Sensitive measurement of free haemoglobin in serum and urine to assess in vivo haemolysis
A new and highly sensitive analytical technique has been developed for the detection and quantification of low concentrations of free haemoglobin in serum or urine. Potential applications include the assessment of in vivo haemolysis during extra-corporal membrane oxygenation (ECMO) in intensive care settings, as well as quality assurance and control in testing of transfusion products.
Situation before
Extracorporeal membrane oxygenation (ECMO) is a lifesustaining treatment used to maintain blood oxygenation during cardiac surgery or in cases of severe respiratory complications, such as COVID-19 pneumonia. In ECMO, the cardiovascular system of the patient is connected to an external circuit that temporarily replaces the heart and lung function. However, ECMO is associated with serious side effects, including in vivo haemolysis, a life-threatening complication.
Free haemoglobin serves as a good biomarker to detect and predict the severity of chronic haemolysis. Together with complete blood count and platelet function tests, free haemoglobin provides critical information for physicians in intensive care departments to assess the status of an unconscious patient.
Current analytical methods for quantifying free haemoglobin at low concentrations are limited. Available sensitive methods are labour-intensive and require manual handling, are prone to interference or rely on toxic reagents such as benzidine. The faster automated methods based on direct spectrophotometry, usually designed for higher rates of in vitro haemolysis, lack analytical sensitivity and precision needed for clinical monitoring at concentrations below <100 mg/L.
Hence there is a clear need for an automated, sensitive, cost-effective safe and reliable technique for quantification of free haemoglobin in clinical and quality assurance applications.
Technology
In contrast to conventional one-step direct spectroscopy, the newly developed technique employs two measurements. First, a baseline absorption of oxyhaemoglobin, the oxygenated form of haemoglobin, is assessed. Following this first measurement, a reagent is added to degrade the oxyhaemoglobin structure. After degradation, a second measurement is performed, and the difference in absorbance before and after degradation is calculated against a calibration curve. This difference directly reflects the concentration of free haemoglobin in the sample.
The method demonstrates high specificity as it avoids breakdown of the sample matrix. This reduces the interference effect of other compounds, such as bilirubin and lipemia.
Our experiments show that the technique can accurately quantify free haemoglobin to a concentration as low as 15 mg/L, representing a 3-8 fold improvement in sensitivity in comparison to leading commercial analysers, e.g. Roche Cobas which is limited to concentrations >60 mg/L. The method exhibits a linear range between 10 and 1000 mg/L, which is physiologically relevant for ECMO and transfusion monitoring.
This enhanced analytical sensitivity provides a one- to two-day earlier warning of haemolytic events, enabling earlier clinical intervention. The technique is free of toxic (carninogenous) compounds, does not suffer from icteric interferences up to 10 mg/dL and is fully REACH-compliant.
Partners we search for
- Industrial partners with expertise in assay development and automation to commercialise the assay into an IVDRcompliant kit compatible with fully automated analysers.
- Clinical partners, such as hospitals, to implement and validate the method within their own laboratories to assess the inter-lab reproducibility and support the broader clinical adoption of this new technique.
About the researchers/research group
The technology has been jointly developed by the University of Antwerp and the Antwerp University Hospital (Department of Laboratory Medicine).
The Department of Laboratory Medicine is responsible for the analysis of human biological samples to support the diagnosis and monitoring of a wide range of diseases. Within this department the subdivision of Clinical Chemistry focuses on the assessment of biomarkers to enhance patient diagnosis and therapeutic follow-up.
IP position
An international patent application with publication number WO2023/036906 was filed on 9/09/2022. This international patent application has entered the national phase in Europe and the United States.
More information
University of Antwerp
Valorisation Office
Middelheimlaan 1
2020 Antwerpen