Pre-analytical sample stability

Pre-analytical stability is essential for high-quality results. Knowledge about analyte stability helps researchers prepare blood samples correctly before shipping, select appropriate handling and storage conditions, and reduce avoidable variation before analysis.

What matters most

We and others have assessed the stability of most biomarkers at room temperature, in frozen samples, during transport in cold containers and in different matrices such as serum and plasma.

−80 °C Most analytes protected from light stay stable for years at −80 °C or colder

EDTA plasma The most stable matrix for many analytes, with notable exceptions

Fast separation Whole blood left at room temperature changes several metabolites within hours

An analyte that is stable under one non-optimal condition is often stable under others, and the same holds for instability. There are exceptions. 5-Methyltetrahydrofolate degrades faster in EDTA plasma than in serum, and methionine is occasionally oxidized to methionine sulfoxide in samples frozen at −25 °C.

Recommended sample processing

Standardized processing keeps degradation to a minimum. These are the steps we recommend for serum and EDTA plasma.

Serum

Collect

Collect whole blood.

15 to 30 min

Let the blood clot at room temperature.

1,000 to 2,000 g

Centrifuge for 10 minutes in a refrigerated centrifuge.

−80 °C

Transfer the supernatant to clean tubes immediately and store at −80 °C.

EDTA plasma

EDTA tubes

Collect whole blood into EDTA tubes.

2 to 8 °C

Keep the tubes cold, for example on a bed of ice, until centrifugation.

1,000 to 2,000 g

Centrifuge for 10 minutes in a refrigerated centrifuge.

−80 °C

Transfer the plasma to clean tubes immediately and store at −80 °C.

What the analyte profile can reveal

The analyte profile of a serum or plasma sample set gives clues about how the samples were handled or stored. These patterns point to a pre-analytical problem rather than a biological effect.

Delayed separation

Median total homocysteine well above 15 µmol/L despite adequate folate or cobalamin suggests whole blood was left at room temperature for hours before centrifugation. Delayed separation also converts arginine to ornithine, giving an Arg/(Arg+Orn) ratio below 0.5.

Vitamin degradation

Low folate and PLP together with low-normal total homocysteine suggest the vitamins have degraded, rather than low folate status in the participants.

Room temperature or repeated freeze-thaw

More than 20% 4-α-hydroxy-5-methyltetrahydrofolate relative to folate, more than 20% methionine sulfoxide relative to methionine, or low 3-hydroxykynurenine and 3-hydroxyanthranilic acid combined with high anthranilic acid.

Samples left at room temperature

High free choline, with median values in the range of 15 to 100 µmol/L, caused by conversion from phosphatidylcholine.

Stability by analyte

A summary of pre-analytical stability for each analyte, with comments on exceptions and the publications behind each assessment. The table is generated from our analyte database.

Stable: no relevant change under non-optimal conditions. Increases or decreases: concentration changes when handling is not optimal. Marked: large changes. Mostly stable: changes only under some conditions, see the comment.

The stability table is generated from the analyte database. Stability details for each analyte are also available in the Analyte catalog.

Stability curves

For researchers who need analyte-specific evidence. Each curve shows an analyte in EDTA plasma and serum, at room temperature and after repeated freeze-thaw cycles.

Left panel: room temperature for the indicated duration. Right panel: increasing number of freeze-thaw cycles. The white zone marks ±15% deviation from the mean of the first three time points for plasma. Data are geometric means with standard error for samples from 15 healthy subjects.

Amino and carboxylic acids Platform B, GC-MS/MS