Relationship between neonatal screening results by HPLC and the number of α-thalassaemia gene mutations; consequences for the cut-off value

Abstract

Objectives To evaluate the relationship between FAST peak percentage by adapted Bio-Rad Vnbs analysis using the valley-to-valley integration and genotypes with the aim to improve differentiation between severe a-thalassaemia forms (HbH disease) and the milder disease types. Method DNA analysis for a-thalassaemia was performed on 91 dried blood spot samples presenting normal and elevated FAST peak levels, selected during three years of Dutch national newborn screening. Results Significant differences were found between samples with and without a-thalassaemia mutations, regardless of the genetic profiles. No significant difference was demonstrated between HPLC in 2a/aa and 2a/2a, between 2a/2a and --/aa and between --/aa and --/2a genotypes. Conclusion This study confirms that the percentage HbBart's, as depicted by the FAST peak, is only a relative indication for the number of a genes affected in a-thalassaemia. Based on the data obtained using the modified Bio-Rad Vnbs software, we adopted a cut-off value of 22.5% to discriminate between possible severe a-thalassaemia or HbH disease and other a-thalassaemia phenotypes. Retrospectively, if this cut-off value was utilized during this initial three-year period of neonatal screening, the positive predictive value would have been 0.030 instead of 0.014.

New approach to accurate interpretation of sickle cell disease newborn screening by applying multiple of median cutoffs and ratios

“…Automation of the first step of SCD neonatal screening offers high throughput with full traceability, a high degree of reproducibility, sensitivity, and a low limit of detection . Precise measurement of Hb fractions in SCD NBS has recently been taken into account mainly in relation to the quantification of Hb Barts in order to discriminate between HbH disease and minor alpha thalassemia . However, to the best of our knowledge, no studies have been carried out to standardize the quantitative interpretation of the different hemoglobin percentages routinely analyzed by HPLC.…”

“…10 Precise measurement of Hb fractions in SCD NBS has recently been taken into account mainly in relation to the quantification of Hb Barts in order to discriminate between HbH disease and minor alpha thalassemia. 11,12 However, to the best of our knowledge, no studies have been carried out to standardize the quantitative interpretation of the different hemoglobin percentages routinely analyzed by HPLC. For this purpose, we needed to determine normal values and establish optimum cutoff values that could be used routinely in NBS.…”

Newborn screening for α-thalassaemia by a capillary electrophoresis method

“…Bouva et al 1 found that the percentage haemoglobin Bart's (HbBart's), as depicted by the FAST peak, was only a relative indication for the number of a genes affected in a-thalassaemia. No significant difference was demonstrated between HPLC in 2a/aa and 2a/2a, between 2a/2a and --/aa or between --/aa and --/2a genotypes.…”

Response to Goodness et al.

“…In our hands, using the Vnbs system, the percentage HbF ('total F') is calculated by adding the F1 peak percentage with the F peak percentage. To determine the effect of the new gradient, we re-analysed the samples from our previous paper 2 with both method 1.08 (old) and 1.013 (new). We also analysed 186 random fresh samples of the routine screening programme, using both methods.…”