Stability of biomarkers during sample collection, processing, transportation and storage varies and is an important source of preanalytical error.

We or others have assessed stability of most biomarkers, at room temperature, in frozen samples, during transportation in cold container, and in various matrices like serum and plasmas. The results are summarized in qualitative terms in the table below.
Biomarker stability

Most biomarkers protected from light are stable for years at temperature ≤ -80 °C. In general, biomarkers that are stable under certain non-optimal conditions are often relatively stable under other conditions. Likewise, biomarker instability is also a feature demonstrated under a variety of conditions. But there are notable exceptions to this general rule. For example, many biomarkers are most stable in EDTA plasma, but this is not the case for 5-methyltetrahydrofolate, which degrades faster in EDTA plasma than in serum. Methionine is rather stable, but is occasionally oxidized to methionine sulfoxide in samples frozen at -25 °C. The table below summarizes stability of biomarkers.

Biomarker profile may reflect non-optimal sample handling or storage

The biomarker profile of a serum/plasma sample set gives some indications of how the samples have been handled or stored. Delayed separation of serum/plasma may cause changes in concentrations of metabolites explained by cellular export (homocysteine), uptake (PL) or metabolism (arginine to ornithine conversion). Improper handling or storage of serum/plasma samples may cause degradation, oxidation, metabolism including formation from precursor(s) (choline) of numerous metabolites. Changes in selected metabolites in isolated serum/plasma are indicated in the figure to the right.

High median total homocysteine (>>15 µmol/L) in combination with adequate circulating folate or cobalamin suggest that whole blood has been left at room temperature for hours before centrifugation to isolate the serum/plasma fraction. Delayed separation also causes an arginine to ornithine conversion giving an Arg/(Arg+Orn) ratio less than 0.5. Low folate and PLP in combination with low-normal total homocysteine suggest vitamin degradation rather than low folate status. Serum/plasma samples that have been exposed to room temperature or repeated freezing-thawing cycles have high (>20%) 4-alfa-hydroxy-5-methyltetrahydrofolate relative to (micriobiological active) folate, high methionine sulfoxide (>20%) relative to methionine and/or low 3-hydroxykynurenine and 3-hydroxyanthranilic acid in combination with high anthranilic acid. Sample left at room temperature also have high free choline with median values in the range 15 – 100 µmol/L.

Stability according to biomarker category
B-vitamins and related nutrients
Biomarker Stability
(Change)
Comments Publ
Folate 5-Methyltetrahydrofolate degrades fast in EDTA plasma, but is relatively stable in chilled whole blood
Pyridoxal 5'-phosphate Converted to pyridoxal; the conversion is inhibited by EDTA. Relatively stable in chilled whole blood.
Pyridoxal Increase in pyridoxal is not observed in chilled whole blood, possibly because of cellullar uptake.
4-Pyridoxic acid  
Thiamine Probably stable.  
Riboflavin FMN is converted to riboflavin; the conversion is inhibited by EDTA.
Flavin mononucleotide  
Cobalamin  
Choline, free Phosphatidylcholine is converted to free choline; the conversion is inhibited by ETDA.
Choline, total    
Trimethylamine-N-oxide Stable.
Betaine  
Dimethylglycine  
Sarcosine  
Amino acids and related metabolites
Biomarker Stability
(Change)
Comments Publ
Total homocysteine Stable in isolated serum or plasma, but is increased in whole blood, because of export of homocysteine from intact cells.
Total cysteine  
Cystathionine  
Methionine Relatively stable, but oxidized to methionine sulfoxide after storage for years at –25 oC.
Methionine sulfoxide  
Serine  
Glycine  
Arginine Relatively stable in isolated serum or plasma, but decreases in (chilled) whole blood.
Ornithine Relatively stable in isolated serum or plasma, but increases in whole blood, leading to an increased Orn/Arg ratio
Histidine  
Tryptophan  
Glutamine Moderate decay due conversion to glutamic acid in improperly stored serum/plasma samples, leading to a marked increase in the Glu/Gln ratio.
Glutamic acid Marked increase due formation from glutamine in improperly stored serum/plasma samples, leading to a marked increase in the Glu/Gln ratio.
Aspargine Moderate decay due conversion to aspartic acid in improperly stored serum/plasma samples, leading to an increase in the Asp/Asn ratio.
Aspartic acid Increase due formation from aspargine in improperly stored serum/plasma samples, leading to an increase in the Asp/Asn ratio.
Alanine  
Homoarginine  
ADMA  
SDMA  
Trimethyllysine  
Kynurenines, B3 vitamers
Biomarker Stability
(Change)
Comments Publ
Kynurenine  
3-Hydroxy-kynurenine  
Kynurenic acid  
Xanthurenic acid  
Anthranilic acid  
3-Hydroxy-anthranilic acid  
Picolinic acid Assumed to be stable.  
Quinolinic acid Assumed to be stable.  
Nicotinic acid  
Nicotinamide  
N1-Methyl-nicotinamide  
Fat soluble vitamins
Biomarker Stability
(Change)
Comments Publ
Vitamin A Stable in whole blood and serum.
25-OH vitamin D3 Stable in whole blood and serum.
Vitamin E Stable in whole blood and serum.
Vitamin K Stable.
Others
Biomarker Stability
(Change)
Comments Publ
Methylmalonic acid  
Neopterin, total Neopterin plus 7,8-dihydroneopterin are measured. The latter is converted to 7,8-dihydroxanthopterin during non-optimal sample handling.
hsCRP  
Serum amyloid A  
Calprotectin  
Creatinine  
Creatine  
Cystatin C  
HbA1c  
Cotinine