Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays

Belouski, S. S.*, Wilkinson, J.^, Thomas, J.*, Kelley, K.*, Wang, S.-W.*, Suggs, S.* and Ferbas, J.* (2010), . Cytometry, 78B: 59–64.

doi: 10.1002/cyto.b.20492

The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.

Author Information:

* Amgen Inc., Department of Medical Sciences

^ Beckman Coulter Inc., Custom BioPharma Group, Miami, Florida (author Julie Wilkinson current address is ImmunoSite Technologies, Fort Lauderdale, FL)

Email: John Ferbas (jferbas@amgen.com)

*One Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks 91320-1799, CA

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Until the investment is made to identify these surrogates, the marketplace will be condemned to relive the current paradigm of high failure rates, very high overall costs associated with vaccine and drug development, few therapies reaching patients with needs, and very slow changes in prevention, morbidity and mortality rates.  Identification of a few surrogate markers in this field would have a profound effect on all of the above and would be a wise investment.

As the search continues for surrogates to determine vaccine efficacy and therapeutic response,   the utility of cell mediated immunity (CMI) assays in the assessment of immune response or immunogenicity is increasing significantly.

Once critical assay reproducibility and robustness of data has been established, what needs to be done in relevant clinical trial settings to identify immune markers that can be used as surrogates of efficacy?

ELISPOT, ICS assays or both to determine CMI?

There are several advantages to using Enzyme-linked Immunosorbent Spot (ELISPOT) assays to interrogate cell functionality.  ELISPOT assays do require fewer cells compared to intracellular cytokine staining (ICS) assays and there have been studies that have shown comparability of fresh to frozen PBMCs in ELISPOT validation studies. Both these parameters are critical for the logistics of running clinical trials. ELISPOT is also an easier assay to run compared to ICS (ELISA with cells), however, there continues to be issues with precision in both assays being run manually with acceptable CVs being as high as 70%.  (Ref: Clin Vaccine Immunol. 2009 February; 16(2): 147–155 Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents (CVs of 30% or less but some as high as 70% for positivity 50spots/10 6 cells), and J Immunol Methods. 2011 Jan 5;363(2):143-57. Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing. (CVs of less than 35% for 0.2% and higher positivity).

A key disadvantage of ELISPOT is the inability for polyspectral immunophenotyping using lineage-specific markers to identify the responding cell populations. Given the publications in the past five years on the association of long-term non-progression in HIV positive individuals with higher polyfunctionality of the cellular response, it is anticipated that these are the kinds of studies that will need to be conducted in large scale clinical trials to determine if vaccine and therapeutic strategies elicit similar responses. (Ref: HIV nonprogressors preferentially maintain highly functional HIV-specific CD8 T cells; Blood 2006;107:4781-4789)

Recent publications in the field have identified biomarkers of tolerance in transplant patients that have required a holistic systems biology approach (combining gene expression-secreted protein profiling, polyfunctional flow cytometry evaluations as well as ELISPOT evaluations). (Ref: J Clin Invest. 2010 Jun 1;120(6):1848-61, Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans)

Can CMI assays be performed in a more cost conscious manner?

Both ELISPOT and polyspectral flow cytometry (PSFC) assays are more complex than most of the assays used in current clinical trial settings.  First, both assay formats are configured to assess function and not just presence of cell types.  And, although we would like to identify a single marker as a surrogate, many studies now predict that there will be a mosaic signature, whether it is a cellular or gene expression assay.  In some initial studies with clinical trial organizations, ImmunoSite Technologies (IST) used up to 60 different five-color combinations as screening tools to dissect the immune response and to find the candidate markers of choice.

Once the candidate marker cocktails have been identified and validated, the cost associated with testing can be significantly reduced.  Plus, the automation of these assays has not only reduced associated labor costs, but has improved reproducibility and has significantly reduced the cost of retesting of samples.  All of these progressive steps have reduced sample testing costs while at the same time improved assay results.

Can the paradigm be changed?

Given critical assay reproducibility and robustness of data, would not a holistic approach be best to identify immune markers that can be used as surrogates of efficacy in relevant clinical trial settings?  And given that the right screening can identify the immune response and find candidate surrogate markers of efficacy, should not investment be made to identify these markers?

Tuesday, March 29, 2011 – Please join in with the topical discussions about the newest trends in clinical drug development, and learn strategies to avoid costly mistakes and improve efficiency.  The focus of this BioFlorida SE Chapter-sponsored event is on advancing biomedical products through the clinical development process, and is being led by Planning Committee Chair Mike Keller of McDermott Will & Emery.  A nationally recognized panel of speakers will be providing an overview of clinical drug development for the novice, as well as providing high level insights for the experienced life sciences professional.

ImmunoSite Technologies, LLC will be participating at this important event.  Contact two of the IST owners attending, Dr. Carlos Aparicio and Julie Wilkinson, to arrange one-on-one discussions about the criticality of immune monitoring and immune surveillance during drug development, or just meet them there for impromptu discussions on other thought-provoking topics.

Tuesday, March 29, 2011

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What Do You Seek In A Contract Research Partnership?

In a time of tight budgets and careful examination of the cost/benefit of partnering with a contract research service provider, ImmunoSite Technologies, LLC (IST) offers a rare combination of experience, knowledge, responsiveness, and trustworthiness.  IST knows what clients need to be successful, and how to provide the highest quality in service.  Clients can depend on ImmunoSite Technologies to be the ideal partner that helps accelerate their success without encumbering their resources.

Cost efficiency – As R&D costs escalate, you need a CRO partner that fits well with your business from a cost/value point-of-view, as well as a shared values point-of-view.  IST understands this and is ready to work with you to craft a partnership that works for you from a time, resource, and cost perspective, all the while delivering the results you need.  IST demonstrates value in the services they provide, by getting the work done quickly and on budget without sacrificing quality.

Knowledge and experience – Backed by years in pharmaceutical, biotech, and diagnostics product development, IST knows the challenges of developing the right assay to get the accuracy, precision and reproducibility needed to succeed.

Compatibility and communication – Essential to a great partnership, is a high level of compatibility and great communications.  IST’s professionals understand this.  You need a team that understands your company and your goals, your particular research objectives and challenges.  You need professionals that will work with you to plan, implement and work through all challenges along the way.  The team at IST has partnered with organizations from around the globe so they know the value of accurate communications and a shared vision to bring creative solutions to fruition, while fostering long-term and productive relationships.

Quality – Outsourcing your assay automation project to a qualified partner like IST is a key strategy for ensuring quality.  In a business where service, precision, accuracy, and timeliness are the key deliverables, IST stands apart by putting the “service” back into the service provider partnership.  Quality is central to their work and the unique value they deliver.  The IST quality system is in compliance with GxP guidelines and international safety and regulatory requirements.

Specialization in assay automation – This is IST’s niche and they provide the unique service that validates the automated assay performance with the client’s samples before the system ever leaves the ImmunoSite Technologies facility.  This offers great peace of mind to their clients who are investing significantly in automation to enhance their productivity, but don’t have the time or people to dedicate to implementing a new automated assay project.  IST has designed and developed automated systems for handling biological specimens from live cells to protein to nucleic acids.  Automation doesn’t have to fill a room to meet your needs.  Engineers at IST are cognizant of space and budget requirements when designing the best system for a client so the solution can be a simple benchtop or hood-sized system.

Explore a Partnership With ImmunoSite Technologies

If great service from a dependable, trustworthy, and experienced partner is what you are seeking for your assay automation project, then contact IST now. The technical experts and management team at ImmunoSite Technologies will meet with you to explore the fit between your company, your project needs and IST capabilities.  When it is a great fit, everybody knows.  IST assay automation services

IST quite literally helped “write the book” on cell analysis testing.  They were asked to contribute a chapter of a book titled Validation of Cell-Based Assays in the GLP Setting: A Practical Guide, by authors/editors Uma Prabhakar and Marian Kelley (Publisher John Wiley and Sons, 2008, ISBN 0470028769, 9780470028766).  Chapter 8 of this book entitled “Intracellular cytokine detection by flow cytometry” was authored by IST scientists Julie G. Wilkinson, Carlos L. Aparicio, and Wade E. Bolton.

Readers gain an understanding of the details and the high level considerations of assay qualification for difficult cell-based assays.  The level of optimization described for cell-based assays lends itself to biomarker assay automation which can be used for biomarker qualification and validation studies to the satisfaction of the FDA, EMEA, and other regulatory agencies.   Cell-based assay platforms covered are flow cytometry, intracellular cytokine ICS, immunophenotyping, elispot, IHC, cylex, neutralization bioassays, and endpoint assays.