Handling serum and plasma samples appropriately is crucial for obtaining accurate and reliable results in immunoassays. The following guidelines cover best practices for serum and plasma handling targeted for immunoassays like ELISA, western blot, and multiplex immunoassays:
Blood Draw: Blood should be drawn following best practices to avoid hemolysis. Hemolyzed samples may affect quantification of certain proteins. Some of the common mistakes leading to hemolysis include the use of needles with too small diameter, transport of blood to other containers with a syringe, shaking or freezing the sample, centrifugating at too high speed or delayed separation from time of collection.
Anticoagulant: For plasma, use appropriate anticoagulants such as EDTA, citrate, or heparin, depending on the requirements of your immunoassay. For serum, the use of collection vials containing clotting agents is advisable for ensuring clotting and reducing processing time.
Immediate Handling: For serum, wait for coagulation to occur for at least 30 min before proceeding to centrifugation. Centrifuge the blood samples within 3 hrs from blood draw. Typically, centrifugation at 1,500-2,000 g for 10 minutes at 18-25°C is recommended.
Aliquoting: Immediately aliquot serum or plasma into clean, pre-labeled tubes to minimize freeze-thaw cycles.
Transportation Temperature: Serum & Plasma can be transported at 4°C immediately upon collection with same-day or next-day delivery courier services if to be analyzed fresh. Alternatively, they can be frozen immediately upon collection and shipped in dry ice.
Storage Temperature: Serum or plasma samples should be kept at -80°C for long-term storage. Short-term storage at -20°C or 4°C may be possible depending on the stability of the biomarkers of interest.
Freeze-Thaw Cycles: Minimize the number of freeze-thaw cycles to maintain the integrity of the proteins.
Test Facility Inspection: Samples should be carefully inspected by the Test facility and any deviations from the standard handling procedures should be recorded. Examples include inspection for hemolytic or icteric samples, samples thawed during transport, incorrect identification or labelling etc.
Inventory: A detailed inventory for easy sample tracking should be kept and linked to a LIMS system where applicable.
Samples should be thawed in a controlled manner on ice or in a refrigerated setting to minimize protein degradation. Gently mix the serum or plasma by inversion or pipetting to ensure homogeneity.
Filtration: Some protocols may require the removal of particulates by centrifugation or filtration.
Dilution: Dilutions of serum or plasma samples may be required and should be stated in the assay inserts. Make sure you use appropriate buffers/diluents for sample dilutions (normally provided with the assay). Sample diluents have been optimized to reduce any non-specific binding, cross reactivities or matrix effects related to antibody-based immunoassays.
Temperature: Assays are conducted at recommended temperatures. Most ELISAs, for example, are performed at room temperature.
Timing: Incubation times must be strictly followed as they can affect results and generate irreproducible data.
Shaking: Gentle shaking is normally required during incubation steps to ensure solution homogeneity; adhere to the protocol guidelines.
Serum and plasma samples should be handled as hazardous biological material and should be disposed of in biohazard waste bags, which are then sealed and labeled. Depending on departmental protocols, biohazard waste is either autoclaved or incinerated.
CLSI. Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests; Approved Guideline – Fourth Edition. CLSI document GP44-A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.