accessibility in certain research settings.