Sample collection and handling
Please note that metabolic profiles consist of metabolites which are the end products of biological processes and can therefore be modified by enzymes and proteins. When a biological sample is collected, the metabolism is not quenched until the activity of the enzymes is stopped. That is why it is important to keep the samples at low temperature as soon as they are collected and they should be frozen as soon as possible. Samples should also remain frozen and stored with dry ice during transportation.
Some recommendations for standard biofluid collection and handling are provided below.
Protocols
- Sample collection, handling and storage consistency is critical. When possible, the manipulation variability between samples should be minimised (i.e. collection protocol and tubes or freeze-thaw cycles should be identical across all samples). Any preservatives added to the samples should be noted.
- For the aliquoting step, polypropylene tubes (2.0 mL cryovials or 1.5/2.0 mL Eppendorf tubes) are preferred.
- Ideally, samples should be snap frozen as soon as possible after collection. Alternatively, placement of samples in a -80°C freezer is acceptable. Samples should be kept frozen (using dry ice) during any transportation and the freeze-thaw cycles should be minimised when possible (as they are detrimental to many metabolites).
- Sample tubes should be labelled clearly with unique identifiers. Ideally, IDs should be written in the tubes using a permanent marker before freezing the sample. Note that unless freeze-safe labels are used, stickers might fall off from the tubes once they are frozen.
- All the available clinical data (including gender, age, time of collection, drugs intake and any other relevant clinical data for the project, etc) should be collated in an Excel file. Personal identifying information should not be included.
- Collect 10 ml from arterial line/central venous catheter in a syringe (to remove saline from the line), in a red top vacutainer tube (additive is a clot activator) or in a tube with no additives.
- Allow the whole blood collected to clot by leaving it undisturbed at room temperature for 15 minutes.
- Remove the clot by spinning down at 2,000 g for 10 minutes in a refrigerated centrifuge (set the temperature to 4⁰C). Remember to balance the centrifuge.
- Pipette out the resulting supernatant (serum) into cryo-tubes or Eppendorf tubes in 500 μl aliquots (usually get 3-4 replicates). This operation should be done in the safety cabinet. 1000 μl will allow us to perform both NMR and MS metabolic profile analysis.
- Freeze immediately at -80 ⁰C.
- Collect 10 ml from arterial line/central venous catheter in a syringe (to remove saline from the line), in a green top vacutainer tube (Li-heparin). Note: Heparin is preferred over EDTA or citrate anticoagulants as they can interfere with metabolic profiling (Nature Protocols 6, 1060–1083, 2011).
- Blood does not clot in the plasma tube; cells are removed by centrifugation. Spin down the blood collected at 2,000 g for 15 minutes in a refrigerated centrifuge (set the temperature to 4⁰C). Remember to balance the centrifuge.
- Pipette out the resulting supernatant (plasma) into cryo-tubes in 500 μl aliquots (usually get 3-4 replicates). This operation should be done in the safety cabinet. 1000 μl will allow us to perform both NMR and MS metabolic profile analysis.
- Freeze immediately at -80 ⁰C.
- Collect the urine sample in an MSU universal container.
- Aliquot the sample as quickly as possible post passing urine. Samples can be aliquoted into 15 mL Falcon tubes, if the appropriate centrifuge rotor is available; alternatively, they can be directly aliquoted into cryo-tubes/Eppendorf tubes.
- Keep samples refrigerated or on ice if you are not able to spin them down immediately and note down how much time the samples have stayed on the ice.
- Spin the sample down for 15 mins at 2,000 g in a refrigerated centrifuge (set the temperature to 4⁰C). Remember to balance the centrifuge.
- Pipette out the supernatant into cryo-tubes or Eppendorf tubes in 1 mL aliquots (never less than 700 μL) while paying attention to avoid collecting solid residue in the bottom. Usually, get 3-4 aliquots. 1 mL will allow us to perform both NMR and MS metabolic profile analysis.
- Freeze immediately at -80 ⁰C.
It is essential that there are no barriers to accessing the clinical metadata of the project at the time of the analysis as it is used to ensure that the sample randomisation prior analysis will not compromise the statistical analysis later on.
Samples analysed
Samples collected must be fit for purpose. Differing biofluids provide unique metabolic signatures due to their organ-specific biological functions and outputs. For example, tissue samples not only provide an opportunity to study metabolism in the target pathology but from an analytical perspective, molecular imaging technologies can also be deployed for studying spatially resolved metabolic processes in histologically validated specimens. Where possible, metabonomic studies should seek to capture multiple biofluid types, which allows the phenome to be interrogated across biological compartments and it thus provides greater insight into whole organism metabolic signalling and system function.
The CPC routinely analyses urine, serum or plasma samples but also can analyse other biological fluids, including cerebrospinal fluid or faecal water, as well as tissue samples (either intact or after metabolite extraction).
Please note that further sample handling development might be required depending on the sample matrix collected.
Sample matrices
- Urine
- Blood serum/plasma
- Faecal water
- Cerebrospinal fluid
- Gastric fluid
- Colostrum
- Vaginal fluid/swabs
- Liver and liver extracts
- Cell extracts