Logistics & Shipments, Sample Preparation
The assessment of specimen stability starts with a process to determine stability based on the intended assay performance. Stability must be evaluated over an appropriate time course beginning with specimen collection through sample acquisition. Each stability parameter that is measured requires an evaluation during assay development and validation. The following is a list that can affect specimen stability: specimen type, specimen collection methods, and specimen storage and shipping conditions.
Peripheral whole blood is the most frequent type of specimen collected for analysis using flow cytometry. However, some assays require the preparation and freezing of peripheral blood mononuclear cells (PBMCs) rather than whole blood. Specimens such as bone marrow, spleen, thymus, lymph nodes and tumors can also be collected and analyzed for flow cytometry. These tissues must be disrupted into single cell suspensions prior to this type of analysis. If solid tissues are to be shipped then the logistics of when, where and how the cell suspension are to be prepared need to be evaluated during the assay development phase.
Whole blood and bone marrow may be collected into blood collection tubes containing some sort of preservative or anti-coagulant. The choice of anticoagulant is driven by both specimen stability and the type of flow cytometry analysis that is employed (i.e., phenotyping, functional assays, etc). In general, EDTA and Sodium Heparin are the most versatile anti-coagulants and may be used for multiple applications. If extended specimen stability is required, due to shipping restrictions, cell stabilizations tube such as CytoChex, that combine both an anticoagulant with a cell preservative may enable longer periods of sample stability.
Since samples collected during clinical trials are generally shipped from multiple remote clinical sites to a central lab for processing and analysis, the effect of shipping conditions on these samples should be fully evaluated. The samples can be packed in a shipping container containing temperature buffering reagents like ambient or refrigerated gel packs, along with temperature sensors to record any extreme shipping conditions.
There are several factors that should be considered when establishing validation acceptance criteria for stability. Precision of the assay is often used for this purpose. Relative percent change (i.e. the percent change between data acquired from the fresh specimen versus the stored specimen) may also be helpful in determining sample stability. The observed light scatter properties along with surface marker stability may be affected by the temperature that the samples are exposed to prior to processing. For example, granulocyte populations in a whole blood samples collected in EDTA and Sodium Heparin are very labile and can show degradation of light scatter properties within 24 hours of collection when held at room temperature.
Keep in mind that there is no single solution for retaining optimal specimen stability across varying collection, storage and shipping scenarios. It is therefore best to incorporate a standard process for the assessment of specimen stability and ensure optimal flow cytometry analysis to support your clinical trials.
