Pick Preferred Specimens to Perfect Immune Profiling

Simple, underappreciated solutions for reliable flow cytometry data and longitudinal study design

Complex problems require custom and creative solutions. Immune profiling is essential in developing many therapeutics, including cell and gene therapies. Consider the lightning-fast development of mRNA therapeutics and vaccines to treat and slow the spread of COVID-19. Clinical and translational researchers employed countless immune profiling experiments to describe a detailed picture of how the SARS-CoV-2 virus, the immune system, and various treatments interact. Accordingly, the growing prevalence of immune profiling over the last decade is evidenced by an ever-increasing number of publications and the allocation of a $180 million research budget for immune profiling studies by National Institutes of Health (NIH) research in 2020 [Pushparaj 2020].


The Gold Standard of Immune Profiling

Fortunately, researchers have several techniques at their disposal for profiling, characterizing, and interrogating various components of the immune system and their interrelationships. Commonly employed techniques include next-generation sequencing (bulk and single-cell approaches), multiplex assays, and flow cytometry. 

Flow cytometry may be considered the gold standard of immune profiling due to its unique ability to identify, sort, and quantitatively characterize subsets of fluorescently labeled immune cells within heterogeneous cell populations. These features have established flow cytometry as a critical tool in discovery, translational, and clinical biomarker research, as well as for evaluating the quality of cell & gene therapy manufactured products. As flow cytometry continues to be adopted across the drug development pipeline, its market size is projected to increase from $6.2 billion in 2022 to a whopping $16.6 billion by 2032 [Shubham 2023]. 

Given the importance of flow cytometry in immune profiling applications, researchers must improve the quality and variability of flow data and optimize study design to allow for more robust and informative conclusions. Emphasizing consistency in sample collection, handling, and processing can reduce the number of study participants needed for biomarker elucidation and validation, particularly when longitudinal and multiple sample collection study designs are incorporated. 

While the advent of increasingly sensitive and accurate instruments for flow cytometry assays has improved data quality and reduced expert hands-on time, an often overlooked and underlying “real” source of variability is from the samples themselves. As a result, there has been a push among researchers and regulatory agencies such as the National Institute of Standards and Technology (NIST) to adopt standardized approaches to sample preparation for flow cytometry experiments within therapeutic development.


Quality Samples, Quality Data

High-quality flow cytometry data starts with the source; your data are only as good as the samples you evaluate. Inconsistencies in sample quality due to uncontrolled differences in acquisition, delivery, storage, and processing methods can have unintended effects on the accuracy and variability of results, leading to weaker conclusions and limited translational potential.

Recent studies have documented how inconsistencies in sample collection represent valid concerns with real consequences impacting flow cytometry results. For instance, the method and duration of peripheral blood mononuclear cell (PBMC) cryopreservation, a common practice for identifying immune cell clinical biomarkers, alter immune cell population, viability, and reactivity in subsequent flow cytometry analysis, degrading the accuracy of results [Li 2022, Ticha 2021]. Additionally, PBMC processing delays as short as 24 hours can contribute to increased granulocyte contamination in flow cytometry analysis, highlighting the need for consistent and timely sample processing post-collection [Yi 2023]. In this case, Sanguine’s onsite prospective collection services enabled researchers to process samples in as little as 2 hours post-collection, showcasing the utility of prospective collection methods that circumvent processing delays and prolonged storage of PBMCs. 

Despite the clear benefits of prospective sample collection for subsequent flow cytometry analysis, most researchers procure specimens from biobanks, limiting standardization and control over collection and processing parameters. Moreover, such indirect approaches to procurement are less convenient for patients and healthy donors, requiring them to donate at medical facilities rather than in their homes or work locations, limiting their ability to reach a geographically and demographically diverse donor network. Taken together, the direct-to-patient strategy benefits translational research by considering the needs of both the researcher and the patient in producing more informative and meaningful conclusions.


Longitudinal Study Design for More Informative Immunoprofiling

Employing longitudinal studies in therapeutic development offers greater statistical power from fewer study participants while allowing researchers to draw more powerful conclusions based on real-world data resembling subsequent clinical trials. Longitudinal studies have also proven helpful for flow cytometry-based immune profiling methods in therapeutic development. For example, a recent study utilized a longitudinal approach to characterize changes in B and T-cell responses in multiple sclerosis (MS) patients following a booster dose of a COVID-19 mRNA vaccine. In analyzing whole blood from MS and healthy control subjects by flow cytometry at six months post primary vaccine and 4-6 weeks post booster, researchers showed that MS patients presented higher levels of terminally differentiated CD4+ and CD8+ effector memory cells compared to healthy controls [Aiello 2023]. These observations demonstrated mRNA vaccine efficacy in preventing COVID-19 in MS patients, shedding light on the potential value of administering such therapies in MS patient populations.

A major challenge in conducting adequate longitudinal studies is limited access to recallable donors. In response, Sanguine developed a direct-to-patient approach, including onsite and at-home collection services, to provide a more streamlined and convenient sample procurement experience, making patient retention and recallability more feasible. Moreover, the flexibility of this approach enables the engagement of extensive networks of patient donors by putting their needs first, further improving the robustness of longitudinal studies.


Simultaneous Sample Collection

The immune system is a complex orchestration of various cells and cytokines that cannot be interrogated with a single assay or sample type. While flow cytometry of blood samples and PBMCs is one of the most common approaches to immune profiling, researchers typically require complementary assays to characterize immune profiles for therapeutic development. For example, Yi and colleagues in the study above on PBMC processing delays utilized single-cell RNA sequencing and ELISpot to identify changes in gene expression and interferon-γ secreting cells from stimulated PBMCs ex vivo, respectively [Yi 2023]. 

Immunoprofiling analysis often requires multiple types of samples in addition to PBMCs. A team from Sana Biotechnology published a 2023 study focused on editing primary pancreatic islet cells (p-islets) from type 1 diabetes mellitus donors (T1DM) to improve their suitability for therapeutic engraftment. While allogeneic engraftment of p-islets has been explored to treat T1DM, such treatments often elicit an immune response from the host, leading to rejection. In this case, donor p-islets were engineered to overexpress CD47, rendering the cells hypoimmune and mitigating rejection upon transplantation. To evaluate host immune response and function to the engineered allogeneic p-islets, the team generated a preclinical, humanized T1DM mouse model using PBMCs and sera from T1DM patients in Sanguine’s donor community [Hu 2023]. Interestingly, the researchers used flow cytometry to confirm a greater abundance of CD47+ cells within engineered p-islets compared to autologous controls following transplantation, indicating their potential to retain hypoimmune properties after engraftment.


Bringing the Patient Experience to Flow Cytometry Assays

To improve the value of immune profiling experiments, Sanguine has developed a series of innovative yet simple solutions that give researchers greater confidence in their data and results. Sanguine addresses concerns related to unintended sample variability by empowering researchers to dictate and standardize rapid and flexible sample procurement and processing procedures of various sample types, including blood, PBMCs, leukopaks, saliva, stool, and urine. Moreover, Sanguine provides unparalleled access to patient data and sample procurement from engaged, large, and diverse healthy and disease-state donor populations, giving researchers the flexibility and capability to conduct robust prospective and longitudinal immune profiling studies.


Find out more about how Sanguine can help you power more precise and practical flow cytometry and immune profiling assays for your research needs here. 


By: William Lawrence, Ph.D.; Geocyte 



[1] Pushparaj, PN. (2020) Chapter 11 – Translational interest of immune profiling. Precision Medicine for Investigators, Practitioners and Providers. 105-122 DOI: https://doi.org/10.1016/B978-0-12-819178-1.00011-3

[2] Shubham, S. (2023). Flow Cytometry Market Research 2032. Allied Market Research Group https://www.alliedmarketresearch.com/flow-cytometry-market

[3] Li, B. (2022) Comprehensive evaluation of the effects of long-term cryopreservation on peripheral blood mononuclear cells using flow cytometry. BMC Immunology DOI: 10.1186/s12865-022-00505-4

[4] Ticha, O. (2021) Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations. Journal of Immunological Methods. DOI: 10.1016/j.jim.2021.113081

[5] Yi PC. (2023) Impact of delayed PBMC processing on functional and genomic assays, Journal of Immunological Methods. Volume 519, 113514, ISSN 0022-1759, DOI: https://doi.org/10.1016/j.jim.2023.113514

[6] Aiello A. (2023) Longitudinal characterisation of B and T-cell immune responses after the booster dose of COVID-19 mRNA-vaccine in people with multiple sclerosis using different disease-modifying therapies. Journal of Neurology, Neurosurgery & Psychiatry. Volume 94:290-299 DOI: 10.1136/jnnp-2022-330175

[7] Hu X. (2023) Human hypoimmune primary pancreatic islets avoid rejection and autoimmunity and alleviate diabetes in\allogeneic humanized mice. Sci Transl Med. Vol 12;15(691) DOI: 10.1126/scitranslmed.adg5794