Over the years, REPROCELL USA has worked on several projects involving blood and blood components. The company has experience with collection, biobanking, nucleic acid extraction, among other services. Blood derived plasma and serum are two materials which are useful in clinical/experimental settings and have been at the forefront of many REPROCELL USA activities. Both biofluids contain molecules such as microRNAs (miRNAs), cell-free DNA (cfDNA), and useful metabolites.1,2,3 Plasma is the liquid portion collected when an anticoagulant is added before cell depletion (sodium citrate, EDTA, and heparin are commonly used).4 Serum, however, is obtained when one allows the blood sample to clot then removes both blood cells and clots/coagulation factors.4 As a result, the contents and utility of each biomaterial differ in a way which merits comparison. It has been noted that serum is the gold standard for clinical testing, but the informative value of plasma should not be underestimated.5 Here, we highlight three studies which capture some differences between the two.
Varying Metabolite Profiles
When collecting serum, blood cell-derived metabolites released during the coagulation cascade can get into one’s sample. This, along with other differences in the collection processes of either may result in a discrepancy between plasma and serum analytes. Many studies have been published comparing the contents of both fluids.
Vignoli et al. evaluated the influence of collection tubes on the metabolomic and lipoprotein profiles of blood samples using NMR.5 The study used blood collected from six healthy volunteers. The authors collected several samples from each donor at five time points (baseline, 2 weeks, 4 weeks, 27 weeks, and 29 weeks). Blood was collected in EDTA plasma, sodium citrate plasma, and serum tubes each time, and the serum samples were prepared in sterile vacutainers containing a gel separator and clot activator (which reduces clotting time). They found that 18 out of 24 metabolites had statistically different concentrations in different tubes (for example, serum tubes had significantly higher concentrations of alanine and glutamine compared to plasma citrate and EDTA tubes). They summarized these findings in Figure 5 of their paper (of which an excerpt is shown below).
Figure 1. Subplots depicting a statistically significant difference (***p < 0.001) in metabolite concentration between serum and both plasma citrate/EDTA tubes. The full figure contains 18 box plots. Selected here are subplots where the comparison between serum and both variants of plasma tubes have p<0.001. “Serum or Plasma (and Which Plasma), That Is the Question” by Vignoli et al., used under CC-BY 4.0.
Of note is how the metabolites which have the greatest discrepancy between serum and plasma samples are amino acids. The authors note that these results are consistent with other published literature which uses different analytic methods. They agree that the anticoagulants used in plasma collection inhibits proteolysis, and state that some metabolites could have been released by platelets during the clotting step in serum collection. As for lipoprotein content, they say that results were less clear.
Mass Spectrometry confirms findings
Another study assessed the impact of collection tube on metabolomics through liquid chromatography mass spectrometry.6 This study looked at 189 metabolites in three types of tubes (EDTA plasma, sodium citrate plasma, and serum) where blood was donated from 80 subjects. Participants donated blood in the early morning after fasting overnight. To prepare the serum samples, the authors let the samples clot for 30 minutes at room temperature and centrifuged at room temperature (RT) for 15 minutes at 1500 x g. For the plasma samples, the EDTA and citrate tubes were mixed for 10 minutes at RT. Then, the citrate tubes were centrifuged for 15 minutes at 1500 x g, at RT. Samples were stored in the Biobank of Bozen/Bolzano, at -80°C, in 120 µL aliquots until analysis.
Unlike the previous study, participants were separated into two age groups. The young group consisted of individuals less than 35 years old, and the elderly group was composed of individuals older than 60. This allowed them to make a comparison between metabolites which vary based on age.
These authors concluded that serum contains higher amino acid concentrations. The results of an ANOVA comparison between the collection tube groups revealed 9 specific amino acids which differ significantly (including Phenylalanine and Glutamine, supporting the study by Vignoli et al.). Of the non-amino acid molecules, the authors say that biogenic amines, glycerophospholipids, and one acylcarnitine vary. They also found that age-dependent metabolomic changes are revealed through serum better than plasma.
MicroRNAs in Maternal Serum and Plasma
In a third study, authors investigated the microRNA (miRNAs) profiles found in serum and plasma obtained from pregnant mothers.7 They applied RT-qPCR and next generation sequencing to assess differences. They state that the selection between plasma and serum in clinical and academic investigations has been (for the most part) random. They justify their work by noting that miRNAs are important in post-transcriptional regulation and that the data they collected could help distinguish the two molecularly.
The authors collected plasma samples from 12 pregnant women and serum from 20 pregnant subjects where both groups were in the second trimester. All patients had normal, uncomplicated, singleton pregnancies, without fetal malformation. Plasma samples were obtained from 2mL of blood in EDTA tubes. To get the plasma, samples were centrifuged twice at 4°C for 10 minutes. The first time using 1,600 x g and the second time at 16,000 x g to remove blood cells from the plasma. To get serum, these authors let whole blood clot for 30 minutes at room temperature, then spun the samples at 4°C, at 3000 x g for 10 minutes to remove the clot. Both plasma and serum were stored at -80°C. Plasma and serum were pooled before further work.
The results obtained from SOLiD high throughput sequencing implied that there was both upregulation and downregulation of miRNA expression levels in serum relative to plasma. These expression level results are summarized in a hierarchical clustering diagram (which the reader can find in the paper). Also, the kinds of microRNAs found in serum and plasma differ. They mention that 329 miRNAs were detected in serum and 193 miRNAs were detected in plasma. After filtering out the ones with less than 20 counts, they determined that there were 77 unique to maternal serum. Table 1 from the paper lists specific miRNAs whose expression levels vary between the two sample types (reproduced here for convenience).
Table 1. miRNAs which are up/downregulated in serum compared to plasma from pregnant women and the top 20 miRNAs expressed in both sample types. Table by Ge et al., used under CC BY_NC 3.0 / Cropped from original.
Out of those listed, 12 were selected for validation using RT-qPCR. With a Pearson’s correlation of 0.974, the authors found that there was a strong agreement between the sequencing results and RT-qPCR data.
Clinical Use
Plasma and serum have countless uses in the clinical setting. In oncology specifically, plasma and serum are sources of circulating tumor DNA (ctDNA) which can be used for liquid biopsies.8 Unlike surgical biopsies, a liquid biopsy involves isolating cancer derivatives from body fluids.8 ctDNA is a form of cell-free DNA released by tumors and reflects disease status. ctDNA can enable a better understanding of cancer biology through epigenetic and genetic analyses. It has found use as a noninvasive cancer detection method, as a means of assessing treatment impact, or even in tumor profiling.9 Plasma and serum both contain ctDNA to varying degrees, but it is usually isolated from plasma (in fact, ReproCELL USA has experience doing just that). A literature review published to Frontiers in Molecular Biosciences describes what is known about using this nucleic acid for disease monitoring in pediatric cancers.9 The authors mention that advances in understanding have pushed forward ctDNA clinical use via detection tests for adult solid and hematological cancers but the same cannot be said for pediatric cancers. They note that ctDNA analysis relies on understanding which somatic mutations are indicative of cancer. For example, they say that rearrangements of the EWSR1 gene are good markers for Ewing sarcoma. Another example is how mutations in the CTNNB1 gene are connected to sporadic hepatoblastoma.10
According to the literature review, clinically useful tumor DNA detection methods include:
- Single Target PCR
- Panels defined by tumor subtype
- Patient Specific Panels using Next Generation Sequencing.
The review also lists some liquid biopsy tests that have made it to the commercial stage.
While the authors do not specifically address differences between ctDNA obtained from serum and plasma, they do say that ctDNA levels can be much higher in plasma. They also state that sample type standardization has been an overlooked issue.
Choose Carefully
Among the papers discussed here, one can find countless examples of how plasma and serum collection procedures differ subtly. However, the defining characteristic of serum is that clotting is performed. Our discussion suggests that this can influence experimental outcomes. Others have described how plasma versus serum selection can alter clinical outcomes.11 This means that anyone working with information derived from these biofluids should take care to review relevant literature and make an informed decision on which of the two is best suited for one’s application.
References
- Parker VL, Gavriil E, Marshall B, Pacey A, Heath PR. Profiling microRNAs in uncomplicated pregnancies: Serum vs. plasma. Biomed Rep. 2021;14(2):24. doi:10.3892/br.2020.1400
- Trulson I, Stahl J, Margraf S, et al. Cell-Free DNA in Plasma and Serum Indicates Disease Severity and Prognosis in Blunt Trauma Patients. Diagnostics (Basel). 2023;13(6):1150. Published 2023 Mar 17. doi:10.3390/diagnostics13061150
- Liu X, Hoene M, Wang X, et al. Serum or plasma, what is the difference? Investigations to facilitate the sample material selection decision making process for metabolomics studies and beyond. Anal Chim Acta. 2018;1037:293-300. doi:10.1016/j.aca.2018.03.009
- Sotelo-Orozco J, Chen SY, Hertz-Picciotto I, Slupsky CM. A Comparison of Serum and Plasma Blood Collection Tubes for the Integration of Epidemiological and Metabolomics Data. Front Mol Biosci. 2021;8:682134. Published 2021 Jul 8. doi:10.3389/fmolb.2021.682134
- Vignoli A, Tenori L, Morsiani C, Turano P, Capri M, Luchinat C. Serum or Plasma (and Which Plasma), That Is the Question. J Proteome Res. 2022;21(4):1061-1072. doi:10.1021/acs.jproteome.1c00935
- Paglia G, Del Greco FM, Sigurdsson BB, et al. Influence of collection tubes during quantitative targeted metabolomics studies in human blood samples. Clin Chim Acta. 2018;486:320-328. doi:10.1016/j.cca.2018.08.014
- Ge Q, Shen Y, Tian F, Lu J, Bai Y, Lu Z. Profiling circulating microRNAs in maternal serum and plasma. Mol Med Rep. 2015;12(3):3323-3330. doi:10.3892/mmr.2015.3879
- Lone SN, Nisar S, Masoodi T, et al. Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Mol Cancer. 2022;21(1):79. Published 2022 Mar 18. doi:10.1186/s12943-022-01543-7
- Doculara L, Trahair TN, Bayat N, Lock RB. Circulating Tumor DNA in Pediatric Cancer. Front Mol Biosci. 2022;9:885597. Published 2022 May 12. doi:10.3389/fmolb.2022.885597
- Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020;67(11):e28594. doi:10.1002/pbc.28594
- Mannello F. Serum or plasma samples? The "Cinderella" role of blood collection procedures: preanalytical methodological issues influence the release and activity of circulating matrix metalloproteinases and their tissue inhibitors, hampering diagnostic trueness and leading to misinterpretation. Arterioscler Thromb Vasc Biol. 2008;28(4):611-614. doi:10.1161/ATVBAHA.107.159608