Search
START TYPING AND PRESS ENTER TO SEARCH

The Impact of PBMC Isolation Precision on Study Outcomes

Peripheral blood mononuclear cells (PBMCs) are a fundamental component of many important fields of study, such as immunology, and are central to modern treatment strategies like CAR T-cell therapy1,2. These cells are also used for drug screening, diagnostics, and biomarker discovery, however, variations in PBMC isolation and processing can significantly impede research efforts. Almost all stages of PBMC processing, from transport and storage to isolation and analysis, can introduce confounding variables that yield unreliable results. Furthermore, PBMCs must be isolated from blood, a complex, hazardous mixture of bioactive compounds and other cell types. These factors make obtaining a viable and uncontaminated pool of PBMCs a significant challenge and threaten research success3,4. Here, we discuss the problems that arise during different stages of PBMC processing and highlight the ability of Sampled’s Global Integrated Analytical Biorepository (GIAB) to solve them.

How the Sampled’s GIAB Meets the Challenges of PBMC Isolation

As living cells, PBMCs are vulnerable to many environmental variables. Temperature and time before processing can affect cell viability and impact immunological phenotypes such as cytokine production, which can hamper studies that require statistically reproducible results. Here, we break down the different stages of PBMC isolation and discuss how mismanagement of these processes can ruin studies and lead to long and costly delays in the drug development pipeline. For each challenge discussed, we provide a description of the robust solutions that Sampled’s GIAB offers.

Logistics and Processing

PBMCs are best isolated from whole blood within twenty-four hours of phlebotomy, and that has not been frozen. In multicentre trials, whole blood samples can be subjected to temperatures ranging from  -1 °C to 35 °C, affecting viability4, the effectiveness of cryopreservation5, and important immunological phenotypes4. Time prior to analysis is also an important factor6. Contamination with other cell types such as granulocytes can occur during PBMC isolation and affect T-cell activation7. Thus, robust and reproducible isolation protocols are essential.

How the Sampled’s GIAB enhances PBMC logistics and processing

Streamlined Logistics and Data Storage

We provide optimal transport materials through our custom kitting service and organize a trusted courier to ensure all samples arrive on time and are maintained in optimal conditions. We also ensure accurate and secure data collection and storage and allow clients to view and audit their data via our cloud platform, SampledSphere.

Automated and Manual Processing

At Sampled, we use the Hamilton EasyBlood, a high-capacity automated instrument for PBMC isolation. Automation minimizes human errors and allows samples to be processed simultaneously, preventing inter-run variation and ensuring the isolation of a pure pool of PBMCs. We also have on-site experts to perform manual isolation for challenging, low-volume, or low-cell number samples. This combination allows us to maximize study success.

PBMC Isolation
Automated PBMC Isolation

Sample Handling and Storage

Handling and storage conditions can introduce variables that impact PBMC viability and function. In fact, temperature prior to cryopreservation of PBMCs can significantly impact immunological phenotypes after thawing, such as interferon-gamma production by T-cells5. Lack of suitable storage capacity can mean that precious samples are left to deteriorate and cannot be used for follow-up studies. Large longitudinal studies can require decades of secure dedicated storage space that many facilities cannot provide. Furthermore, having reliable backup plans for power outages and freezer breakdowns can make the difference between study success and needing to start from scratch.

Sampled’s GIAB Provides Handling and Storage Capacity to Meet the Requirements of any Study

Storage

The Sampled GIAB is home to 600 -80 °C freezers with a capacity for 1,000 freezers and adheres strictly to the International Society for Biological and Environmental Repositories standards8. This means no sample is left to deteriorate at suboptimal conditions and we can accommodate large-scale studies that require high-volume, secure storage.

Study Management

We work with clients to immortalize samples, ensuring, where possible, that there is enough left over for future studies. Our broad processing capabilities mean we can process and store multiple important biomolecules from PBMCs, such as DNA, protein, and serum.

Disaster Recovery

Sometimes disasters like freezer malfunctions are unavoidable, and having a detailed disaster recovery plan can help to mitigate risk. Sampled offers an on-call team of technical experts who can retrieve your samples and store them in our freezers with dedicated alarms and multiple backup generators, helping to secure your samples should the worst happen.

Analytical Considerations

Data analysis is where all of the variables introduced in PBMC processing appear, and it can be challenging to determine what results are genuine and which are artifacts. Quality control (QC) is important to determine cell viability and number and can help identify confounding variables that may have been introduced. As such, failure to perform QC can waste time and resources.

How Sampled’s GIAB Enhances Data Analysis

QC

For PBMC counting and viability tests, Sampled uses standardized pre-calibrated single-use cassettes and automated fluorescence microscopy (ChemoMetec Nucleocounter®). We perform plasma QC with the Lunatic spectrophotometer from Unchained Labs and cDrop analysis software. QC saves time and resources by ensuring only good-quality samples are brought forward for analysis.

Dedicated Bioinformatics Team

Sampled has a team of expert bioinformaticians who can use sophisticated analyses that take confounding factors into account. Even if damaging variables have been introduced, our team can salvage your data, allowing you to achieve study success even where best practices were not followed.

PBMCs are pivotal in immunological research and many drug discovery pipelines. However, PBMC processing is fraught with challenges, including variability in isolation and environmental sensitivity, which can compromise research integrity. Sampled’s GIAB addresses these obstacles through streamlined logistics, robust quality control, and advanced storage solutions, ensuring sample viability and purity. By mitigating variables at each processing stage, the Sampled GIAB enhances the reproducibility of immunological research, accelerating drug development and improving study outcomes.

If you want to learn more about how Sampled’s GIAB can help you succeed with PBMC studies, you can check out our latest white paper[1] .

References

  1. Lim WA, June CH. The Principles of Engineering Immune Cells to Treat Cancer. Cell. 2017;168(4):724-740. doi:10.1016/j.cell.2017.01.016
  2. Speiser DE, Chijioke O, Schaeuble K, Münz C. CD4+ T cells in cancer. Nat Cancer. 2023;4(3):317-329. doi:10.1038/s43018-023-00521-2
  3. Serra V, Fiorillo E, Cucca F, Orrù V. Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function. Int J Mol Sci. 2022;24(1):634. doi:10.3390/ijms24010634
  4. Olson WC, Smolkin ME, Farris EM, et al. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. J Transl Med. 2011;9(1):26. doi:10.1186/1479-5876-9-26
  5. Owen RE, Sinclair E, Emu B, et al. Loss of T cell responses following long-term cryopreservation. J Immunol Methods. 2007;326(1-2):93-115. doi:10.1016/j.jim.2007.07.012
  6. Yi PC, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK. Impact of delayed PBMC processing on functional and genomic assays. J Immunol Methods. 2023;519:113514. doi:10.1016/j.jim.2023.113514
  7. Agashe C, Chiang D, Grishin A, et al. Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. J Immunol Methods. 2017;449:23-27. doi:10.1016/j.jim.2017.06.004
  8. ISBER. Accessed February 16, 2024.
  9. Lim WA, June CH. The Principles of Engineering Immune Cells to Treat Cancer. Cell. 2017;168(4):724-740. doi:10.1016/j.cell.2017.01.016
  10. Speiser DE, Chijioke O, Schaeuble K, Münz C. CD4+ T cells in cancer. Nat Cancer. 2023;4(3):317-329. doi:10.1038/s43018-023-00521-2
  11. Serra V, Fiorillo E, Cucca F, Orrù V. Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function. Int J Mol Sci. 2022;24(1):634. doi:10.3390/ijms24010634
  12. Olson WC, Smolkin ME, Farris EM, et al. Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. J Transl Med. 2011;9(1):26. doi:10.1186/1479-5876-9-26
  13. Owen RE, Sinclair E, Emu B, et al. Loss of T cell responses following long-term cryopreservation. J Immunol Methods. 2007;326(1-2):93-115. doi:10.1016/j.jim.2007.07.012
  14. Yi PC, Zhuo L, Lin J, Chang C, Goddard A, Yoon OK. Impact of delayed PBMC processing on functional and genomic assays. J Immunol Methods. 2023;519:113514. doi:10.1016/j.jim.2023.113514
  15. Agashe C, Chiang D, Grishin A, et al. Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. J Immunol Methods. 2017;449:23-27. doi:10.1016/j.jim.2017.06.004
  16. ISBER. Accessed February 16, 2024. https://isber.org