By Dr. Kurt Sales, CSO
Peripheral Blood Mononuclear Cells (PBMC) are critical for the pharmacodynamic assessment of new drug products. They provide crucial information on how immunomodulatory drugs, designed to target immune cells, function in a clinical trial setting and provide drug developers with that crucial mode of action data required to prove efficacy.
In a clinical trial setting, quality and consistency in PBMC preparation are paramount for the generation of quality data. Agilex Biolabs has a network of laboratories throughout Australia to support PBMC processing from whole blood using a single Standard operating procedure (SOP). Recently Agilex participated in the Quality Assurance Program (QAP) coordinated by the Immunovirology Research Network (IVRN) of the Australian Centre for HIV and Hepatitis Virology (ACH4) and has attained certification for the isolation and cryopreservation of PBMCs.
What are PBMCs and why are they important?
PBMCs are the proportion of immune cells in circulating whole blood immune cells that have a single round nuclei. This population is heterogenous, comprising mainly of T cells, B cell, Natural Killer (NK) cells and monocytes and derivatives of each of these cell types. PBMC are typically used for flow cytometry and Elispot analysis of immune function, however may also be used for genetic analysis and, after lysates are made from them, ligand binding and Western blot analysis.
PBMC isolation from whole blood is a relatively uncomplicated process, unchanged over decades, typically involving the harvest of diluted whole blood after it has been layered over a medium of known density (e.g. Ficoll). The density medium separates erythrocytes (red blood cells) and cells with multi-lobed nuclei (e.g .neutrophils) from lymphocytes and monocytes. Several adaptions have more recently been implemented that can make the isolation process even quicker and easier, however it is important to note that these may lead to reduced quality in terms of viability and / or total cell number. PBMC viability and cell count are best when they are isolated relatively quickly from whole blood. However a primary benefit of PBMC is their amenability to long term storage, and there are well established protocols that permit PBMC to be stored in liquid nitrogen for years after isolation. This long-term storage permits batch analysis of PBMC from longitudinal clinical trials, as we have previously demonstrated (1-5). A further benefit of working with PBMC includes reduction of background that can be evident with certain assays such as long- term cultures and some ligand binding assays.
It is important to note that not all studies are suited to PBMC collection for several reasons as outlined below:
A) Cell types: By definition, cells that have multiple nuclei such as granulocytes (e.g. neutrophils, basophils and eosinophils) and cells that do not have a nuclei (e.g erythrocytes and platelets) are not included in PBMC. In practice basophils are closer to the density of lymphocytes than granulocytes and may be collected when harvesting PBMC, but are very rare in healthy subjects.
B) Activation / proliferation status: The activation and proliferation status of cells is readily measured by analysis of markers including CD69 and Ki67 by flow cytometry. Once stored and thawed, PBMC are quiescent and expression of these markers is low. Often, we need to culture and stimulate the PBMCs after thawing in order to drive signal.
C) Marker downregulation / degradation: PBMC isolation has been shown to reduce expression of some markers (6).
D) Loss of drug: Drug is typically washed from the matrix during the PBMC isolation process. This loss has obvious implications for receptor occupancy type analysis, but may also affect activation markers on immune cells.
E) Blood volume: PBMC isolation by conventional means typically requires relatively high blood volumes as harvesting involves visualization of the PBMC layer with the naked eye. While flow cytometry analysis can get by with as little as 50µL of whole blood, PBMC isolation typically requires at least 9mL whole blood. This can limit their use in longitudinal clinical trials where there are multiple timepoints requiring blood draws. We have recently demonstrated that ELISpot can be performed with cells isolated from whole blood, reducing the requirement for PBMC for this type of assay. Furthermore, newer technologies are being developed that promise to substantially reduce the volume of blood required for PBMC isolation (7). However, optimisation of processes downstream of PBMC isolation, such as freezing, are yet to be developed and these technologies are likely some time away from use in clinical trials.
Agilex Scientists have decades of experience in PBMC isolation, preservation and downstream analysis of immunological markers by Flow Cytometry, ELISpot and PCR, to name but a few. Our recent certification through the IVRN QAP solidifies our expertise in handling PBMCs and supporting clinical studies in Australia. As outlined above, whilst PBMCs can provide useful data for determining efficacy and pharmacodynamics of the drug in a clinical study, there are limitations associated with their use. It is therefore critical to engage with a knowledgeable partner like Agilex Biolabs when planning the PBMC and pharmacodynamics assessments in your clinical study to ensure that the data will be fit for purpose and robust.
Contact Agilex Biolabs
To learn more about the applications that Agilex can support downstream of PBMC isolation, please contact our Team today for a confidential discussion at BD@agilexbiolabs.com.
References
1. Mavrangelos C, Campaniello MA, Andrews JM, Bampton PA, Hughes PA. Longitudinal analysis indicates symptom severity influences immune profile in irritable bowel syndrome. Gut. 2018;67(2):398-9. doi: 10.1136/gutjnl-2017-314308.
2. Costello SP, Hughes PA, Waters O, Bryant RV, Vincent AD, Blatchford P, Katsikeros R, Makanyanga J, Campaniello MA, Mavrangelos C, Rosewarne CP, Bickley C, Peters C, Schoeman MN, Conlon MA, Roberts-Thomson IC, Andrews JM. Effect of Fecal Microbiota Transplantation on 8-Week Remission in Patients With Ulcerative Colitis: A Randomized Clinical Trial. JAMA. 2019;321(2):156-64. doi: 10.1001/jama.2018.20046.
3. Mikocka-Walus A, Hughes PA, Bampton P, Gordon A, Campaniello MA, Mavrangelos C, Stewart BJ, Esterman A, Andrews JM. Fluoxetine for Maintenance of Remission and to Improve Quality of Life in Patients with Crohn’s Disease: a Pilot Randomized Placebo-Controlled Trial. J Crohns Colitis. 2017;11(4):509-14. doi: 10.1093/ecco-jcc/jjw165.
4. Hughes PA, Moretta M, Lim A, Grasby DJ, Bird D, Brierley SM, Liebregts T, Adam B, Blackshaw LA, Holtmann G, Bampton P, Hoffmann P, Andrews JM, Zola H, Krumbiegel D. Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients. Brain Behav Immun. 2014;42:191-203. doi: 10.1016/j.bbi.2014.07.001.
5. Hughes PA, Harrington AM, Castro J, Liebregts T, Adam B, Grasby DJ, Isaacs NJ, Maldeniya L, Martin CM, Persson J, Andrews JM, Holtmann G, Blackshaw LA, Brierley SM. Sensory neuro-immune interactions differ between irritable bowel syndrome subtypes. Gut. 2013;62(10):1456-65. doi: 10.1136/gutjnl-2011-301856.
6. Capelle CM, Cire S, Ammerlaan W, Konstantinou M, Balling R, Betsou F, Cosma A, Ollert M, Hefeng FQ. Standard Peripheral Blood Mononuclear Cell Cryopreservation Selectively Decreases Detection of Nine Clinically Relevant T Cell Markers. Immunohorizons. 2021;5(8):711-20. doi: 10.4049/immunohorizons.2100049.
7. Alsved J, Rezayati Charan M, Ohlsson P, Urbansky A, Augustsson P. Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing. Sci Rep. 2024;14(1):8748. doi: 10.1038/s41598-024-59156-7.
