Preanalytical Phase

Due to its ease of access and its central role in biological functions, much emphasis has been placed on analyzing blood specimens for biomarker discovery. The main clinical blood specimens are plasma and serum. One critically important, and often underestimated factor modulating the likelihood of success in discovering relevant biomarkers in blood specimens are the procedures used in sample collection and specimen generation.

This preanalytic phase can significantly alter the results derived from the analysis of blood-derived samples by introducing unwanted systematic biases. The preanalytical phase comprise the processes prior to the actual analysis of the sample and include steps needed to obtain the primary sample (e.g. blood) and to obtain the analytical specimen (e.g. plasma, serum, cells). Standardization of blood sample collection is a prerequisite to avoid systemic biases.

For Peptidomics® analyses EDTA plasma is the preferred specimen, since the proteolytic activity in comparison to serum is markedly reduced due to inhibition of ion-dependent enzymes via chelate formation. Avoiding ex-vivo degradation of proteins and peptides is beneficial, since the complexity of the resulting sample is reduced and for the analysis of in vivo protease cleavage sites it is mandatory to preserve the integrity of the peptidome.

Blood collection and specimen generation for Peptidomics analyses

The provision of adequate SOP’s is crucial to guarantee a high quality sample. Since in the discovery phase the number of samples to be analyzed is limited provision of high quality samples should be feasible. But more importantly this phase determines the outcome of all subsequent stages.

Disclaimer: Legal or ethical issues (e.g. importance of informed consents) or potential risks of phlebotomy (e.g. bleeding) are not covered.

These short lists provide only the basic procedures. For an in-depth description and a protocol do not hesitate to contact us.

Blood collection

• favoured site of collection is the median cubital vein (easily found and accessed, comfortable for the patient)
• collect from fasting patients in the morning between 7-9 am (circadian rhythms)
• clean skin with an alcohol (2-propanol)
• allow alcohol to evaporate (danger of hemolysis, interference)
• apply tourniquet 3-4 inches above the site of venipuncture
• release as soon as blood begins flowing into the collection device (hemoconcentration)
• free flow has to be assured to avoid haemolysis
• centrifugation has to be started within 30 min after blood collection

Specimen generation

• centrifugation for 10 min at 2000 x g
• temperature should generally be 15-24 °C to prevent activation of platelets (unless recommended differently for distinct analytes like gastrin)
• a significant number of platelets (~25%) is still present in the sample, which requires an additional centrifugation step
• plasma is transferred into a second vial
• centrifugation for 15 min at 2500 x g at room temperature.
• supernatant is transferred in aliquots of 1.5 mL into cryo vials

Frequently made mistakes

• The blood sample tubes were put on ice or in a refrigerator.
• A cooling centrifuge was adjusted below room temperature.
• The centrifugation speed was wrong (e.g. rounds per minute were set instead of g-force).
• The removal of blood plasma by pipetting was done without proper caution (contamination with cells)

Further reading:

Specimen collection and handling: standardization of blood sample collection
Methods Mol Biol. 2008;428:35-42

Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics
Comb Chem High Throughput Screen. 2005 Dec;8(8):725-33