Papers and reviews
Cacho-Navas, C., et al. Simultaneous Quantification of Multiple Immune Checkpoint Interactions in Melanoma. Journal of Clinical Medicine. 2026
Gumuzio, J., et al. Immune Checkpoint Crosstalk: CTLA-4/CD80 Engagement as a Predictor of Anti-PD-1/PD-L1 Therapy Outcome in NSCLC. Frontiers in Immunology. 2026
Sánchez-Magraner, L., et al. Functional Engagement Of The PD-1/PD-L1 Complex But Not PD-L1 Expression Is Highly Predictive Of Patient Response To Immunotherapy In Non–Small-Cell Lung Cancer. Journal Of Clinical Oncology. 2023
Whitepapers
Immune Checkpoint Interaction Quantification By QF-Pro® Whitepaper
Quantitative Analysis Of Β-Catenin / E-Cadherin Interaction
Post-translational modification. Identification of Phosphorylation Activated States in Protein
Scientific Posters
Advance Translational Research By Quantifying Protein Functions In Tissue With QF-PRO
Immune Checkpoint Crosstalk: CTLA-4/CD80 Engagement as a Predictor of Anti-PD-1/PD-L1 Therapy Outcome in NSCLC
Preliminary results on PD-1/PD-L1 axis engagement among PD-L1–negative NSCLC patients receiving chemoimmunotherapy
Got any questions about QF-Pro®
What does the QF-Pro ® Score actually measure?
The QF-Pro® Score is a quantitative measurement expressed as percentage FRET (% FRET). It represents the proportion of labelled molecules within a region of interest in which the two target proteins are physically interacting — within a distance of 1–10 nm — at the moment of measurement.
A high score indicates a high degree of functional interaction between the two targets, regardless of their individual expression levels. This makes it a true functional biomarker — not a measure of how much protein is present, but of whether that protein is actively engaged.
Why measure fluorescence lifetime instead of fluorescence intensity?
Intensity-based readouts — including standard IHC and IF — measure how much light a labelled sample emits. That signal is inherently affected by antibody concentration, staining efficiency, tissue thickness, and photobleaching, making it difficult to standardise across samples, cohorts, and laboratories.
Fluorescence lifetime measures how long a fluorophore remains in its excited state — a physical property of the molecule that is independent of how much label is present. It only changes detectably when a donor and acceptor fluorophore are within 1–10 nm of each other, as occurs during FRET. This is why lifetime is the right signal for detecting molecular interactions: it cannot be mimicked by high expression alone.
In practice, this means QF-Pro® data is reproducible across tissue preparations, cohort sizes, and laboratory settings — a prerequisite for any biomarker intended for clinical use.
How does QF-Pro ® compare to the proximity ligation assay (PLA)?
PLA detects proximity between proteins, not confirmed molecular interaction. The distances it measures can exceed 40 nm — well above the 1–10 nm range of genuine protein-protein interactions — which can produce false positive results. PLA also generates a semi-quantitative, dot-count readout that is difficult to standardise across cohorts.
QF-Pro® uses fluorescence lifetime to confirm actual binding at 1–10 nm resolution and delivers a continuous quantitative score (% FRET) that is reproducible across samples, sites, and operators.
How does QF-Pro ® compare to homogeneous time-resolved FRET (HTRF)?
HTRF measures FRET in the time domain within homogenised samples. By destroying tissue architecture, it cannot spatially map protein interactions or functional events across a sample — making it unsuitable for translational research or clinical applications that require spatial context.
QF-Pro® quantifies protein functionality with spatial resolution directly in intact FFPE tissue, preserving the cellular and microenvironmental context that is essential for biomarker validity in patient cohorts.
What sample types does QF-Pro ® work with?
QF-Pro® is validated for use with formalin-fixed paraffin-embedded (FFPE) tissue sections and cell pellets, as well as 96-well plate cell models. Its compatibility with archival FFPE material is a key advantage — enabling analysis of both retrospective clinical cohorts and prospective clinical trial
samples without the need for fresh or frozen tissue.
Do the QF-Pro ® reagent kits come with primary antibodies?
No. Kits include the secondary binding reagents and all other reagents required for QF-Pro® labelling. The choice of primary antibodies is left to the end user based on the biomarker(s) of interest. Our customer support team and distributor network can assist in selecting appropriate antibodies for any QF-Pro® assay.
What primary antibodies can I use with QF-Pro ®?
QF-Pro® secondary binding reagents are compatible with mouse and rabbit primary antibodies. One binding site must be labelled with a mouse primary antibody (donor labelling) and the other with a rabbit antibody (acceptor labelling). Monoclonal antibodies are recommended where possible, though polyclonal antibodies have been used successfully. Antibodies optimised for IF/ICC/IHC are preferred, but others can also work with additional end-user validation.
Can I multiplex with QF-Pro ®?
Yes. Each QF-Pro® assay delivers up to three protein expression readouts (e.g., PD-1, PD-L1, and CD3) alongside one functional quantification (e.g., PD-1/PD-L1 interaction score) within the same sample. QF-Pro® also supports up to four sequential rounds of multiplexing on a single sample, enabling up to twelve biomarker expression readouts and four independent functional interaction profiles from one tissue section.
Is QF-Pro ® affected by tissue autofluorescence and background?
No. A proprietary amplification step raises the signal-to-noise ratio of QF-Pro® samples to 4 or above, enabling accurate detection of QF-Pro® scores even in tissues with significant autofluorescence. The software also incorporates automated thresholding algorithms that remove autofluorescence artefacts from the final dataset.
How long does the QF-Pro ® workflow take?
The complete workflow — labelling, acquisition, and analysis — is typically completed within 2–3 days. Labelling follows a standard immunofluorescence protocol: either two days (primary antibody incubation overnight at 4°C) or one day (2 hours at room temperature). Acquisition on Violet 3.0 takes approximately 8 minutes per FFPE slide or 3 minutes per well. Analysis is guided and automated by the QF-Pro® Software.
How many samples can I process with a QF-Pro ® Kit?
Each kit contains reagents to label 100 FFPE slides — equivalent to 50 patients, each requiring one donor slide and one donor-acceptor slide — or one 96-well plate.
Can QF-Pro ® support companion diagnostic (CDx) development?
Yes. QF-Pro® is designed to support the full CDx co-development pathway alongside new therapeutics. The platform delivers a CDx-grade quantitative readout — the QF-Pro® Score — that supports patient stratification inside clinical trials and carries through to CDx approval and routine
diagnostic deployment on the same assay.
The fully integrated workflow — one reagent kit, one acquisition system, one analysis software — ensures the measurement is standardisable across sites and operators, meeting the reproducibility requirements of regulatory bodies such as FDA and EMA. HAWK works directly with pharma and biotech partners to co-develop CDx assays aligned with their therapeutic programs.
Do I need to purchase the Violet 3.0 imaging system to use QF-Pro ®?
Violet 3.0 is the recommended and fully integrated solution for QF-Pro® assays. It is purpose-built around frequency-domain FLIM (FD-FLIM) and validated end-to-end with the QF-Pro® Reagent Kits and Software.
Customers with existing FD-FLIM hardware — such as systems using PCO.flim-compatible detectors — may be able to run QF-Pro® measurements using third-party equipment, as the software is partially decoupled from the hardware. However, most standard microscopes do not include the FD-FLIM detectors or light sources required, making Violet 3.0 the practical choice for most labs.
Can I use Violet 3.0 for standard immunofluorescence experiments?
Yes. Violet 3.0 functions as a widefield fluorescence microscope with a multichannel LED system, and is fully compatible with standard immunofluorescence protocols across four excitation channels (365 nm, 470 nm, 550 nm, and 635 nm). This means it can serve as the lab’s primary imaging platform for both QF-Pro® assays and conventional IF experiments.
Does Violet 3.0 require any special laboratory conditions?
No. Violet 3.0 is designed for standard laboratory bench installation. All required cooling and light-sealing is built into the system, and a water-cooled detection sensor ensures consistent performance under any environmental conditions.
How automated is the QF-Pro ® workflow?
The QF-Pro® Software guides users through the entire process from sample loading to final data report. Once samples are loaded and regions of interest selected, Violet 3.0 acquires them automatically and sequentially. Data processing — including automated thresholding and artefact removal — runs without manual intervention, with the option to fine-tune parameters if needed.
The output is a structured report ready for export and statistical analysis.
Do I need bioinformatics expertise to analyse QF-Pro ® data?
No. The QF-Pro® Software is designed to be accessible to any life science researcher, with no coding, scripting, or bioinformatics background required. The analysis pipeline is guided and largely automated, and the final output is a structured data report compatible with standard tools such as GraphPad Prism or Excel.