In Wisconsin (WI), the nomination to add X-linked adrenoleukodystrophy (X-ALD) to the WI newborn screening (NBS) panel was reviewed and recommended to be accepted by the Metabolic Subcommittee on September 21, 2021, and then reviewed and also recommended to accept by the Umbrella Committee on December 3, 2021. On March 4, 2022, the Secretary’s Advisory Committee on Newborn Screening (SACNBS) reviewed the nomination, and voted unanimously in favor of adding X-ALD to the WI NBS panel. The next steps are the Secretary’s acceptance of the recommendation from the SACNBS committee, and then the Governor’s approval. After the final approval, a rulemaking process will occur, may take up to 2 years.
We request funds to increase our public NBS lab’s capacity and capability in order to add X-ALD. These funds will allow us to implement NBS for X-ALD in WI through a pilot process before the administrative rule becomes effective. We aim to implement NBS for X-ALD, and to re-structure the tandem mass spectrometry (MS/MS) NBS testing process not only to ensure smoothly integrating NBS for X-ALD into the current ongoing routine NBS workflow, but also to enhance overall MS/MS NBS testing.
Specific Aim 1: Identify newborns with X-ALD using a fully validated novel flow injection analysis with negative ion mode tandem mass spectrometry in a two-tier testing setting.
C26:0-Lysophosphatidylcholine (C26:0-LPC) is the primary biomarker used in NBS for X-ALD. We recently developed an X-ALD screening assay to measure C26:0-LPC levels in dried blood spot NBS specimens using a flow injection analysis (FIA), coupled with electrospray ionization (ESI) MS/MS performed in negative ion mode. The presented FIA method shortens analysis run-time to 1.7 minutes, while maintaining the previously established advantage of utilizing negative mode MS/MS to eliminate isobaric interferences that could lead to screening false positives. Specimens with a C26:0-LPC value greater than the cutoff set for the first tier testing, a multiplexing MS/MS assay in positive mode, will undergo this second tier X-ALD screening test. This method was recently published.
If we implemented NBS for X-ALD as a standalone testing process, we would need separate and additional instruments and personnel. Here we propose a two-tier approach. Using FDA-approved and commercially available multiplexing as a first-tier test to measure C26:0-LPC (NeoBase™ 2 Non-derivatized MSMS kit). Only specimens above the cutoff in the first-tier test would undergo the second-tier assay that utilizes negative mode MS/MS to eliminate isobaric interferences that could lead to screening false positives. This two-tier testing approach will lead to a highly sensitive and specific outcome for X-ALD testing.
Specific Aim 2: Improve MS/MS NBS testing efficiency and performance through multiplexing and two-tier testing algorithm.
The two-tier testing strategy also provides an opportunity to multiplex additional succinylacetone (SUAC) with NeoBase™ 2 Non-derivatized MSMS kit. In WI, a standalone process to measure SUAC is currently done in NBS for tyrosinemia type I. Similar to NBS for X-ALD, only specimens with a SUAC value above the cutoff will undergo SUAC measurement using a calibration curve based quantitation. Multiplexing strategy will certainly provide the efficacy needed in a high throughput public health NBS laboratory, but only if coupled with the second tier testing will it not compromise the screening performance with low false positive rates.
By the end of this two-year funding cycle, we will achieve the following:
1. Establish a highly sensitive and highly specific two-tier NBS for X-ALD test algorithm with 90,000 newborns screened for X-ALD.
2. Implement an updated MS/MS NBS testing strategy: first-tier testing simultaneously measures multiple analytes to achieve high screening sensitivity efficiently; second-tier testing measures disorder specific analytes to achieve high specificity confidently.
