Clinical method evaluation of hemoglobin S and C identification by top-down selected reaction monitoring and electron transfer dissociation

Kaveh Samii,Didia Coelho Graça,Alexander Scherl,Wolfgang Jabs,Nicolas Vuilleumier,Pierre Lescuyer,Denis Hochstrasser,Lorella Clerici,Ralf Hartmer,Olivier Lassout,Yury O. Tsybin

doi:10.1186/s12014-019-9261-1

Abstract

BackgroundBiological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C.MethodsWe adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples.ResultsThe developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C.ConclusionsThis top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

Highlights

Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample
The process starts with patient information, clinical history and hematological data (i.e. Hb levels, red blood cell morphology) which serves as a first indicator for a hemoglobin disorder [4]
We evaluated the concordance of the TD electron transfer dissociation (ETD) mass spectrometry (MS) method with standard CEX-high-performance liquid chromatography (HPLC) diagnostic procedures to identify Hb A, Hb C, Hb E variants, as the most frequent Hb disorders encountered in routine hematology diagnostics

Summary

Introduction

Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Hemoglobin disorders diagnosis is a complex process based on the combination of clinical and biological data. The process starts with patient information (i.e. ethnic origin), clinical history and hematological data (i.e. Hb levels, red blood cell morphology) which serves as a first indicator for a hemoglobin disorder [4]. Identification of the most common and clinically significant Hb variants (i.e. Hb S, Hb C, Hb E, Hb D-Punjab and Hb O-Arab) faces the same problem. They are all the result of a single point mutation on the Hb β chain. Hb S and Hb C result from the substitution of valine or lysine, respectively, instead of glutamic acid at position 6 on β chain; Hb E from the substitution of lysine instead of glutamic acid at position 26 on β chain, Hb D-Punjab and Hb O-Arab from the substitution of glutamine or lysine, respectively, instead of glutamic acid at position 121 on β chain

Methods

Results

Conclusion