REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 252

however, a preliminary evaluation including additional temperature conditions (i.e.,
chilled or frozen) and expected shipment duration can be illuminating. Stability of
markers stored at ambient temperature should be compared to chilled conditions
where samples are shipped with ice packs, if appropriate. Samples can be shipped in
the original collection container or may be processed prior to shipment to extend
marker stability before arriving at the analysis laboratory. Validation of postcollection
stability becomes increasingly important in global clinical trials, where samples are
shipped to a central laboratory and may be received next day, or second or third day,
after blood draw. In some instances, cellular samples may be fixed prior to shipping to
extend stability up to 7 days (Streck cell preservation media) [59]. Additional value is
gained when the assay is transitioned to clinical trials and markers are stable more
than 24 h postcollection. Extended stability facilitates batch sample analysis andmore
efficient sample management in clinical trials.
To evaluate PBMC marker stability in the collection container, samples should be
processed immediately after collection (as baseline), as well as at different post-
collection time points that would mimic clinical conditions. PBMC samples collected
in Vacutainer CPT tubes offer several options for shipping in clinical studies;
therefore, marker stability in all sample storage conditions should be evaluated. CPT
tubes can be shipped as anticoagulated whole blood or PBMC can be separated via
centrifugation and resuspended in the plasma layer above the gel barrier and remain in
the collection container prior to shipping. PBMC can also be isolated from the plasma
and transferred to a secondary container for processing, storage, or shipping. Finally,
PBMC can be isolated and frozen for shipping, especially in global studies where it is
not feasible to transport samples in the original CPT collection tube to the laboratory
for processing within the marker stability time frame. In this case, stability of markers
after cryopreservation must also be investigated. Finally, if processed and stained
samples cannot be acquired immediately after fixation, the fixed sample stability
should be evaluated to permit sample storage for later acquisition.
Documentation A validation summary report becomes the reference document
describing assay performance and limitations as defined by validation. It should
describe the assay method, include raw data from the validation, and summarize
results and assay limitations. Assay acceptance criteria are a very important element
of this overall exercise; the validation fails if assay acceptance criteria are not met. In
addition, the report should cite the laboratory notebooks where validation is
documented and identify binders or electronic records of raw data. Accurate
validation experiment documentation can be valuable for performing revalidation
experiments and assay adaptation for future drug development studies, as required. It
is this document that will be used within a clinical trial to determine if results are an
effect of treatment or expected assay variability. Validation reports are considered
permanent records and are reviewed and approved by senior management.
Documentation and implementation of a quality assurance plan focused on assay
and instrument quality during validation are useful tools to ensure reliable flow
cytometry data are generated at every stage in the drug development process. A
validation plan should be considered to outline experimental procedures and