Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter February 23, 2022

Comparison of three different protocols for obtaining hemolysis

Nora Nikolac Gabaj ORCID logo, Marijana Miler ORCID logo, Alen Vrtaric ORCID logo, Ivana Celap ORCID logo, Marina Bocan, Petra Filipi, Vanja Radisic Biljak ORCID logo, Ana-Maria Simundic ORCID logo, Vesna Supak Smolcic ORCID logo and Marija Kocijancic ORCID logo



Hemolysis is associated with erroneous or delayed results. Objectives of the study were to compare four different methods for obtaining hemolysis in vitro on three different analyzers.


Hemolysis was prepared with addition of pure hemoglobin into serum pool, osmotic shock, aspiration through blood collection needle, freezing/thawing of whole blood. Biochemistry parameters were measured in duplicate at Architect c8000 (Abbott, Abbott Park, USA), Beckman Coulter AU680 (Beckman Coulter, Brea, USA) and Cobas 6000 c501 (Roche, Mannheim, Germany), according to manufacturers’ declarations. Cut-off value was defined as the highest value of H index with corresponding bias lower than acceptance criteria.


We were not able to obtain results with freezing protocol. On all three platforms, lowest number of analytes were sensitive to hemolysis at H=0.5 using method of adding free hemoglobin. When osmotic shock was used, cut-off values for the most analytes were generally met at lower values. Hemolysis significantly interfered with measurement of potassium and lactate dehydrogenase (LD) at H=0.5 on all platforms. The most of the tested analytes had the lowest acceptable H index when aspiration method was used. At the low level of hemolysis (H=0.8) glucose, sodium, potassium, chloride, phosphate, and LD were affected on all analyzers, with some additional analytes depending on the manufacturer.


Hemolysis interference differs on different analyzers and according to protocol for obtaining hemolysis. Aspiration method was generally the most sensitive to hemolysis interference, while addition of free Hb was the most resistant.

Corresponding author: Marijana Miler, Working Group for Preanalytical Phase of the Croatian Society of Medical Biochemistry and Laboratory Medicine, Zagreb, Croatia; and Department of Clinical Chemistry, Sestre milosrdnice University Hospital Center, Zagreb, Croatia, E-mail:

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Not applicable.

  5. Ethical approval: The study was approved by the Ethical Committee of the Sestre milosrdnice University Hospital Center. For the study, samples were prepared from the remaining portions of serum and whole blood pools after routine laboratory testing was completed. Demographic, anamnestic or clinical data on patients were not collected.


1. Lippi, G, Salvagno, GL, Montagnana, M, Brocco, G, Guidi, GC. Influence of hemolysis on routine clinical chemistry testing. Clin Chem Lab Med 2006;44:311–6. in Google Scholar

2. Marques-Garcia, F. Methods for hemolysis interference study in laboratory medicine - a critical review. EJIFCC 2020;31:85–97.Search in Google Scholar

3. Dolci, A, Panteghini, M. Harmonization of automated hemolysis index assessment and use: is it possible? Clin Chim Acta 2014;432:38–43. in Google Scholar

4. Nikolac Gabaj, N, Miler, M, Vrtarić, A, Hemar, M, Filipi, P, Kocijančić, M, et al.. Precision, accuracy, cross reactivity and comparability of serum indices measurement on Abbott Architect c8000, Beckman Coulter AU5800 and Roche Cobas 6000 c501 clinical chemistry analyzers. Clin Chem Lab Med 2018;56:776–88. in Google Scholar

5. von Meyer, A, Cadamuro, J, Lippi, G, Simundic, AM. Call for more transparency in manufacturers declarations on serum indices: on behalf of the Working Group for Preanalytical Phase (WG-PRE), European Federation of Clinical Chemistry and Laboratory Medicine (EFLM). Clin Chim Acta 2018;484:328–32. in Google Scholar

6. Clinical and Laboratory Standards Institute (CLSI). Hemolysis, icterus, and lipemia/turbidity indices as indicators of interference in clinical laboratory analysis; approved guideline. CLSI document C56-A. Wayne, PE, USA: Clinical and Laboratory Standards Institute; 2012.Search in Google Scholar

7. Clinical Laboratory Standards Institute. Interference testing in clinical chemistry; approved guideline, 3rd ed. CLSI document EP07. Wayne, Pennsylvania, USA: Clinical Laboratory Standards Institute; 2018.Search in Google Scholar

8. Lippi, G. Interference studies: focus on blood cell lysates preparation and testing. Clin Lab 2012;58:351–5.Search in Google Scholar

9. Lippi, G, Salvagno, GL, Montagnana, M, Brocco, G, Cesare Guidi, G. Influence of the needle bore size used for collecting venous blood samples on routine clinical chemistry testing. Clin Chem Lab Med 2006;44:1009–14. in Google Scholar

10. Lippi, G, Musa, R, Aloe, R, Mercadanti, M, Pipitone, S. Influence of temperature and period of freezing on the generation of hemolysate and blood cell lysate. Clin Biochem 2011;44:1267–9. in Google Scholar

11. Gidske, G, Sølvik, UØ, Sandberg, S, Kristensen, GBB. Hemolysis interference studies: freeze method should be used in the preparation of hemolyzed samples. Clin Chem Lab Med 2018;56:e220–2. in Google Scholar

12. Ceriotti, F, Fernandez-Calle, P, Klee, GG, Nordin, G, Sandberg, S, Streichert, T, et al.. Criteria for assigning laboratory measurands to models for analytical performance specifications defined in the 1st EFLM Strategic Conference. Clin Chem Lab Med 2017;55:189–94. in Google Scholar

13. American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2021. Diabetes Care 2021;44(1 Suppl):S15–33. in Google Scholar

14. National Kidney Foundation. KDOQI clinical practice guideline for hemodialysis adequacy: 2015 update. Am J Kidney Dis 2015;66:884–930. in Google Scholar

15. Ricós, C, Alvarez, V, Cava, F, García-Lario, JV, Hernández, A, Jiménez, CV, et al.. Current databases on biological variation: pros, cons and progress. Scand J Clin Lab Invest 1999;59:491–500. 10.1080/00365519950185229.10.1080/00365519950185229Search in Google Scholar PubMed

16. Gidske, G, Aakre, KM, Rustad, P, Sandberg, S, Norling, A, Pelanti, J, et al.. Handling of hemolyzed serum samples in clinical chemistry laboratories: the Nordic hemolysis project. Clin Chem Lab Med 2019;57:1699–711. in Google Scholar

17. Dimeski, G. Effects of hemolysis on the Roche ammonia method for Hitachi analyzers. Clin Chem 2004;50:976–7. in Google Scholar

18. Delgado, JA, Morell-Garcia, D, Bauça, JM. Hemolysis interference studies: the particular case of sodium ion. EJIFCC 2019;30:25–34.Search in Google Scholar

19. Koseoglu, M, Hur, A, Atay, A, Cuhadar, S. Effects of hemolysis interferences on routine biochemistry parameters. Biochem Med 2011;21:79–85. in Google Scholar

20. Perović, A, Dolčić, M. Influence of hemolysis on clinical chemistry parameters determined with Beckman Coulter tests - detection of clinically significant interference. Scand J Clin Lab Invest 2019;79:154–9. in Google Scholar

Received: 2021-11-23
Accepted: 2022-02-11
Published Online: 2022-02-23
Published in Print: 2022-04-26

© 2022 Walter de Gruyter GmbH, Berlin/Boston