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Abstract
Background
Intravascular hemolysis and in vitro hemolysis are prevalent contributors to failed blood sample analysis in the routine hospital laboratory. Interferences by hemoglobin in spectrophotometric and certain enzyme activity assays is the major causative factor.
Methods
By exploiting the hemoglobin-binding properties of the iron-regulated surface determinant H (IsdH) protein from Staphylococcus aureus we have developed a new method to instantly remove hemoglobin and hemoglobin-haptoglobin complexes from plasma in vitro thereby enabling the measurement of hemoglobin-sensitive analytes in hemolyzed plasma. In the present study we used an engineered IsdH mutant form conjugated to Sepharose for the efficient removal of plasma hemoglobin in concentrations up to 15 mg/mL. The high abundance of haptoglobin, which forms a tight complex with hemoglobin in plasma, did not affect the hemoglobin removal by IsdH Sepharose.
Results
Applying the method on plasma samples that beforehand were spiked with blood hemolysate re-enabled measurement of the hemolysis sensitive parameters: alkaline phosphatase, conjugated bilirubin, iron, ferritin, γ-glutamyltransferase, total thyroxine and troponin T. IsdH Sepharose-mediated hemoglobin removal also enabled measurement of hemolysis sensitive parameters in hemolyzed samples from anonymized patients.
Conclusions
In conclusion, IsdH Sepharose is a simple cost-effective pretreatment of hemolyzed samples correcting and enabling the measurement of several important hemoglobin-sensitive parameters in a way compatible with standard procedures in routine laboratories.
Intravascular hemolysis and in vitro hemolysis are prevalent contributors to failed blood sample analysis in the routine hospital laboratory. Interferences by hemoglobin in spectrophotometric and certain enzyme activity assays is the major causative factor.
Methods
By exploiting the hemoglobin-binding properties of the iron-regulated surface determinant H (IsdH) protein from Staphylococcus aureus we have developed a new method to instantly remove hemoglobin and hemoglobin-haptoglobin complexes from plasma in vitro thereby enabling the measurement of hemoglobin-sensitive analytes in hemolyzed plasma. In the present study we used an engineered IsdH mutant form conjugated to Sepharose for the efficient removal of plasma hemoglobin in concentrations up to 15 mg/mL. The high abundance of haptoglobin, which forms a tight complex with hemoglobin in plasma, did not affect the hemoglobin removal by IsdH Sepharose.
Results
Applying the method on plasma samples that beforehand were spiked with blood hemolysate re-enabled measurement of the hemolysis sensitive parameters: alkaline phosphatase, conjugated bilirubin, iron, ferritin, γ-glutamyltransferase, total thyroxine and troponin T. IsdH Sepharose-mediated hemoglobin removal also enabled measurement of hemolysis sensitive parameters in hemolyzed samples from anonymized patients.
Conclusions
In conclusion, IsdH Sepharose is a simple cost-effective pretreatment of hemolyzed samples correcting and enabling the measurement of several important hemoglobin-sensitive parameters in a way compatible with standard procedures in routine laboratories.
| Original language | English |
|---|---|
| Journal | Clinical Chemistry and Laboratory Medicine |
| Volume | 57 |
| Issue number | 11 |
| Pages (from-to) | 1690-1698 |
| ISSN | 1434-6621 |
| DOIs | |
| Publication status | Published - Nov 2019 |
Keywords
- haptoglobin
- hemoglobin
- hemolysis
- iron-regulated surface determinant H (IsdH)
- plasma
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Danmarks Grundforskningsfond – Centers of Excellence - ATLAS - DG Center for Functional Genomics and Tissue Plasticity
Mandrup, S. (Head coordinator), Grøntved, L. (PI), Ravnskjær, K. (PI), Krag, A. (PI), Moestrup, S. K. (PI), Graversen, J. H. (Co-PI), Blagoev, B. (PI), Madsen, J. G. S. (Co-PI), Lauridsen, M. E. M. (Project participant), Thiele, M. (Project participant), Wernberg, C. W. (PhD student), Nielsen, R. (Project participant), Marcher, A.-B. (Project participant), Siersbæk, M. (Project participant), Mortensen, T. P. (Project participant), Sarvari, A. K. (Project participant), van Hauwaert, E. L. (PhD student), Avolio, F. (Project participant), Boel, F. (PhD student), Skytthe, M. K. (PhD student), Terkelsen, M. K. (PhD student), Bendixen, S. M. (PhD student), Dam, T. V. (PhD student), Hansen, D. (PhD student) & Elmelund-Præstekær, L. C. B. (Coordinator)
01/10/2017 → 30/09/2023
Project: Research