Newborn Blood Spot Screening for Sickle Cell Disease by Using Tandem Mass Spectrometry: Implementation of a Protocol to Identify Only the Disease States of Sickle Cell Disease

Abstract

BACKGROUND The currently recommended technologies of HPLC and isoelectric focusing for newborn blood spot screening for sickle cell disease (SCD) identify both the disease and carrier states, resulting in large numbers of infants being followed up unnecessarily. Analysis of blood spot tryptic peptides performed by using tandem mass spectrometry (MS/MS) is an alternative technology to detect hemoglobin (Hb) variant disorders. METHODS We analyzed 2154 residual newborn blood spots and 675 newborn blood spots from infants with Hb variants by using MS/MS after trypsin digestion. Screening cutoffs were developed by using the ratio between the variant peptide–to–wild-type peptide abundance for HbS, C, DPunjab, OArab, Lepore, and E peptides. A postanalytical data analysis protocol was developed using these cutoffs to detect only the disease states of SCD and not to identify carrier states. A parallel study of 13 249 newborn blood spots from a high-prevalence SCD area were analyzed by both MS/MS and HPLC. RESULTS Screening cutoffs developed distinguished the infants with the disease states of SCD, infants who were carriers of SCD, and infants with normal Hb. In the parallel study no false-negative results were identified, and all clinically relevant cases were correctly identified using the MS/MS protocol. Unblinding the data revealed a total of 328 carrier infants that were successfully excluded by the protocol. CONCLUSIONS The screening protocol developed correctly identified infants with the disease states of SCD. Furthermore, large numbers of sickle cell carrier infants were successfully not identified, thereby avoiding unnecessary follow-up testing and referral for genetic counseling.

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

“…However, the latter hypothesis cannot be ruled out, since we do not have access to the volume of blood transfused. Identification of newborns who received transfusions, by applying the HbA/HbF ratio as described elsewhere, was compared to our approach. In our study, the cutoff HbA/HbF ratio > 0.6, corresponding to 99th percentile, observed in the normal no transfusion group, could be used as the threshold (Table ).…”

“…We will analyze this threshold prospectively in order to confirm its utility. Moreover, the need for a specific ratio (HbA/abnormal Hb) to distinguish carriers from SCD disease has been reported, especially with tandem mass spectrometry (MS/MS) . The HbA/abnormal Hb ratio was constant whatever the GA (data not shown), and indicated the ability of the method to detect Hb variants even in extremely preterm infants.…”

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

“…The same group then screened Ͼ40,000 newborns in Belgium and successfully detected 16 patients with severe hemoglobin disorders (26). Moats et al used a similar approach to screen Ͼ13,000 newborns in the UK for Sickle Cell disease and correctly identified all seven disease occurrences (27). Because hemoglobin is the most abundant protein in whole blood (ϳ150 mg/ml), these four studies achieved adequate sensitivity by simply infusing the trypsin digested samples into a triple-quadrupole MS. To move beyond hemoglobin, additional sensitivity can be provided by using liquid chromatography (LC) separations coupled online with MRM.…”

Newborn screening for sickle cell disease in Europe: recommendations from a Pan‐European Consensus Conference

“…It was also acknowledged that other methods are emerging, e.g. matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS) and DNA‐based methods (Hachani et al , ; Moat et al , , ; Daniel & Henthorn, , ; Theberge et al , ; Detemmerman et al , ). There was consensus that new methods should be demonstrated to be at least as sensitive and as specific as HPLC and CE before they be adopted for routine screening.…”