Data from clinical assays—biomarkers, PK, PD, and immunogenicity—are often key outcomes from clinical trials, and implementing these endpoints is very costly, and time- and resource consuming. Therefore, ensuring that appropriate measures are taken, starting from sample collection until the completion of laboratory testing is paramount. This article explains how to optimize bioanalytical procedures so that reliable results are obtained in clinical studies.

In the past, the usual procedure within clinical trials was to test a number of drug samples. These included samples for pharmacokinetics (PK) and a small number of samples for other assessments, such as, e.g., pharmacodynamics (PD). Each sample was taken individually, and the amount of time needed for processing each sample was limited. Typical techniques used for sample preparation included centrifugation, aliquoting and freezing. Occasionally sample testing using larger sample preparation flows would have to be performed, and therefore other preparative techniques would be required.

These requirements and procedures have now evolved to the extent that modern clinical studies routinely employ larger and more complex sample treatments. These include PK assessments, more specific sample treatments for PD studies, and the undertaking of multiple steps during these specific treatments and processes. This additional work means that it may now take up to 5 hours to process a sample, or batch of samples, and may include large batches of samples or aliquots, and up to 10 different assays being performed on a sample at a time. Each of these assays is performed under its own specific conditions, resulting in time-consuming treatment schedules. To cope with this additional complexity of processes, and the implementation of new techniques, pilot studies will need to be undertaken for training and qualification of staff before performing the full series of analyses.

One consequence of this is that laboratory manuals are getting more specific and demanding. Alongside typical specific conditions are increased numbers of requests, such as defining the timelines of a sample up to centrifugation, as well as those after centrifugation, specifying the storage conditions required, the matrix used, any additional handling steps that are needed, and any other special requirements. As a result, it is more challenging to maintain repetitious and consistently good sample quality and to plan resources.

So the questions we need to answer are: Why do all these parameters need to be set? How are these more complex sample treatments implemented? What are the thoughts and reasoning behind this approach? How is it proven to be effective, and how will information be passed from lab to site?

Setting the right parameters
Stepping momentarily away from the clinical trials setting, a case study entitled, “The effects of anticoagulant choice and sample processing time on hematologic values of juvenile whooping cranes,” from 20101 describes the collection of blood from these birds and the dependence of test results on a number of factors. Two anti-coagulants were used in the study, K3 EDTA and lithium heparin (LiHep), and slides were made either immediately or after periods of 4-6 hours, with the test results being analyzed to determine whether there was any correlation between the anti-coagulant used and the sample processing time.

Table 1 shows the total granulocyte concentration—heterophils and eosinophils; H/E concentration—of each of the divided samples. Table 2 shows the relative (%) leukocyte counts of each of the divided samples.

In this specific case study, no effect on results was seen depending on anti-coagulant if immediate sample processing was feasible. However, a time delay in sample processing did have an impact on the results.

Validation procedures: Allocation of time and resources
As demonstrated by the case study, it is important that validated laboratory testing techniques, and specific parameters must be checked regularly if the goals of the test are to be reached. This is a multistep process and is key to ensuring that the final results provided are fully “appropriate and correct” in terms of the demands of the analysis and the quality of the results.

For example, when validating a method for the testing of peripheral blood mononuclear cells (PBMCs), the first step of the process is the implementation and qualification of the method. This starts with the set-up and testing of the protocol, including testing PBMC stimulation and labeling procedures. In addition lysis and fixation processes are also determined and the permeabilization buffer selected. When acquisition and analysis templates and a gating strategy] have been put in place, preliminary testing of the reproducibility of the method can be carried out.

Part 2 of the method validation procedure comprises in-vitro feasibility testing of the effect of the product on key T cell functions, including CD4 Th1/Th2 and T Regulatory response. Culture conditions and stimulation parameters are chosen, such as reagents, stimulus, duration of culture and duration of stimulation, the dose- and time-effect of product determined (n= up to 6 subjects), and the in-vitro effect on both CD4 Th1/ Th2 and T Regulatory responses tested. Identical culture conditions are used to prepare a duplicate sample for fluorescence-activated cell sorting (FACS) testing. The final step in the in-vitro feasibility study is the data processing and statistical analysis.

Part 3 of method validation consists of the validation of the FACS method for analysis of CD4 Th1/Th2 subset (CD3, CD4, IFN-Gamma, IL-4) and T Regulatory (CD3, CD4, CD25, FoxP3, CD127) response. This includes the determination of method sensitivity, and its reproducibility between replicates, runs, analysts, donors and FACS systems. In addition stability testing, e.g. storage of PBMC and the effect of cryopreservation, has to be carried out. The inherent stability of reagents should also be determined. Finally, the FACS method chosen needs to be shown to be robust.

Part 4 of the method validation procedure comprises the transition of the method to the clinical site. This includes providing the procedure itself and training of the clinical staff in handling procedures, test conditions, and “go’s and don’t go’s.” A pilot study needs to be performed using replicate samples and employing several analysts to provide data for staff evaluation, method and training validation. The results are then reviewed and the clinical trial can be performed.

Each of these four steps would typically take up to one month, with the first three steps carried out in the bioanalytical laboratory, and the fourth step at the clinical site undertaking the trial. The fifth and final part of the method validation involves much more work in the bioanalytical laboratory and may take up to several months to complete. PBMC stimulation studies, CD4 Th1/ Th2 (CD3, CD4, IFN-Gamma, IL-4) FACS analysis, and T Regulatory (CD3, CD4, CD25, FoxP3, CD127) FACS analysis are undertaken on more than200 samples.

Additional testing
Additional steps may need to be taken to ensure the stability, reproducibility and robustness of bioanalytical samples, depending on the needs for the specific individual studies. For example, with urine collections there may be the need to add specific products, such as Tween, a surfactant, or bovine serum albumin to avoid interference in tubes; and in the determination of cytokines it may be desirable or necessary to use a non-standard blood collection tube. In test procedures that require sputum induction, the effect of using Sputolysin, which may alter the constituents and viscosity of mucus, on end parameters during handling needs to be determined; and when choosing the matrices, the effect of the presence of binding factors on specific compounds and parameters needs to be assessed. When cold storage of samples is required, any effects of snap freezing at -20°C or -70°C on sample quality have to be determined.

Conclusion
When performing bioanalytical test procedures, it is important to choose wisely when considering parameters such as the sampling tubes, anti-coagulant used, the test conditions, the sample processing times prior to the test, and the sample processing and test methods used. The validation of techniques is crucial to ensure the reliability of test results. The implementation of these procedures may appear to be time-consuming, but this work is invaluable, especially when supported by good communication and training between the bioanalytical laboratory and the clinical site. This is the key to getting good final results and understanding any potential pitfalls in attempting to achieve a successful bioanalytical outcome.

The choice of partner is therefore key to the success of the clinical trial, and experience and capabilities should be evaluated fully. To achieve a good technique that delivers very good data when running a clinical trial, specialists working on the bioanalytical aspect of the trial need to determine the experimental set-up and validation techniques and liaise with the specialists at the clinical research site who are responsible for the execution of test procedures. Pilot studies run between the clinical research site and the bioanalytical laboratory providing the best procedures for handling samples to be determined, identification of the optimal conditions for running the tests, and give the analysts experience in performing the tests. By establishing an effective and efficient communication flow between the laboratory and the clinical site, synergies between the two can be established and exploited, ensuring the optimal outcome for clients. 

References

1. Maurer, Joan; Reichenberg, Betsy; Kelley, Cristin; and Hartup, Barry K., “The Effects of Anticoagulant Choice and Sample Processing Time on Hematologic Values of Juvenile Whooping Cranes” (2010). North American Crane Workshop Proceedings. Paper 125.