Whole blood analysis and its role in pre-clinical drug development

Low translatability and reproducibility of preclinical studies result in project delays and increased costs for drug developers. It’s estimated that between 51-89% of all preclinical research is irreproducible. The resulting cost is $ 28B in the United States alone (1). That’s an alarming number.

In this post we’re covering challenges and solutions on:

• Identifying and characterizing potential toxicities in pre-clinical drug development, including human-specific immune reactions that can be hard to predict
• Assessing and modeling desired biological effects
• Characterizing pharmacokinetic properties to determine a safe initial starting dose of a drug

During the past decades, there has been a revolution in the possibilities to cure or alleviate disease with modified biologics or by targeting the body’s immune system. While this has increased the potential to find new and more effective therapies, it also poses new challenges. The necessity of administering drugs by infusion instead of oral delivery can trigger unexpected infusion reactions. Modulation of the immune system can have unpredicted effects and vary significantly from individual to individual. Other challenges are associated with the lack of suitable animal models to accurately predict human immune responses. This is especially true for biologics of human origin and stems from the differences in immunology across species. These factors pose a potential dilemma when drug developers attempt to translate non-clinical studies into clinical reality. There are numerous examples in which animal studies could not predict severe human toxicity (2).

One of the most infamous examples of poor translatability is the TGN1412 trial. Six healthy volunteers suffered severe inflammatory reactions and chronic organ failure after receiving an immunomodulatory drug known as TGN1412. The healthy volunteers were given a sub-clinical dose, 500 times lower than what was considered safe based on animal data (3). Neither in vitro nor in vivo experiments, including toxicology studies in non-human primates, provided evidence for the adverse side effects seen in this trial. The trial was halted, but the six participants were left with life-long injuries. This resulted in a paradigm shift in the safety prediction of biological drugs. 

Blood handling and freshness of the blood are essential for obtaining qualitative results, especially when analyzing sensitive cells.

Choosing the most suitable test system 

In the aftermath of TGN1412, the search for more physiologically relevant test systems began. Today, both in-vitro platforms and animals are used to identify and characterize potential toxicities. The choice of a particular system is highly dependent on the availability of resources, including access to animals or blood donors. As late as February 2022, the FDA updated its guidance on non-clinical toxicology, urging sponsors to find alternatives to non-human primates (NHPs) due to a major constraint in the supply of animals. Whole blood (WB) assays based on human blood are more relevant from an immunological perspective compared to animals,  and they are also a cost-efficient alternative. WB assays have a broad application area in pre-clinical testing, including immunogenicity and toxicology studies, bioactivity testing for in-blood targets, investigating modulation of inflammation, cross-species reactivity, cytokine release, and in the development of vaccines judging adjuvant and antigen-specific immune responses. Blood handling and freshness of the blood are essential for obtaining qualitative results, especially when analyzing sensitive cells such as neutrophils, platelets, and monocytes. Furthermore, extraction methodologies, methods of blood storage, and the use of additives need to be carefully managed.

Maximizing a qualitative outcome when using whole blood
To accurately predict immunotoxicity, it’s important to use a whole blood test system that avoids some of the most common pitfalls;  for practical reasons, whole blood or blood components are commonly stored before use (but not always). Vacutainer tubes collect blood by force, resulting in the activation and lysis of blood cells. During separation and storage, whole blood is usually combined with relatively high levels of anticoagulants to prevent clotting. Anticoagulants prevent functional platelet analysis and inactivate critical cascade systems, such as complement, that play an essential role in the initial immune response. Furthermore, platelet function rapidly decreases when whole blood is held in a refrigerator or on ice. As a result, the activity of coagulation factors, particularly Factors V and VIII, diminishes. Keeping these parameters in mind when using whole blood, increases the accuracy of test results.

PBMCs are less suitable for answering scientific questions related to platelet activation and complement-dependent immune reactions. 

While isolated cell components and preparations may be beneficial in answering specific scientific questions related to effects on individual cell types, they are less valuable in answering questions on overall effects on the immune system and translating them to the in vivo situation. The interactions among and between whole blood components are complex and interrelated, which means that tests performed using only a single blood component risk being unable to detect downstream effects on other cells or compensatory mechanisms. It is much more challenging to translate dose-effect relationships from a cell-based assay into maximum human plasma concentrations in a cell culture medium than in a WB assay where all individual serum proteins and cells are present. A recent study comparing isolated PMBCs and two different whole blood assays revealed significant differences in background noise and sensitivity. This appears to be due to different effects on the isolated cells by the array of other preparation methods and the use of high levels of anticoagulants, making PBMCs less suitable for answering scientific questions related to platelet activation and complement-dependent immune reactions. 

Although isolated blood components have a place in specific settings, whole blood analysis in pre-clinical drug development is the model of choice for reliable translation of overall concentration-effect relationships into clinical trials. If the objective is to achieve a relevant picture of the risk for unexpected immune reactions or clotting upon infusion of a drug, the blood needs to be carefully collected, fresh and free from unnecessary additives.

1. https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165 
2. https://www.sciencedirect.com/science/article/pii/S2452302X1930316X#bib15
3. https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165