Short communication3-years experience review of neonatal screening for hemoglobin disorders using tandem mass spectrometry
Introduction
Newborn screening programs for hemoglobin disorders have become common in North America and in some European countries. Most of them presently use IEF or HPLC as an initial testing method [1], [2]. The reliability of both techniques is acceptable when analysis is performed on whole blood or cord blood. But neonatal screening policies recommend the use of dried blood spots and the interpretation of the results obtained on such samples may sometimes be challenging.
During the last decade, tandem mass spectrometry has evolved as a reference technique in neonatal screening laboratories for the identification of inherited metabolic diseases [3], [4], [5], [6]. Therefore, it could be convenient to use the same equipment for hemoglobinopathy screening. Mass spectrometry has been previously described for the characterization of hemoglobin mutations [7], [8], [9], [10], [11] and technically, TMS is a more sensitive and specific method than IEF or HPLC. Theoretically, an optimized method could allow the identification of about all hemoglobin mutants, but getting such specificity requires fastidious and complex interpretative algorithm [9]. Therefore, in opposite to IEF or HPLC, most TMS routine protocols mainly focus on the detection of specific variants.
Considering that, we have developed a mass spectrometry approach to screen for hemoglobin mutations. Most variants have poor clinical significance and the objectives of the United Kingdom NHS Sickle Cell & Thalassaemia Screening Programme mainly focused on the identification of infants at risk of sickle cell disorders and cases of β-thalassemia [12]. Therefore, we centered our method to the detection of HbS, HbC and HbE, and to the identification of β globin production defaults. Validation results and comparison with IEF results have been previously documented [13]. Identification of HbDPunjab, HbOArab or HbLepore had initially not be considered in our screening method since prevalence of those variants in our population is insignificant and clinical interest in terms of prevention is limited to sickle cell syndromes (i.e. Hb SDPunjab, Hb SOArab or Hb SLepore) [14], [15], [16].
We report here the results of our three year experience using tandem mass spectrometry for the detection of the major Hb variants in East-Belgian newborns.
Section snippets
Samples
All samples (dried blood spot on filter paper, Whatman 903) were obtained from three to five day-old neonates. Sample collection was part of the mandatory neonatal screening program of the French Community of Belgium.
Tryptic digestion
Technical specifications of the MS method were described previously [13]. Briefly, 3.2-mm diameter disc was punched into 96-well microtiter plates and elution was achieved by gentle rotation with deionized water (200 μL) for one hour. 100 μL aliquots were transferred to fresh
Results
During the last three years, 43,736 newborns have been tested for hemoglobin disorders. Among the results yielded, and according to the calculated MRM ratios, 444 samples were considered positive. Confirmatory testing on hemoglobinopathy mutations was performed on all positive samples by genotyping the whole β globin gene and we did not detect any discrepancy between the initial screening method and the molecular confirmation throughout the period study. According to the previous comparison
Discussion
This report illustrates the use of tandem mass spectrometry to identify the major Hb variants in a European region with a significant immigrated population from Southern Europe and Africa. It allowed us to detect 16 patients with severe hemoglobin disorders during this three year period (12 Hb SS, 1 Hb SC, 1 Hb S/β0-thalassemia and 2 β0-thalassemia), which represents 0.04% of the 43,736 analyzed newborns. All identified patients were referred to pediatricians for early medical care and
Abbreviations
- IEF
Isoelectric Focusing
- HPLC
High Performance Liquid Chromatography
- TMS
tandem mass spectrometry
- Hb
hemoglobin
- ACN
acetonitrile
- MRM
multiple reaction monitoring
Acknowledgments
The authors thank A.M. François and E. Nandrin for their excellent technical assistance.
References (18)
- et al.
Hot electron capture dissociation distinguishes leucine from isoleucine in a novel hemoglobin variant, Hb Askew, Beta54(D5)Val–>Ile
J Am Soc Mass Spectrom
(2009) - et al.
Rapid identification of hemoglobin variants by electrospray ionization mass spectrometry
Blood Cells Mol Dis
(2001) - et al.
Hemoglobin H disease: not necessarily a benign disorder
Blood
(2003) - et al.
Evaluation of cation-exchange HPLC compared with isoelectric focusing for neonatal hemoglobinopathy screening
Clin Chem
(1999) - et al.
Neonatal screening for haemoglobin variants using filter paper-dried blood specimens
Clin Lab Haematol
(1991) - et al.
Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry
Pediatr Res
(1995) - et al.
Newborn screening with tandem mass spectrometry: 12 months' experience in NSW Australia
Acta Paediatr Suppl
(1999) Using tandem mass spectrometry for metabolic disease screening among newborns
- et al.
Expanded newborn screening for inborn errors of metabolism by electrospray ionization–tandem mass spectrometry: results, outcome, and implications
Pediatrics
(2003)
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