Skip to main content

Main menu

  • Home
  • About
    • JALM
    • Editorial Board
    • Most Read
    • Most Cited
    • Alerts
  • Articles
    • Current Issue
    • Early Release
    • Future Table of Contents
    • Archive
    • Browse by Subject
    • Special Issues & Collections
  • Info for
    • Authors
    • Reviewers
    • Subscribers
    • Advertisers
    • Permissions & Reprints
  • Abstracts
  • JALM Talk
  • Submit
  • Feedback
  • Other Publications
    • Clinical Chemistry

User menu

  • Subscribe
  • My alerts
  • Log in

Search

  • Advanced search
The Journal of Applied Laboratory Medicine
  • Other Publications
    • Clinical Chemistry
  • Subscribe
  • My alerts
  • Log in
The Journal of Applied Laboratory Medicine

Advanced Search

  • Home
  • About
    • JALM
    • Editorial Board
    • Most Read
    • Most Cited
    • Alerts
  • Articles
    • Current Issue
    • Early Release
    • Future Table of Contents
    • Archive
    • Browse by Subject
    • Special Issues & Collections
  • Info for
    • Authors
    • Reviewers
    • Subscribers
    • Advertisers
    • Permissions & Reprints
  • Abstracts
  • JALM Talk
  • Submit
  • Feedback
Research ArticleArticles

Sample Type and Storage Conditions Affect Calprotectin Measurements in Blood

Lise Pedersen, Erling Birkemose, Charlotte Gils, Sara Safi, Mads Nybo
DOI: 10.1373/jalm.2017.024778 Published April 2018
Lise Pedersen
Department of Clinical Biochemistry, Holbæk Hospital, Holbæk, Denmark;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Erling Birkemose
Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Charlotte Gils
Department of Clinical Immunology and Biochemistry, Lillebaelt Hospital, Vejle, Denmark.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: charlotte.gils@rsyd.dk
Sara Safi
Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mads Nybo
Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Background: Calprotectin, a complex of calcium-binding proteins, is abundant in granulocytes. Increased levels of plasma calprotectin have been found in patients with inflammatory and autoimmune diseases. However, a number of preanalytical factors may affect calprotectin measurement in blood samples.

Methods: Twelve blood samples [4 tubes, 1 each of lithium-heparin (Li-heparin), EDTA, and serum] were drawn from each of 14 healthy individuals. To evaluate the effect of temperature and storage time in the lag time between collection and centrifugation, samples were kept for 2 h at 4 °C, 20 °C, or 37 °C, before centrifugation. Leukocyte, neutrophil, and monocyte counts were measured in EDTA samples on a Sysmex XN-10 hematology analyzer to investigate the relationship between calprotectin concentrations and the granulocyte count.

Results: Calprotectin measurements in EDTA samples were not influenced by temperature or time lag between collection and analysis. Compared to EDTA plasma, significantly higher calprotectin concentrations were found in serum and Li-heparin plasma samples. Furthermore, calprotectin concentrations increased in serum and Li-heparin samples when stored at higher temperatures. There was a linear relationship between the serum calprotectin concentration and neutrophil count in EDTA whole blood.

Conclusions: EDTA is the most suitable anticoagulant for determination of calprotectin in plasma, as this sample matrix does not seem to be affected by temperature or time between sample collection and analysis. Of particular note, neutrophil activation by either clotting or centrifugation should be avoided during the preanalytical process.

Impact Statement

Calprotectin is used as a marker of disease activity in patients with many inflammatory conditions and autoimmune diseases. However, influence of different preanalytical variables on calprotectin measurements has not previously been investigated. Results from this study show a temperature-dependent increase in calprotectin concentrations in serum and heparin samples left for some time before centrifugation. Use of EDTA plasma for determination of calprotectin in blood is not affected by temperature or time between collection and analysis. This manuscript advances knowledge in the preanalytical testing phase of calprotectin measurement, an assay that is widely used in clinical settings.

Calprotectin is a calcium-binding cytosolic protein that constitutes up to 40% of the cytosolic protein or 5% of the total protein content of both neutrophils and monocytes (1). The protein belongs to the S100 family, a group of multifunctional proteins with various regulatory roles in different cellular processes (2).

Calprotectin is released from neutrophils and monocytes during their activation, and increased systemic levels of the protein have been linked with increased immunological activity and inflammation (3). Clinically, calprotectin in blood has been investigated as a marker of disease activity in many inflammatory conditions and autoimmune diseases, such as inflammatory bowel disease, rheumatoid arthritis, and cardiovascular disease (4–6). However, calprotectin measurements in blood may be affected by preanalytical variables such as type of anticoagulant, and temperature and time of storage. Platelet activation in vitro affects tests in the coagulation laboratory (7) and type of anticoagulant; temperature and storage time are known to influence the platelet activation (8–10).

Activation of leukocytes is influenced by platelets (11) and therefore it can be hypothesized that calprotectin levels may be dependent on the same preanalytical factors that affects platelet activation. The aim of the study was to investigate the difference in calprotectin concentration collected in different tubes processed under different conditions. We furthermore investigated the potential relationship between calprotectin levels and the leukocyte count in whole blood.

METHODS

Subjects and samples

Blood samples were collected from 10 female and 4 male nonfasting volunteers 20–61 years of age. From each participant 12 tubes were collected: 4 serum tubes (Catalog No 369032, lot No 5027328), 4 Lithium Heparin (Li-heparin) tubes (Catalog No 367526, lot No 5271003) and 4 EDTA tubes (Catalog No 368861, lot No 5306579), all from BD Vacutainer System. One set of tubes from each volunteer were centrifuged immediately, while the other tubes were stored for 2 h at different temperatures before centrifugation as described in Table 1. After storage, all tubes were centrifuged for 9 minutes at 3600g at the same temperature used for storage. The supernatant from all samples was transferred to storage tubes immediately after centrifugation and stored at −80 °C for 3 weeks until calprotectin measurements were performed in a single run with use of the thawed samples. The study was approved by the Regional Ethical Committees for the Region of Southern Denmark, and informed consent was obtained from all participants.

View this table:
  • View inline
  • View popup
Table 1.

Calprotectin measurements at different storage conditions (time and temperature) prior to centrifugation.

Calprotectin assay

Analysis of serum/plasma calprotectin was performed as previously described (6) by use of the automated ELIA Calprotectin for ImmunoCap250 (Thermo Fisher Scientific). The established reference interval for serum calprotectin (2.5–97.5%) is 1040–4262 μg/L. Establishment of the reference interval for the serum calprotectin assay was conducted with use of serum samples obtained from a cohort of 120 healthy adult Danish blood donors (62 females and 58 males, 19–66 years of age) at Odense University Hospital. Participants were excluded if any of the following was present: pregnancy, autoimmune diseases, AIDS/HIV, infectious diseases, cancer, allergy, high (diastolic BP > 90 mmHg) or low (systolic BP < 110 mmHg) blood pressure, known cardiovascular disease (including thromboses), diabetes requiring treatment, severe skin diseases treated with steroids, ulcer, current use of narcotics or medicine, recent operation/tattoo/piercing, or treatment with hormones. All samples were aliquoted immediately after sampling and stored at −80 °C until analysis (6).

Blood count

Enumeration of leukocytes, neutrophils, and monocytes was determined in the EDTA whole blood samples immediately after sampling (tube: EDTA) with a Sysmex XN-10 hematology analyzer.

Statistical analysis

Variables are presented as medians and ranges. Because data tested with the D'Agostino-Pearson omnibus test did not follow a normal distribution, nonparametric tests were used. The Mann–Whitney U-test was used to evaluate differences between 2 samples, while Friedman's test was used for more than 2 samples.

Correlation between variables was evaluated with Spearman's rank correlation and interpreted according to the guidelines by Colton (12). Data were analyzed by use of GraphPad Prism and differences were considered statistically significant at P < 0.05.

RESULTS

Effect of storage temperature

The results of the calprotectin measurements for different storage conditions are shown in Table 1. Calprotectin concentrations in EDTA samples stored for 2 h before analysis at 4 °C, 20 °C, or 37 °C did not differ (P = 0.15). After storage, calprotectin concentrations in serum and Li-heparin samples increased with increasing temperature, and the differences between all storage temperatures were significant for both sample tubes (P < 0.001).

Effect of storage time

Calprotectin concentrations did not differ between samples centrifuged immediately and samples stored for 2 h at 20 °C in EDTA, Li-heparin, or serum samples (P = 0.95, P = 0.26, and P = 0.74, respectively).

Effect of anticoagulant

The results of the calprotectin measurements for different types of anticoagulant are shown in Table 2. Pairwise comparison of samples stored for 2 h revealed no differences between serum and Li-heparin when stored at 4 °C and 20 °C. At 37 °C serum calprotectin concentrations were significantly higher than in Li-heparin samples (P < 0.001). Calprotectin levels in EDTA tubes were significantly lower than calprotectin concentrations in serum at all temperatures. For samples stored at 4 °C, the median concentrations in EDTA samples were approximately 2 times lower than in serum (P < 0.001). At 20 °C the EDTA plasma concentrations were 8.6-fold lower than in serum (P < 0.001), and in samples stored at 37 °C EDTA plasma concentrations were 10.3-fold lower than in serum (P < 0.001).

View this table:
  • View inline
  • View popup
Table 2.

Calprotectin measurements in samples with different types of anticoagulant stored for 2 h.

Correlation between calprotectin and cell count

The results of the cell counts for the 14 volunteers are shown in Table 3.

View this table:
  • View inline
  • View popup
Table 3.

The results of cell count for the 14 volunteers.

There was a moderately strong linear relationship between serum calprotectin concentrations at 20 °C and the leukocyte count (Spearman's r = 0.58, P = 0.032), as well as the neutrophil count (Spearman's r = 0.70, P < 0.001; Fig. 1), but not between serum calprotectin and monocyte count (Spearman's r = −0.12, P = 0.7). The correlation between the total leukocyte count and calprotectin levels in Li-heparin and EDTA samples did not achieve statistical significance (Spearman's r = 0.47, P = 0.09) and Spearman's r = 0.29, P = 0.33 respectively).

Fig. 1.
  • Download figure
  • Open in new tab
Fig. 1. Correlation between serum calprotectin in samples clotted at room temperature versus total leucocyte and neutrophile count in matched EDTA samples.

DISCUSSION

The key findings in this study were 1) a significant effect of storage temperature of uncentrifuged samples on the calprotectin concentration in serum and Li-heparin samples, 2) a significantly lower concentration of calprotectin in EDTA samples compared to serum at all temperatures, 3) a stable calprotectin concentration in EDTA samples regardless of storage temperature or time, and 4) a moderately strong linear relationship between serum calprotectin and the leukocyte and neutrophil count.

A previous study compared calprotectin levels in matching EDTA-plasma and serum samples, and revealed no difference in fresh nonfrozen samples (6). This suggests that the difference between matrices happens during sample processing after sampling. Also, this indicates that the level of calprotectin is stable in EDTA regardless of postsampling processing. We therefore believe the concentration in EDTA samples reflects the patient level the most accurately (at sampling).

During aggregation and activation platelets release a wide variety of proteins (e.g., proinflammatory cytokines) into serum. Exposure to proinflammatory cytokines causes activation of leukocytes and subsequent release of factors involved in an inflammatory response from the cells (11). Our finding of increased calprotectin concentrations due to neutrophil activation is also consistent with earlier reports (13). On the basis of this, we hypothesize that in-vitro leukocyte activation, and thereby calprotectin levels, may depend on anticoagulant, storage time, and storage temperature.

The temperature-dependent increase in serum calprotectin concentrations and the positive correlation between serum calprotectin and the leukocyte and neutrophil counts support the hypothesis that granulocytes are activated during serum clotting and release of granulocyte-activating substances leads to release of calprotectin.

Li-heparin samples also displayed a temperature-dependent increase in calprotectin concentrations suggesting that blood clotting is not the only factor necessary for release of calprotectin in vitro. Heparin has been widely investigated as an antiinflammatory agent, (14) and low molecular weight heparin has been shown to cause release of cytokines from human leukocytes (15). It is possible that heparin from the sample tubes stimulates release of calprotectin from granulocytes.

Because the volunteers included here were not selected on the basis of health status, we cannot be sure of absence of diseases contributing to increased calprotectin levels in some of the blood samples. However, this does not conflict with the aim of the study, which is an investigation of preanalytical factors influencing calprotectin measurements independent of calprotectin concentrations pretesting. Thus, our findings contribute information to the characteristics of the preanalytical phase in laboratory testing of calprotectin, which is important information because this analysis is widely used in various clinical settings.

The study has some limitations that should be noted. We only examined the effect of increasing temperatures over a 2-h period, which does not allow general conclusions to be drawn regarding temperature and time effects on calprotectin measurements. Also, we did not compare the levels of calprotectin before and after freezing, so we cannot eliminate that the freezing procedure had an impact on measurements. As mentioned earlier, a previous comparison of calprotectin levels in fresh, nonfrozen serum and EDTA samples did not show any difference (6), suggesting that freezing of serum/plasma may affect the calprotectin levels differently in the 3 samples types. If serum calprotectin is investigated in biobanked samples, further study of a possible freeze–thaw effect in other sample types is warranted.

In conclusion, the temperature-dependent increase in calprotectin concentrations in serum and heparin tubes left for some time before centrifugation indicate these collection conditions are suboptimal for clinical purposes. We therefore recommend use of EDTA plasma for determination of calprotectin in blood, as this sample matrix does not appear to be affected by temperature or time between collection and analysis when investigated over a 2-h period. Also, neutrophil activation due to clotting or centrifugation should be avoided during the preanalytical process.

Acknowledgments

The authors thank Birgit Lene Pedersen and Lone Sørensen for excellent technical assistance.

Footnotes

  • Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

  • Received July 24, 2017.
  • Accepted September 18, 2017.
  • © 2017 American Association for Clinical Chemistry

References

  1. 1.↵
    1. Ehrchen JM,
    2. Sunderkotter C,
    3. Foell D,
    4. Vogl T,
    5. Roth J
    . The endogenous Toll-like receptor 4 agonist S100A8/100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 2009;86:557–66.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Sedaghat F,
    2. Notopoulos A
    . S100 protein family and its application in clinical practice. Hippokratia 2008;12:198–204.
    OpenUrlPubMed Order article via Infotrieve
  3. 3.↵
    1. Naess-Andresen CF,
    2. Egelandsdal B,
    3. Fagerhol MK
    . Calcium binding and concomitant changes in the structure and heat stability of calprotectin (L1 protein). J Clin Pathol 1995;48:278–84.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Østgård RD,
    2. Deleuran BW,
    3. Dam MY,
    4. Hansen IT,
    5. Jurik AG,
    6. Glerup H
    . Faecal calprotectin detects subclinical bowel inflammation and may predict treatment response in spondylarthritis. Scand J Rheumatol 2017;26:1–8.
    OpenUrl
  5. 5.
    1. Bae SC,
    2. Lee YH
    . Calprotectin levels in rheumatoid arthritis and their correlation with disease activity: a meta-analysis. Postgrad Med 2017;129:531–7.
    OpenUrl
  6. 6.↵
    1. Pedersen L,
    2. Nybo M,
    3. Poulsen MK,
    4. Henriksen JE,
    5. Dahl J,
    6. Rasmussen LM
    . Plasma calprotectin and its association with cardiovascular disease manifestations, obesity and the metabolic syndrome in type 2 diabetes mellitus patients. BMC Cardiovasc Disord 2014;14:196.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  7. 7.↵
    1. Magnette A,
    2. Chatelain M,
    3. Chatelain B,
    4. Ten Cate H,
    5. Mullier F
    . Pre-analytical issues in the haemostasis laboratory: guidance for the clinical laboratories. Thromb J 2016;14:49.
    OpenUrl
  8. 8.↵
    1. Semple JW,
    2. Freedman J
    . Platelets and innate immunity. Cell Mol Life Sci 2010;67:499–511.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  9. 9.
    1. Blok SL,
    2. Engels GE,
    3. van Oeveren W
    . In vitro hemocompatability testing: the importance of fresh blood. Biointerphases. 2016;11:029802
  10. 10.↵
    1. Ho CH,
    2. Chan IH
    . The influence of time of storage, temperature of storage, platelet number in platelet-rich plasma, packed cell, mean platelet volume, hemoglobin concentration, age, and sex on platelet aggregation test. Ann Hematol 1995;71:129–33.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  11. 11.↵
    1. Bazzoni G,
    2. Dejana E
    , Del Maschio. Platelet-neutrophil interactions: possible relevance in the pathogenesis and inflammation. Haematologica. 1991;76:491–9.
    OpenUrlPubMed Order article via Infotrieve
  12. 12.↵
    1. Colton T
    . Statistics in Medicine. 1st ed. Boston: Little, Brown; 1974.
  13. 13.↵
    1. Cotoi OS,
    2. Dunér P,
    3. Ko N,
    4. Hedblad B,
    5. Nilsson J,
    6. Björkbacka H,
    7. Schiopu A
    . Plasma S100A8/A9 correlates with blood neutrophil counts, traditional risk factors, and cardiovascular disease in middle-aged healthy individuals. Arterioscler Thromb Vasc Biol 2014;34:202–10.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Koller M,
    2. Kutscha-Lissberg F,
    3. Brom J,
    4. Weidinger G,
    5. Muhr G
    . Influence of low molecular weight heparin (certoparin) and unfractionated heparin on the release of cytokines from human leukocytes. Inflammation 2001;25:331–7.
    OpenUrlCrossRefPubMed Order article via Infotrieve
  15. 15.↵
    1. Young E
    . The anti-inflammatory effects of heparin and related compounds. Thromb Res 2008;122:743–52.
    OpenUrlCrossRefPubMed Order article via Infotrieve
View Abstract
PreviousNext
Back to top

In this issue

The Journal of Applied Laboratory Medicine: 2 (6)
Vol. 2, Issue 6
May 2018
  • Table of Contents
  • About the Cover
  • Index by author
Print
Share
Sample Type and Storage Conditions Affect Calprotectin Measurements in Blood
Lise Pedersen, Erling Birkemose, Charlotte Gils, Sara Safi, Mads Nybo
The Journal of Applied Laboratory Medicine May 2018, 2 (6) 851-856; DOI: 10.1373/jalm.2017.024778
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Article Alerts
Sign In to Email Alerts with your Email Address
Citation Tools
Sample Type and Storage Conditions Affect Calprotectin Measurements in Blood
Lise Pedersen, Erling Birkemose, Charlotte Gils, Sara Safi, Mads Nybo
The Journal of Applied Laboratory Medicine May 2018, 2 (6) 851-856; DOI: 10.1373/jalm.2017.024778

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • METHODS
    • RESULTS
    • DISCUSSION
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

Articles

  • Who Gets a Laboratory Test in Hospital, Why, and How Often? A Retrospective Observational Study of 4 Australian Hospitals
  • Europium Nanoparticle-Based Sialyl-Tn Monoclonal Antibody Discriminates Epithelial Ovarian Cancer–Associated CA125 from Benign Sources
  • Comparison of Transcription-Mediated Amplification and Real-Time PCR Assays for Hepatitis B Virus DNA Quantitation in Serum
Show more Articles

Laboratory Management

  • Utilization Review of Paraneoplastic Neurological Syndrome Antibody Screening Panels: Experience at a Tertiary Academic Health Center
  • Reengineering Critical Laboratory Testing for Timely Chemotherapeutic Management
Show more Laboratory Management

Similar Articles

Subjects

  • Laboratory Management

Navigate

  • Home
  • About
  • Articles
  • Information for Authors
  • Submit

Other Publications

  • Clinical Chemistry
Footer logo

© 2019 American Association for Clinical Chemistry

Powered by HighWire