Skip to main content

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

An Erratum to this article was published on 21 April 2016


Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2



Cerebrospinal fluid


Follicular fluid




Red blood cell


Matrix assisted laser desorption ionisation time-of-flight mass spectrometry


Ultraviolet visible spectrometry


  1. 1.

    You JS, Gelfanova V, Knierman MD, Witzmann FA, Wang M, Hale JE (2005) The impact of blood contamination on the proteome of cerebrospinal fluid. Proteomics 5(1):290–296. doi:10.1002/pmic.200490084

    CAS  Article  Google Scholar 

  2. 2.

    Zhang J (2007) Proteomics of human cerebrospinal fluid—the good, the bad, and the ugly. Proteomics Clin Appl 1(8):805–819. doi:10.1002/prca.200700081

    CAS  Article  Google Scholar 

  3. 3.

    Levay PF, Huyser C, Fourie FL, Rossouw DJ (1997) The detection of blood contamination in human follicular fluid. J Assist Reprod Genet 14(4):212–217

    CAS  Article  Google Scholar 

  4. 4.

    Jozwik M, Wolczynski S (1998) Technical note: blood contamination of aspirated follicular fluid. J Assist Reprod Genet 15(7):409–410

    CAS  Article  Google Scholar 

  5. 5.

    Romeo MJ, Espina V, Lowenthal M, Espina BH, Petricoin EF 3rd, Liotta LA (2005) CSF proteome: a protein repository for potential biomarker identification. Expert Rev Proteomics 2(1):57–70. doi:10.1586/14789450.2.1.57

    CAS  Article  Google Scholar 

  6. 6.

    Sarapik A, Velthut A, Haller-Kikkatalo K, Faure GC, Bene MC, de Carvalho Bittencourt M, Massin F, Uibo R, Salumets A (2012) Follicular proinflammatory cytokines and chemokines as markers of IVF success. Clin Dev Immunol 2012:606459. doi:10.1155/2012/606459

    Article  Google Scholar 

  7. 7.

    Liu AX, Zhu YM, Luo Q, Wu YT, Gao HJ, Zhu XM, Xu CM, Huang HF (2007) Specific peptide patterns of follicular fluids at different growth stages analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Biochim Biophys Acta 1770(1):29–38. doi:10.1016/j.bbagen.2006.06.017

    CAS  Article  Google Scholar 

  8. 8.

    Jarkovska K, Kupcova Skalnikova H, Halada P, Hrabakova R, Moos J, Rezabek K, Gadher SJ, Kovarova H (2011) Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization. Mol Hum Reprod 17(11):679–692. doi:10.1093/molehr/gar047

    CAS  Article  Google Scholar 

  9. 9.

    Zhang J, Goodlett DR, Quinn JF, Peskind E, Kaye JA, Zhou Y, Pan C, Yi E, Eng J, Wang Q, Aebersold RH, Montine TJ (2005) Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease. J Alzheimers Dis 7(2):125-133; discussion 173-180

  10. 10.

    Bruegel M, Planert M, Baumann S, Focke A, Bergh FT, Leichtle A, Ceglarek U, Thiery J, Fiedler GM (2009) Standardized peptidome profiling of human cerebrospinal fluid by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Proteomics 72(4):608–615. doi:10.1016/j.jprot.2008.11.018

    CAS  Article  Google Scholar 

  11. 11.

    Piras AM, Dessy A, Chiellini F, Chiellini E, Farina C, Ramelli M, Della Valle E (2008) Polymeric nanoparticles for hemoglobin-based oxygen carriers. Biochim Biophys Acta 1784(10):1454–1461. doi:10.1016/j.bbapap.2008.03.013

    CAS  Article  Google Scholar 

  12. 12.

    Rezeli M, Vilaghy B, Kilar F, Kanyo K, Torok B, Torok A (2002) Significant differences in capillary electrophoretic patterns of follicular fluids and sera from women pre-treated for in vitro fertilization. J Biochem Biophys Methods 53(1–3):151–156

    CAS  Article  Google Scholar 

  13. 13.

    Stoop MP, Coulier L, Rosenling T, Shi S, Smolinska AM, Buydens L, Ampt K, Stingl C, Dane A, Muilwijk B, Luitwieler RL, Sillevis Smitt PA, Hintzen RQ, Bischoff R, Wijmenga SS, Hankemeier T, van Gool AJ, Luider TM (2010) Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples. Mol Cell Proteomics 9(9):2063–2075. doi:10.1074/mcp.M900877-MCP200

    CAS  Article  Google Scholar 

  14. 14.

    Biroccio A, Urbani A, Massoud R, di Ilio C, Sacchetta P, Bernardini S, Cortese C, Federici G (2005) A quantitative method for the analysis of glycated and glutathionylated hemoglobin by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Anal Biochem 336(2):279–288. doi:10.1016/j.ab.2004.10.002

    CAS  Article  Google Scholar 

  15. 15.

    Greco V, Pieragostino D, Piras C, Aebersold R, Wiltfang J, Caltagirone C, Bernardini S, Urbani A (2014) Direct analytical sample quality assessment for biomarker investigation: qualifying cerebrospinal fluid samples. Proteomics 14(17–18):1954–1962. doi:10.1002/pmic.201300565

    CAS  Article  Google Scholar 

  16. 16.

    Jimenez CR, Koel-Simmelink M, Pham TV, van der Voort L, Teunissen CE (2007) Endogeneous peptide profiling of cerebrospinal fluid by MALDI-TOF mass spectrometry: Optimization of magnetic bead-based peptide capture and analysis of preanalytical variables. Proteomics Clin Appl 1(11):1385–1392. doi:10.1002/prca.200700330

    CAS  Article  Google Scholar 

  17. 17.

    Liu XD, Zeng BF, Xu JG, Zhu HB, Xia QC (2006) Proteomic analysis of the cerebrospinal fluid of patients with lumbar disk herniation. Proteomics 6(3):1019–1028. doi:10.1002/pmic.200500247

    Article  Google Scholar 

  18. 18.

    Leal-Noval SR, Munoz-Gomez M, Arellano-Orden V, Marin-Caballos A, Amaya-Villar R, Marin A, Puppo-Moreno A, Ferrandiz-Millon C, Flores-Cordero JM, Murillo-Cabezas F (2008) Impact of age of transfused blood on cerebral oxygenation in male patients with severe traumatic brain injury. Crit Care Med 36(4):1290–1296. doi:10.1097/CCM.0b013e3181692dfc

    Article  Google Scholar 

  19. 19.

    Hanrieder J, Nyakas A, Naessen T, Bergquist J (2008) Proteomic analysis of human follicular fluid using an alternative bottom-up approach. J Proteome Res 7(1):443–449. doi:10.1021/pr070277z

    CAS  Article  Google Scholar 

  20. 20.

    Norwitz ER, Tsen LC, Park JS, Fitzpatrick PA, Dorfman DM, Saade GR, Buhimschi CS, Buhimschi IA (2005) Discriminatory proteomic biomarker analysis identifies free hemoglobin in the cerebrospinal fluid of women with severe preeclampsia. Am J Obstet Gynecol 193(3 Pt 2):957–964. doi:10.1016/j.ajog.2005.06.055

    CAS  Article  Google Scholar 

  21. 21.

    Raymackers J, Daniels A, De Brabandere V, Missiaen C, Dauwe M, Verhaert P, Vanmechelen E, Meheus L (2000) Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization–mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry. Electrophoresis 21(11):2266–2283. doi:10.1002/1522-2683(20000601)21:11<2266:AID-ELPS2266>3.0.CO;2-Z

    CAS  Article  Google Scholar 

  22. 22.

    Yuan X, Russell T, Wood G, Desiderio DM (2002) Analysis of the human lumbar cerebrospinal fluid proteome. Electrophoresis 23(7–8):1185–1196. doi:10.1002/1522-2683(200204)23:7/8<1185:AID-ELPS1185>3.0.CO;2-G

    CAS  Article  Google Scholar 

  23. 23.

    Sinha A, Srivastava N, Singh S, Singh AK, Bhushan S, Shukla R, Singh MP (2009) Identification of differentially displayed proteins in cerebrospinal fluid of Parkinson’s disease patients: a proteomic approach. Int J Clin Chem 400(1–2):14–20. doi:10.1016/j.cca.2008.09.026

    CAS  Google Scholar 

Download references


The authors are grateful to the patients for their enrollment in the study and to clinicians from the West-Tallinn Central Hospital Neurological Department (Dr. Katrin Gross-Paju) and the Nova Vita Clinic (Agne Velthut-Meikas). The authors also owe their gratitude to Dr. Jüri Laasik for his guidance on biological sample collection. This work was supported by the Estonian Ministry of Education and Research [Grant IUT19-8 (P.P) and IUT34-16 (A.S)], Estonian Science Foundation Grants 8811 (P. P.) and 8385 (T. K.), scholarships from SA Archimedes and World Federation of Scientists (K. L.) and by Enterprise Estonia, Grant No. EU30020.

Author information



Corresponding author

Correspondence to Katrina Laks.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Laks, K., Kirsipuu, T., Dmitrijeva, T. et al. Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS. Protein J 35, 171–176 (2016).

Download citation


  • Biological fluid
  • Blood contamination
  • Mass spectrometry