Endogenous, non-reducing end glycosaminoglycan biomarkers are superior to internal disaccharide glycosaminoglycan biomarkers for newborn screening of mucopolysaccharidoses and GM1 gangliosidosis.

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidoses (MPSs). Our previous publications on glycosaminoglycan (GAG) biomarker levels in DBS for mucopolysaccharidosis type 1 (MPS-I) and MPS-II demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we evaluate two methods for measuring GAG biomarkers in seven MPS types and GM1 gangliosidosis. We obtained newborn DBS from patients with MPS-IIIA-D, -IVA, -VI, -VII, and GM1 gangliosidosis. These samples were analyzed via two GAG mass spectrometry methods: (1) The internal disaccharide biomarker method; (2) The endogenous non-reducing end (NRE) biomarker method. This study supports the use of second-tier GAG analysis of newborn DBS by the endogenous NRE biomarker method, as part of NBS to reduce the false positive rate.

Performance of OC-Auto Micro 80 Fecal Immunochemical Test in an Integrated Academic-Community Health System.

GOALS: We aimed to determine the performance of the OC-Auto Micro 80 fecal immunochemical test (FIT) in an average-risk population receiving care in an integrated, academic-community health system. BACKGROUND: The FIT is the most used colorectal cancer (CRC) screening test worldwide. However, many Food and Drug Administration-cleared FIT products have not been evaluated in clinical settings. STUDY: We performed a retrospective cohort study of patients (50 to 75 y old) in the University of Washington Medicine health care system who were screened for CRC by OC-Auto Micro 80 FIT between March 2016 and September 2021. We used electronic health records to extract patient-level and clinic-level factors, FIT use, colonoscopy, and pathology findings. The primary outcomes were the FIT positivity rate and neoplasms detected at colonoscopy. Secondary outcomes were FIT positivity by sex and safety-net versus non-safety-net clinical settings. RESULTS: We identified 39,984 FITs completed by 26,384 patients; 2411 (6.0%) had a positive FIT result (>100 ng/mL of hemoglobin in buffer), and 1246 (51.7%) completed a follow-up colonoscopy. The FIT positive rate was 7.0% in men and 5.2% in women (P <0.01). Among those who completed a colonoscopy after an abnormal FIT result, the positive predictive value for CRC, advanced adenoma, and advanced neoplasia was 3.0%, 20.9%, and 23.9%, respectively. CONCLUSIONS: In a retrospective analysis of a large heterogeneous population, the OC-Auto Micro 80 FIT for CRC screening demonstrated a positivity rate of 6.0% and a positive predictive value for CRC of 3.0%.

Detecting 22q11.2 Deletion Syndrome in Newborns with Low T Cell Receptor Excision Circles from Severe Combined Immunodeficiency Screening.

OBJECTIVE: Based on experiences and results from newborn screening for severe combined immunodeficiency (SCID), we evaluated the occurrence of chromosome 22q11.2 deletion syndrome (22q11.2DS) in newborns with different T cell receptor excision circles (TREC) results and established a second tier genetic test for 22q11.2DS. STUDY DESIGN: Recalled dried blood spots from 486 newborns with TREC results <90 copies/uL were tested from the SCID newborn screening. Quantitative real-time polymerase chain reaction assay was used to detect the copy number of TBX1 and HIRA genes by simple DNA extraction method. Multiplex ligation dependent probe amplification was used for further confirmation. RESULTS: Four hundred sixty-eight cases were considered negative because their haploid copy number of TBX1 and HIRA genes was >0.75. Eighteen cases with TBX1 and/or HIRA gene copy number <0.75 were suspected as positive, and 13 cases were further confirmed with 22q11.2DS. Detection rates of 22q11.2DS were 10.7% (6/56) in TREC <30 copies, 6.8% (9/132) in <50 TREC copies, 4.6% (12/260) in <70 TREC copies, and 2.7% (13/486) in <90 TREC copies. CONCLUSIONS: 22q11.2DS detection can be incorporated into the second-tier assay in subjects with low TREC copies in SCID screening. The dried blood spot methods were feasible for 22q11.2DS newborn screening.

Taiwan National Newborn Screening Program by Tandem Mass Spectrometry for Mucopolysaccharidoses Types I, II, and VI.

OBJECTIVE: To evaluate the initial cutoff values, rates of screen positives, and genotypes for the large-scale newborn screening program for multiple mucopolysaccharidoses (MPS) in Taiwan. STUDY DESIGN: More than 100 000 dried blood spots were collected consecutively as part of the national Taiwan newborn screening programs. Enzyme activities were measured by tandem mass spectrometry from dried blood spot punches. Genotypes were obtained when a second newborn screening specimen again had a decreased enzyme activity. Additional clinical evaluation was then initiated based on enzyme activity and/or genotype. RESULTS: Molecular genetic analysis for cases with low enzyme activity revealed 5 newborns with pathogenic alpha-L-iduronidase mutations, 3 newborns with pathogenic iduronate-2-sulfatase mutations, and 1 newborn was a carrier of an arylsulfatase B mutation. Several variants of unknown pathogenic significance were also identified, most likely causing pseudodeficiency. CONCLUSIONS: The highly robust tandem mass spectrometry-based enzyme assays for MPS-I, MPS-II, and MPS-VI allow for high-throughput newborn screening for these lysosomal storage disorders. Optimized cutoff values combined with second tier testing could largely eliminate false-positive results. Accordingly, newborn screening for these lysosomal storage disorders is possible.

Functional and biological studies of α-galactosidase A variants with uncertain significance from newborn screening in Taiwan.

Fabry disease is an X-linked disorder resulted from deficiency of α-galactosidase A (GLA) activity. In Taiwan, a total of 792,247 newborns were screened from 2008 to 2014 in two newborn screening centers, and 13 variants of uncertain significance (VOUS) in the GLA gene were identified. To determine whether these variants were pathogenic or not, functional, biochemical, clinical and pedigree analyses were performed. In vitro functional assay was established through site-directed mutagenesis, and four in silico tools were used to predict pathogenesis. The enzyme activity of dried blood spots and plasma metabolite lyso-Gb3 level from subjects with the variants were measured. Additionally, clinical manifestations were evaluated extensively from the subjects and their relatives. Our results revealed that p.G104V, p.I232T, p.D322H, and p.G360C all exhibited relatively low residual enzyme activities and elevated plasma lyso-Gb3 level. These data strongly suggest that these Fabry mutations may cause classical or later-onset phenotypes. In contrast, neither significantly clinical symptoms nor elevated lyso-Gb3 level was found in cases with p.P60S, p.A108T, p.S304T, p.R356Q, and p.P362T variants, which may be non-pathogenic or milder forms of Fabry variants. More data need to be included for the patients with p.N53D, p.P210S, p.M296L, and p.K391T variants. The established system provides us more information to classify these GLA variants.

Lymphocyte Galactocerebrosidase Activity by LC-MS/MS for Post-Newborn Screening Evaluation of Krabbe Disease.

BACKGROUND: Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. Newborn screening for Krabbe disease is ongoing, but improved methods for follow-up analysis of screen-positive babies are needed to better advise families and to optimize treatment. We report a new assay for the enzymatic activity of GALC in lymphocytes. METHODS: T lymphocytes were isolated from venous blood by magnetic bead technology. The assay used a close structural analog of the natural substrate and LC-MS/MS to quantify the amount of product with the aid of a chemically identical internal standard. RESULTS: The analytical range of the assay (ratio of assay response for the QC high standard to that from all non-enzymatic-dependent processes) was 20-fold greater than that for the conventional radiometric GALC assay. The LC-MS/MS could distinguish cells that were null in GALC from those that contained traces of active enzyme (down to 0.3% of normal). There was a good correlation between the level of residual GALC activity in lymphocytes and the severity of Krabbe disease. CONCLUSIONS: The new assay can measure small amounts of residual GALC activity in leukocytes with high accuracy compared to previous assays and can contribute, along with genotyping, biomarker analysis, and neurological imaging, a better plan for post-newborn screening follow-up for Krabbe disease.

Mass Spectrometry but Not Fluorimetry Distinguishes Affected and Pseudodeficiency Patients in Newborn Screening for Pompe Disease.

BACKGROUND: Deficiency of the lysosomal enzyme acid α-glucosidase (GAA) causes Pompe disease. Newborn screening for Pompe disease is ongoing, and improved methods for distinguishing affected patients from those with pseudodeficiency, especially in the Asian population, would substantially reduce the number of patient referrals for clinical follow-up. METHODS: We measured the enzymatic activity of GAA in dried blood spots on newborn screening cards (DBS) using a tandem mass spectrometry (MS/MS) assay. The assay displayed a relatively large analytical range compared to the fluorimetric assay with 4-methylumbelliferyl-α-glucoside. DBS from newborns confirmed to have infantile-onset Pompe disease (IOPD, n = 11) or late-onset Pompe disease (LOPD) (n = 12) and those from patients bearing pseudodeficiency alleles with or without Pompe mutations, or Pompe disease carriers (n = 230) were studied. RESULTS: With use of the MS/MS GAA assay in DBS, 96% of the pseudodeficiency newborns and all of the Pompe disease carriers were well separated from the IOPD and LOPD newborns. The fluorimetric assay separated <10% of the pseudodeficiencies from the IOPD/LOPD group. CONCLUSIONS: The relatively large analytical range MS/MS GAA assay but not the fluorimetric assay in DBS provides a robust approach to reduce the number of referrals and should dramatically facilitate newborn screening of Pompe disease.

Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs. METHODS: We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization. RESULTS: In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis. CONCLUSIONS: This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

Very Early Treatment for Infantile-Onset Pompe Disease Contributes to Better Outcomes.

OBJECTIVE: To evaluate whether very early treatment in our patients would result in better clinical outcomes and to compare these data with other infantile-onset Pompe disease (IOPD) cohort studies. METHODS: In this nationwide program, 669,797 newborns were screened for Pompe disease. We diagnosed IOPD in 14 of these newborns, and all were treated and followed in our hospital. RESULTS: After 2010, the mean age at first enzyme-replacement therapy (ERT) was 11.92 days. Our patients had better biological, physical, and developmental outcomes and lower anti-rh acid α-glucosidase antibodies after 2 years of treatment, even compared with one group that began ERT just 10 days later than our cohort. No patient had a hearing disorder or abnormal vision. The mean age for independent walking was 11.6 ± 1.3 months, the same age as normal children. CONCLUSIONS: ERT for patients with IOPD should be initiated as early as possible before irreversible damage occurs. Our results indicate that early identification of patients with IOPD allows for the very early initiation of ERT. Starting ERT even a few days earlier may lead to better patient outcomes.

Clinical implications of opioid parent-metabolite ratios.

BACKGROUND: The opioid epidemic has underscored the importance of urine drug testing in the management of chronic pain. However, interpreting test results can be challenging, especially in scenarios where medications may have been directly added to urine samples to simulate compliance. METHODS: We conducted a retrospective analysis of 9,690 opioid testing results using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study aimed to define the expected ratios between parent drugs and metabolites for eight commonly prescribed opioids. Cases with a parent-metabolite ratio above the 95th percentile were subjected to chart review. RESULTS: A total of 13 cases appeared likely consistent with simulated compliance with buprenorphine, 2 with methadone, 14 with oxycodone, and one with hydrocodone. The unusual patterns of parent-metabolite ratio can also be associated with hyperacute drug exposures/use, pharmaceutical impurity, or underlying liver enzyme deficiency. Furthermore, patients who failed the decision limits could exhibit other illicit use or aberrant behaviors. CONCLUSION: Laboratories conducting LC-MS/MS-based opioid testing can more objectively identify anomalies by analyzing parent-metabolite ratios. When in consultation with providers, laboratories can point to these data when suggesting the possibility of simulated compliance and help identify cases warranting further investigation.

Unraveling the unexpected: Interference in iron assay leads to diagnosis of multiple myeloma.

BACKGROUND: Iron studies are critical for diagnosing iron deficiency and hemochromatosis. We present a case exhibiting macrocytic anemia with perplexingly high plasma iron concentrations. METHODS AND RESULTS: The initial clinical presentation with significantly elevated iron results raised concerns for hemochromatosis. However, inconsistent results in dilution studies suggested the presence of an interfering substance. Inspection of the reaction curves from the instrument revealed very high background absorption in the 800 nm channel. This, coupled with the observation of an insoluble precipitate upon mixing the acid buffer reagent with the patient's serum, as well as the patient's high total protein and low albumin levels, suggested immunoglobulin overproduction. Serum protein electrophoresis confirmed a monoclonal gammopathy with a subsequent diagnosis of multiple myeloma. CONCLUSION: Excessive monoclonal immunoglobulins can precipitate in acidic buffers and interfere with spectrophotometric measurements in iron testing. Although challenging, investigating an interference and determining its cause can uncover underlying diseases that have yet to be diagnosed.

Benzodiazepine analysis by an improved LC-MS/MS method illustrates usage patterns in Washington State.

BACKGROUND: The standard approach for benzodiazepine detection often includes immunoassay followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The illicit use of non-prescribed benzodiazepines has been trending up nationally. METHODS: We developed and validated an improved LC-MS/MS assay for benzodiazepine detection in urine. We expanded the testing panel by adding five drugs to the previous panel of ten. We determined the prevalence of individual benzodiazepines in our patient population. Immunoassay results were compared with LC-MS/MS to evaluate assay performance. RESULTS: Clonazepam and alprazolam were the most common benzodiazepines present. Etizolam and flualprazolam were also prevalent in Washington State. Compared with the LC-MS/MS assay, the immunoassay had variable cross-reactivity, which explained false negative and false positive immunoassay results. The inclusion of new drugs in the LC-MS/MS panel significantly reduced the incidence of immunoassay results interpreted as falsely positive. CONCLUSION: New illicit benzodiazepines have emerged regionally and nationally. The inclusion of novel drugs in LC-MS/MS assay was helpful in properly characterizing the epidemiology of benzodiazepine use in our patient population. This information will lead to better assay result interpretations and patient care, and our experiences provide a roadmap for other clinical laboratories looking to expand their testing menu or transition to new instrumentation.

Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry.

Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography-mass spectrometry (LC-MS/MS) system.

A Second-generation opioid LC-MS/MS assay improves laboratory workflow and capacity.

The widespread use of opioid drugs has contributed to escalating rates of addiction, overdoses, and drug-related deaths. Targeted urine drug screening plays an important role in supporting the care of patients with chronic pain or addiction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide excellent sensitivity and specificity, and, as a result, remains the definitive choice for confirmatory urine drug testing. However, the complexities of LC-MS/MS operation present major challenges to the clinical laboratory. In this study, we leveraged upgraded instrumentation to develop and validate a simplified "dilute-and-shoot" LC-MS/MS opioid assay. By modifying the chromatographic gradient, isobaric interferences were well-resolved and eliminated. Analytical ranges were expanded by utilizing alternative mass transitions, and updated quality assurance parameters were established. Results from 204 clinical samples correlated well between the new method and a previous version. The upgraded systems provided better sensitivity, greater dynamic ranges, and the new method reduced carryover, which enabled us to eliminate extra injections and chromatogram reviews. The new method also reduced turnaround time and doubled testing capacity. These improvements could serve as a model for other laboratories approaching a similar transition in mass spectrometric instrumentation.

Evaluation of Two Methods for Quantification of Glycosaminoglycan Biomarkers in Newborn Dried Blood Spots from Patients with Severe and Attenuated Mucopolysaccharidosis Type II.

All newborn screening (NBS) for mucopolysaccharidosis-I and -II (MPS-I and MPS-II) is carried out via the measurement of α-iduronidase (IDUA) and iduronate-2-sulfatase (IDS) enzymatic activity, respectively, in dried blood spots (DBS). The majority of low enzyme results are due to pseudodeficiencies, and data from recent MPS-II population screenings and studies from the Mayo Clinic show that the false positive rate can be dramatically reduced by the inclusion of a second-tier analysis of glycosaminoglycans (GAGs) in DBS as part of NBS. In the present study, which focused on MPS-II, we obtained newborn DBS from 17 patients with severe MPS-II, 1 with attenuated MPS-II, and 6 patients with various IDS pseudodeficiencies. These samples were submitted to two different GAG mass spectrometry analyses in a comparative study: (1) internal disaccharide biomarkers and (2) endogenous biomarkers. For both of these methods, the biomarker levels in six patients with pseudodeficiencies were below the range measured in MPS-II patients. One patient with attenuated MPS-II was not distinguishable from severe disease patients, but all MPS-II patients were distinguishable from the reference range using both methods. The minimal differential factor (lowest GAG marker level in MPS-II samples divided by highest level in the reference range of 60 random newborns) was 3.01-fold for the internal disaccharide method. The endogenous biomarker method demonstrated an improved minimum differential of 5.41-fold. The minimum differential factors between MPS-II patients and patients with pseudodeficiencies for the internal disaccharide and endogenous biomarker methods were 3.77-fold and 2.06-fold, respectively. This study supports use of the second-tier GAG analysis of newborn DBS, especially the endogenous disaccharide method, as part of NBS to reduce the false positive rate.

Liquid Chromatography-Tandem Mass Spectrometry in Newborn Screening Laboratories.

Tandem mass spectrometry (MS/MS) is the most universal platform currently available for the analysis of enzymatic activities and biomarkers in dried blood spots (DBS) for applications in newborn screening (NBS). Among the MS/MS applications in NBS, the most common is flow-injection analysis (FIA-) MS/MS, where the sample is introduced as a bolus injection into the mass spectrometer without the prior fractionation of analytes. Liquid chromatography combined with MS/MS (LC-MS/MS) has been employed for second-tier tests to reduce the false-positive rate associated with several nonspecific screening markers, beginning two decades ago. More recently, LC-MS/MS has been applied to primary screening for new conditions for which FIA-MS/MS or other methods, including genomic screening, are not yet adequate. In addition to providing a list of the currently used LC-MS/MS-based assays for NBS, the authors share their experience regarding the maintenance requirements of LC-MS/MS vs. FIA-MS/MS systems. The consensus is that the maintenance of LC-MS/MS and FIA-MS/MS instrumentation is similar, and LC-MS/MS has the advantage of allowing for a larger number of diseases to be screened for in a multiplex, cost-effective fashion with a high throughput and an adequate turnaround time.

Laboratory Test Ordering Patterns in Pediatrics From Naturopaths and General Practitioners.

OBJECTIVES: Naturopathic medicine emphasizes prevention and the self-healing process through natural therapies. Naturopathic doctors (NDs) use clinical laboratories as frequently as traditionally trained physicians. Here we evaluated the test-ordering patterns of NDs and general practitioners (GPs). METHODS: A retrospective analysis was performed from a tertiary pediatric hospital. We analyzed tests ordered by NDs who used laboratory services and compared the test ordering patterns with GPs from adolescent medicine, family medicine, or pediatric clinics. Requests were categorized into 10 groups. We determined the tests with the highest ordering frequencies, as well as the percentage of tests that had an abnormal result. RESULTS: NDs ordered more tests per patient per date of specimen collection compared with GPs. The most frequently ordered tests by NDs were trace elements and toxic metals (23.2% of total), allergens (21.8%), and general chemistry (15.3%). For the same test, the percentage of tests with an abnormal result was significantly lower for NDs than GPs. CONCLUSIONS: We observed different ordering patterns between NDs and GPs. NDs ordered more esoteric tests and had lower rates of abnormal test results compared with GPs. Understanding the patterns of testing from different providers' specialties is useful to choose effective laboratory stewardship interventions.

Galactocerebrosidase activity by liquid-chromatography tandem mass spectrometry for clinical diagnosis of Krabbe disease.

BACKGROUND: Deficiency of galactosylcerebrosidase (GALC) causes Krabbe disease. Historically, a diagnosis is made by measuring GALC enzymatic activity with a radioisotope assay. To improve the workflow and performance, we developed and clinically validated a leukocyte enzymatic assay using liquid chromatography tandem mass spectrometry (LC-MS/MS). MATERIALS: Extracted cell lysates were quantified and incubated with commercially available multiplexed substrates and internal standards. Liquid-liquid extraction was performed, and pre-analytical and analytical variability were evaluated and validated following clinical laboratory regulation guidelines. RESULTS: Enzymatic reaction products were resolved from substrate breakdown products by a 3.5-minute column separation. Intra- and inter- assay imprecision were less than 15%. No matrix effects or carryover were observed. ACD anticoagulant tubes provide the best sample stability. Detection of product was linear with an R2 of 0.99. Small differences in GALC activity were measurable near the anticipated disease range. Confirmed cases of Krabbe disease were well differentiated from carriers and non-Krabbe individuals (normal reference range). CONCLUSION: An LC-MS/MS assay was developed, which can measure trace residual GALC activity in leukocytes and aid in the diagnosis of Krabbe disease. The multiplexed mixture allows for built-in sample quality control and enables a streamlined workflow for evaluation of multiple lysosomal storage diseases.

Very rare condition of multiple Gaucheroma: A case report and review of the literature.

BACKGROUND: This study presented a 3 years old boy with Gaucher disease (GD) who was treated with enzyme replacement therapy (ERT) for 19 months and then developed multiple Gaucheroma. Review of literature was performed simultaneously. METHODS: The medical chart and literature were reviewed. A boy presented at the age of 15 months with anemia, thrombocytopenia, and hepatosplenomegaly. GD was confirmed by enzyme assay and gene mutations. ERT was administered right after the diagnosis. When the boy was 3 years old, multiple masses were discovered during a regular checkup abdominal MRI and biopsy revealed Gaucheroma. We also reviewed 20 GD patients with Gaucheroma and Gaucher cell infiltrated lymphadenopathies. CONCLUSION: Gaucheroma is a rare condition of regularly treated GD patients. This patient even showed poor response to doubled ERT doses. The imaging studies are necessary for Gaucher patients to detect Gaucheroma and determine their malignancy. Regular checkups are recommended in all GD patients even with regular treatment, due to the possibility of having deteriorating change, like Gaucheroma.