Re-evaluation of the measurement of haptoglobin in human plasma samples

Abstract

Haptoglobin (Hp) is an abundant plasma protein scavenging hemoglobin (Hb) via CD163 on macrophages. This process consumes Hp, which therefore negatively correlates to hemolysis. However, exact measurements of Hp plasma levels are complicated by different phenotypes (Hp1-1, Hp2-1, and Hp2-2) forming different oligomeric states with differences in immunoreactivity. In addition, humans have an immune-cross-reactive Hp-related protein. In the present study, we developed Hp-specific monoclonal antibodies for an accurate Hp analysis of the different Hp phenotypes in a panel of 112 anonymous samples from hospitalized individuals subjected to routine Hp immunoturbidimetric measurements. The data revealed immunoturbidimetry as a reliable method in most cases but also that the use of non-phenotype-specific calibrators leads to substantial bias in the measurement of the Hp-concentration, non at least in Hp1-1 individuals. Furthermore, analysis of the Hb-dependence of the CD163 interaction with Hp1-1 and Hp2-2 showed that a higher ‘cost-effectiveness’ in the consumption of dimeric Hp1-1 versus multimeric Hp phenotypes is a likely contribution to the observed differences in the plasma levels of the Hp phenotypes. In conclusion, the determination of Hp phenotype and the use of phenotype-specific calibrators are essential to obtain a precise estimate of the Hp level in healthy and diseased individuals.

Keywords:

• Haptoglobins
• hemoglobins
• blood proteins
• phenotype
• enzyme-linked immunosorbent assay
• immunoassay

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Acknowledgments

We would like to thank Signe Marie Andersen, Malene Bille Jørgensen, Jette Hvelplund, and Maiken Lumby Henriksen for excellent technical assistance. Figure 6 was created with BioRender.com.

Disclosure statement

Authors state no conflict of interest.

Additional information

Funding

This work was supported by the Danish National Research Foundation under Grant DNRF141 to Center for Functional Genomics and Tissue Plasticity (ATLAS); the Danish Council for Independent Research under Grant 4004-001; and Novo Nordisk Foundations under Grant NNF14OC0011537.