Lab Matters Fall 2023 | Page 69

APHL 2023 POSTER ABSTRACTS

MS / MS X-ALD Cutoff Establishment and Onboarding

C . Kirwan , C . Porter , G . Bonn ; Colorado Department of Public Health and Environment

X-linked Adrenoleukodystrophy ( X-ALD ) is a peroxisomal disorder originating in the ABCD1 gene located on the X-chromosome . Producing a transmembrane transport protein , the gene facilitates transport of very long chain fatty acids across the membrane for beta-oxidation and subsequent metabolic production . Impaired function originating from lack of transport proteins or gene mutations leads to the accumulation of very long chain fatty acids ( VLCFAs ), such as C26:0-lysophosphatidylcholine ( C26:0-LPC ) and C24:0-lysophosphatidylcholine ( C24:0-LPC ) analytes . The accumulation of these analytes leads to detectable screening testing through the use of MS / MS mass spectroscopy for initial newborn screening testing conducted by the Colorado Newborn Screening Program ( CONBSP ) at Colorado Department of Public Health and Environment ( CDPHE ). Following the validation of PerkinElmer NeoBase 2 and MS / MS QSight instruments in July 2022 , CONBSP initiated the induction of X-ALD and C26:0-LPC testing , bringing Colorado and Wyoming in line with the new Recommended Uniform Screening Program ( RUSP ) requirements for screening of disorders . A validation plan was designed to address accuracy and precision of establishing C26:0-LPC analyte on two of the PerkinElmer MS / MS Qsight 210MD instruments . Utilizing CDC Quarter 1 and Quarter 4 QC and PT samples , for accuracy and precision , respectively , determination of statistically significant differences and lack thereof established confidence in CONBSP capabilities in C26:0-LPC screening in population testing . Establishment of an initial conservative cutoff of 0.9 μmol / L for C26:0-LPC was validated due to the rarity of the disorder and determination of cutoff , taking into consideration data trends and literature in more than 23,000 patient samples run routinely Sunday – Friday over the course of four months followed with confirmation testing . A 98th percentile cutoff was established from these patient samples and correlated to PerkinElmer ’ s reference study cutoffs . Cutoff adjustments will be made post onboarding through the use of PerkinElmer Biochemical for LC / MS secondary testing in review . Additionally , during the study , the analysis of CDC quality control samples stored (> 3 months ) long term versus short term (< 3 months ) was conducted in addition to those stored in the fridge ( 2-8 ° C ) versus freezer (<-10 ° C ) in regards to the stability of C26:0-LPC for MS / MS testing .

Presenter : Cullan Kirwan , kirwancullan @ hotmail . com

Validation and Implementation of a Custom Second Tier Next-Generation Sequencing Assay for Improved Accuracy of Cystic Fibrosis Screening at DCLS

J . Chavez , L . Lion ; Virginia Division of Consolidated Laboratory Services

The current Cystic Fibrosis ( CF ) screening assay at DCLS includes a biochemical test for elevated Immunoreactive Trypsinogen ( IRT ) followed by a Luminex 39-mutation gene panel for those samples that fall within the daily top 4 % of IRT levels . The Luminex panel currently in use detects 39 common CF variants , enabling us to refer babies for diagnostic testing with a high level of sensitivity .

However , this panel is known to lack the heterogeneity necessary to adequately screen the allelic diversity of CF Transmembrane Conductance Regulator ( CFTR ) gene mutations in Virginia . Variant detection is significantly lower in non-white newborns due to the Eurocentric composition of the Luminex-39 panel . The DCLS Newborn Screening Group and the Sequencing and Bioinformatics Group are developing and validating a targeted next-generation sequencing ( NGS ) assay for the expansion of the current CF second-tier screen . The NGS assay will be run using Illumina MiSeq instruments and the corresponding Illumina library prep kit . This assay will include DNA extraction from dried blood spot cards , library preparation , DNA sequencing and comparison of sequencing results to the variants listed in CFTR2 . This assay aims to identify most variants classified as “ CF-causing ,” “ varying clinical significance ” and “ unknown significance ” in the CFTR2 database , totaling over 400 variants . This presentation will outline gaps in the current assay and the benefits of implementing the NGS assay in its place , then discuss key aspects of the validation of this assay . While the assay is not expected to be implemented before the APHL 2023 Conference , this poster will include a prospective analysis of its accuracy , precision and impact on the screening of CF in the VA population .

Presenter : Joseph Chavez , joseph . chavez @ dgs . virginia . gov

Validation of a Multiplex Assay for SCID / SMA Newborn Screening

C . Weaver , South Carolina Department of Health & Environmental Control Public Health Laboratory

Severe Combined Immunodeficiency ( SCID ) is a group of disorders characterized by reduction of T-cells leading to a fatal loss of adaptive immunity . Molecular-based assays can identify SCID patients prior to symptom onset and expedite therapeutic intervention . The PerkinElmer NeoMDx assay accomplishes this by detecting DNA fragments produced during T-cell maturation called T-cell Receptor Excision Circles ( TRECs ). The multiplexed NeoMDx assay also screens for Spinal Muscular Atrophy ( SMA ) which is a disease characterized by progressive loss of motor neurons leading to immobility , respiratory failure and death . Successful therapies are available but are most effective before symptom onset . As with SCID , newborn screening has allowed for expedited SMA treatment . The NeoMDx assay targets the SMN1 gene because both copies are deleted in > 95 % of SMA patients . The South Carolina Public Health Laboratory ( SCPHL ) recently validated the PerkinElmer NeoMDx assay for SCID and SMA newborn screening . The assay proved to be > 98 % accurate for both targets ( TREC and SMN1 ) and the internal reference ( RPP30 ) over three levels of control material . Intra- and inter-day precision were well within acceptable limits of 15 % coefficient of variance . SCPHL tested 5571 deidentified , residual specimens to define internal controls and diagnostic cutoffs . Using specimens from known SCID and SMA cases , diagnostic sensitivity was found to be 100 % for both SCID and SMA while specificity was 99.8 % for SCID and 100 % for SMA . Based on data presented here , SCPHL approved NeoMDx as a high complexity CLIA regulated test and went live with the method in September 2022 .

Presenter : Cory Weaver , weavercj @ dhec . sc . gov