Evaluation Of Mass Spectrometry Based Approaches For The Diagnosis Of Hemoglobinopathies

Mass spectrometry (MS) is becoming an accepted technique for clinical applications. It has already been shown to be a valuable technique for the characterisation of structural hemoglobin variants, and its applicability for screening purposes has been demonstrated by several research groups. It has not yet been implemented as an approved clinical laboratory technique to screen for hemoglobinopathies, especially the thalassemias. In order to replace existing methods the proposed approach has to provide improved technical, financial and medical benefits. For the purposes of antenatal/neonatal screening laboratories, all methods must at least detect HbS, HbC, HbD-Punjab, HbE, HbO-Arab, Hb Lepore, elevated HbF, and elevated HbA₂ (to distinguish α and β thalassemia).

Three mass spectrometry based approaches have been evaluated against a current standard method (cation exchange liquid chromatography, HPLC, and gel electrophoresis) in a clinical trial incorporating 2017 patient samples, in 12 batches. The MS methods used were:

1. 1.

   Measurement of the mass of intact globin chains and the relative proportion of β and δ globin chains. When any α or β variant was detected, this was followed by tandem MS of tryptic peptides to confirm identity.

2. 2.

   Ultra performance liquid chromatography+MS/MS experiments using pre-determined tryptic peptides to screen for clinically important structural variants.

3. 3.

   Mass measurement of intact globin chains followed by top down electron transfer dissociation (ETD) fragmentation of selected globin chains for confirmation of variant (no complex pre-analytical sample digestion/separation, or second stage analysis).

Variant detection

The clinical samples were all tested using methods 1 and 2. Method 3 has been used on a more limited sample set which, however, included all samples which were identified to have clinically significant variants. All methods agreed with the hospital lab results for >99.5% of samples. However, a number of additional observations could be made using the mass spectrometry data. The current clinical laboratory method was found to give false results on three samples (0.15%), two of which were identified by the existing approach as HbD-Punjab but were in fact characterised unambiguously by MS as HbG-Philadelphia. One sample was identified as containing elevated HbF by the existing method but was characterised as a β-chain variant using MS. In addition, seven patient samples were found by mass spectrometry to contain non-significant Hb variants which were not detected using the existing hospital method.

HbA₂ measurement (based on ratio of δ chains to ‘δ+β' chains)

The correlation coefficient (R²) for HbA2 measurement between HPLC and MS in the 12 batches varied from 0.57-0.90 (method 1), and 0.72-0.90 (method 2, using the T5 tryptic digest peptide pair), but with significant systematic bias requiring either calibration to a standard or adjustment of the normal range. However, a cut-off between normal and abnormal was clearly evident, and there was complete agreement between HPLC and MS methods in categorisation of samples as beta-thalassemia trait. Method 3 has shown potential to measure HbA₂levels but has not yet been tested on a large batch of clinical samples.

The approaches differ in their instrumentation requirements, sample introduction, sample preparation and data interpretation. Key issues to be considered when selecting the most appropriate method to develop for future hospital use include cost, speed, sensitivity, selectivity, potential for automation and diagnostic information provided.