Spectral flow cytometry and the growing array of commercially available fluorophores have led to the development of increasingly complex immunophenotyping panels.  Generating robust data depends not only on a strong panel design but also on the use of appropriate reference controls and a solid understanding of spectral unmixing principles.  Spectral unmixing is a critical component of successful spectral flow cytometry.  So essential, in fact, that KCAS Bio scientist, Nassouh Mourabet, recently presented on this topic, at AAPS NBC, in a presentation entitled “Unmixing Impact Comparison Across Immunophenotyping Panels.”  

In this blog post, we will review critical aspects of spectral unmixing and highlight KCAS Bio studies that evaluated the stability of reference controls for a validated TBMNK and a validated T-subset panel. By mastering and understanding these elements, researchers can maximize the potential of spectral flow cytometry, ensuring comprehensive and accurate multicolor analyses in their studies.

Spectral Overlap and Unmixing

Immunophenotyping by flow cytometry enables researchers to classify and understand different cell populations according to the protein expression on or within cells. This is achieved using fluorochrome-labeled antibodies directed against antigens of interest. A common challenge in immunophenotyping assays run on conventional flow cytometers is spectral overlap, which occurs when fluorescent light from one or more markers overlaps with the emitted light of another fluorochrome-labeled marker.  This overlap reduces the sensitivity of detection and can hinder appropriate distinction of individual cell populations. Careful fluorochrome selection and panel configuration can address some aspects of spectral overlap. In Conventional flow cytometry, compensation is employed to correct for overlap.

However, in spectral flow cytometry, where panels can include up to 40 fluorochromes, the entire emission spectrum is collected allowing unique signatures identification. Spectral overlap is corrected using single color reference controls and unmixing algorithms that separate the overlapping signals. The efficiency and accuracy of these algorithms can vary depending on the panel complexity and data quality.  While a detailed discussion of unmixing algorithms is beyond the scope of this post, it is essential to note that various algorithms can be used to account for complex interactions between fluorophores and can leverage statistical methods to estimate the signal contribution of each fluorophore.

Considerations for Single Color Reference Controls

Several key factors of the method design and reference control setup can affect unmixing accuracy.  These factors include:

Panel Complexity and Fluorophore Section: Panels with more markers or complex configurations may require more sophisticated unmixing techniques.  In panels with higher degrees of overlap between fluorophore emission spectra, the unmixing can be challenging.  Mourabet et. al., explored the impact of fluorophore selection, specifically the use of tandem dyes in the KCAS Bio TBMNK and T-subset panels, which are validated for use with human PBMCs.

Tandem dyes consist of two covalently bound fluorescent molecules (a donor and an acceptor) and can pose challenges in spectral unmixing.  When the tandem dyes break down, the donor fluorochrome bleed-through increases, shifting the spectral signature from a tandem signature to a donor signature. Mourabet’s work evaluated the signal stability of individually stained single color reference controls for PerCP-Cy5.5, Pe-CY7 and, APC-H7.  The tandem breakdown was tracked over time using the MFI ratio of the donor to tandem peak channel.  With proper fixation and storage, the tandem dye reference controls were stabilized for up to five days. In future studies, longer term stability, beyond five days will be evaluated.  This finding highlights an important consideration when incorporating tandem dyes into a panel, as the longevity of the reference control set may be directly impacted by the fluorophores used. 

Use of Fixatives: The fixation method is another essential consideration in preparing single-color reference controls.  The type of fixation can impact the length of time that the single-color reference controls remain effective for use.  Mourabet and his team evaluated reference controls treated with different fixatives, including 0.5% formaldehyde, 4% formaldehyde, Stabilizing Fixative and CytoFix. In total, 16 fluorochrome conjugated antibodies were evaluated, in four fixatives over three timepoints. While all fixatives maintained the integrity of each biomarker on day zero, only 0.5% formaldehyde maintained the integrity of every biomarker in the two panels out to five days.  This finding is significant as it allows one set of reference controls to be saved and reused during a known period.  This may be particularly advantageous during method validations when the processed stability of samples is being evaluated.

Long Term Stability of Reference Controls: Beyond understanding the impact of fluorochrome and fixative selection, the KCAS Bio team was also interested in the long-term stability of the reference controls. To investigate this, reference controls and samples were acquired at a day-zero baseline, unmixed, and analyzed. At one- and two-months post baseline, replicate studies were conducted staining samples and reference controls with the same antibody lot used at baseline. The samples were acquired, and the data was unmixed and analyzed.

When month-one and month-two samples were unmixed with reference controls acquired either at the day-zero baseline or freshly prepared at month one or month two, the data was comparable. This suggests that when the same antibody lots are used within their expiry period, the same reference controls can be effective for unmixing. 

Mourabet and his team are continuing to explore additional timepoints, with plans to evaluate up to nine months from baseline. A firm understanding of reference control stability is important when considering sample acquisition during long-term studies.

Recommendations and Best Practices

Implementing best practices for unmixing in spectral flow cytometry is crucial to ensure the accuracy and reliability of the data generated in spectral flow cytometry. A few takeaways:

  • Choose Appropriate Fluorophores: Selecting fluorophores with minimal spectral overlap can simplify unmixing.  Making informed choices during panel design and optimization should involve balancing the number of markers with the complexity of the unmixing process.
  • Choose the Appropriate Fixative: It is key to select an appropriate fixative for use with your reference control set. Proper fixative selection will ensure that the integrity of the biomarker is maintained and may also impact the stability of the reference set.
  • Regular Instrument Calibration:  Instrument calibration and maintenance can never be overlooked.  While not discussed here, properly calibrating the flow cytometer is crucial for accurate unmixing.  Ensure that your flow cytometer is properly calibrated to maintain data accuracy.
  • Using Advanced Unmixing Algorithms: Apply the most effective unmixing algorithms based on the specific requirements of the study. There are a variety of algorithms for use and selecting the appropriate algorithm is critical for your data analysis.

Final Thoughts

The insights provided by KCAS Bio’s Nassouh Mourabet and team are valuable for anyone involved in immunophenotyping by spectral flow cytometry.  The exploration of fluorochrome and fixative selections highlights the importance of having a well understood reference control set and the importance of unmixing techniques in obtaining accurate and meaningful results.

As spectral flow technology evolves, the need for accurate and reliable unmixing methods will only grow. Ongoing research will be critical for overcoming the challenges associated with spectral overlap and improving the overall quality of immunophenotyping data. By staying informed about the latest advancements and best practices, researchers can ensure their studies yield the most accurate and insightful results.

Want to learn more? Reach out and schedule a discussion today to learn how we can support your spectral flow cytometry needs.