High-sensitivity neonatal urine drug testing has similar positivity rates to meconium for detecting in utero exposure to methamphetamine and cocaine.

Current guidelines recommend universal screening for substance use disorders in obstetric patients, and neonatal drug testing is also frequently performed. Meconium is often the preferred specimen type to detect neonatal drug exposure due to a longer window of detection compared to urine, but most laboratories send out meconium testing to specialized reference laboratories, which can delay results for several days or more. Here, we evaluate a rapid and definitive liquid chromatography-tandem mass spectrometry method for neonatal urine drug testing and compare results obtained using this method to paired meconium drug testing in 1,424 neonates for amphetamines, cocaine, cannabinoids, opiates, oxycodone and phencyclidine. Urine testing showed equivalent sensitivity to current meconium methods for detecting in utero exposure to amphetamines and cocaine.

Estimation of gestational age-specific reference intervals for coagulation assays in a neonatal intensive care unit using real-world data.

BACKGROUND: Interpretation of coagulation testing in neonates currently relies on reference intervals (RIs) defined from older patient cohorts. Direct RI studies are difficult, but indirect estimation may allow us to infer normative neonatal distributions from routinely collected clinical data. OBJECTIVE: Assess the utility of indirect reference interval methods in estimating coagulation reference intervals in critically ill neonates. METHODS: We analyzed first-in-life coagulation testing results from all patients admitted to a level IV neonatal intensive care unit between January 1, 2018, and January 1, 2024. Results obtained after transfusion of any blood product were excluded. Indirect RIs were estimated across gestational age groups using refineR and compared with currently reported intervals for patients less than 1 year of age. RESULTS: Prothrombin times (PTs) and international normalized ratios (INRs) were available for 1128 neonates, while activated partial thromboplastin times (APTTs) were available for 790 neonates. The indirect RI was 10 to 25 seconds in preterm, 10 to 22 seconds in term, and 10 to 24 seconds in all neonates for PT; 0.7 to 2.1 in preterm, 0.8 to 1.8 in term, and 0.8 to 1.9 in all neonates for INR; and 25 to 68 seconds in preterm, 25 to 58 seconds in term, and 25 to 62 seconds in all neonates for APTT. Compared with our current intervals, the indirect RIs would flag 58% fewer PT, 43% fewer INR, and 17% fewer APTT results as abnormal. CONCLUSION: Indirectly estimated RIs in neonates admitted to intensive care show substantial divergence from current, first-year-of-life RIs, leading to an abundance of abnormal flags. The associations between these flags and provider behavior, transfusion practice, or clinical outcomes are areas of future exploration.

Reexamining the Minimum Sweat Rate Requirement for Sweat Chloride Testing.

BACKGROUND: Guidelines for sweat chloride testing endorse a minimum sweat rate for reporting results. Bilateral sweat collection is recommended, but if both sites fail to meet the minimum rate (quantity not sufficient, QNS), the test should be repeated. In this study, we examine the correlation between sweat rate and sweat chloride concentration ([Cl-]), assess the accuracy of specimens collected at suboptimal rates, and investigate the use of pooled bilateral specimens for chloride measurement. METHODS: Pearson correlation was employed to analyze the relationship between sweat rate and chloride concentration, [Cl-], in 674 macroduct collections. Weighted kappa was evaluated to determine cystic fibrosis (CF) diagnostic classification concordance for 18 tests with paired arms above vs below the minimum sweat rate. Deming regression was applied to compare [Cl-] from pooled bilateral specimens vs neat specimens in 27 collections with residual volume available after clinical testing. RESULTS: Pearson correlation of sweat rate vs [Cl-] was minimal (r = -0.0735) across specimens with varying rates and [Cl-]. There was substantial agreement in CF diagnostic classification between arms for bilateral collections with discordant sweat rates. Regression analysis of [Cl-] in pooled vs nonpooled specimens revealed a slope of 0.984 and an intercept of 0.796. CONCLUSIONS: Negligible correlation of sweat rate and [Cl-] suggests the minimum sweat rate for macroduct collectors may be overly stringent. Reporting of [Cl-] in specimens with ≥10 µL (rate ≥0.3 µL/min) may reduce QNS rates without compromising diagnostic accuracy. Preliminary data suggests pooling of bilateral collections may be a feasible option to achieve the required volume for testing.

Raising the Dead Volume: Analysis of Microsamples Diluted and Corrected with Near Infrared Tracer.

BACKGROUND: Sample processing robotics require large liquid volumes to operate efficiently. Robotics are impractical in settings that deal in small specimen volumes such as pediatric laboratories. Short of manual sample handling, remedies for the current state include a redesign of current hardware or specialized adaptation for submilliliter specimens. METHODS: We blindly increased the volume of plasma specimens with diluent containing a near infrared dye, IR820, to assess the change to the original specimen volume. Diluted specimens were analyzed using a variety of assay formats/wavelengths (sodium, calcium, alanine aminotransferase, creatine kinase, cholesterol, HDL cholesterol, triglyceride, glucose, total protein, creatinine), and results were compared to neat specimens. Recovery of analyte in the diluted specimens vs neat was the primary outcome measure. RESULTS: Mean analytic recovery from the diluted specimens across all assays ranged from 93% to 110% after correction using IR820 absorbance. Absorbance correction compared favorably to mathematical correction using known volumes of specimens and diluents (93%-107%). Pooled mean analytic imprecision across all assays ranged from 2% using the neat specimen pool to 8% when plasma pool was diluted to 30% of its original concentration. No interference from dye addition was noted, indicating the diluent was broadly applicable and chemically inert. The greatest variability in recovery was observed when respective analyte concentrations were present near the lower limits of assay detectability. CONCLUSIONS: Addition of a chemically inert diluent containing a near-infrared tracer is a feasible way to raise specimen dead volume and potentially automate processing and measurement of clinical analytes in microsamples.

Biochemical characterization of patients with dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD; E3) oxidizes lipoic acid. Restoring the oxidized state allows lipoic acid to act as a necessary electron sink for the four mitochondrial keto-acid dehydrogenases: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain α-keto-acid dehydrogenase, and 2-oxoadipate dehydrogenase. DLD deficiency (DLDD) is caused by biallelic pathogenic variants in DLD. Three major forms have been described: encephalopathic, hepatic, and myopathic, although DLDD patients exhibit overlapping phenotypes. Hyperlactatemia, hyperexcretion of tricarboxylic acid cycle (TCA) metabolites and branched-chain keto acids, increased plasma branched-chain amino acids and allo-isoleucine are intermittent metabolic abnormalities reported in patients with DLDD. However, the diagnostic performance of these metabolites has never been studied. Therefore, we sought to systematically evaluate the diagnostic utility of these biomarkers for DLDD. We retrospectively analyzed the results of biochemical testing of six unrelated DLDD patients, including values obtained during both well visits and acute decompensation episodes. Elevation of branched-chain amino acid concentrations was not consistently observed. We found that five of six patients in our cohort had a maximum lifetime value of allo-isoleucine of 6 µmol/L, showing that alloisoleucine elevations even during illness may be subtle. Urine organic acid analysis (UOA) during acute decompensation episodes was abnormal in all cases; however, the pattern of abnormalities had high intersubject variability. No single biomarker was universally present, even in patients experiencing metabolic decompensation. We also observed novel biochemical associations: three patients had hyperexcretion of TCA cycle metabolites during crisis; in two patients, 2-ketoadipic and 2-hydroxyadipic acids, by products of lysine degradation, were detected. We propose that these result from 2-oxoadipate dehydrogenase deficiency, an underappreciated biochemical abnormality in DLD. Given the diversity of biochemical profiles among the patients with DLDD, we conclude that accurate biochemical diagnosis relies on a high index of suspicion and multipronged biochemical analysis, including both plasma amino acid and urine organic acid quantitation during decompensation. Biochemical diagnosis during the well state is challenging. We emphasize the critical importance of multiple simultaneous biochemical tests for diagnosis and monitoring of DLDD. We also highlight the under-recognized role of DLD in the lysine degradation pathway. Larger cohorts of patients are needed to establish a correlation between the biochemical pattern and clinical outcomes, as well as a genotype-phenotype correlation.

GPT-4 Underperforms Experts in Detecting IV Fluid Contamination.

BACKGROUND: Specimens contaminated with intravenous (IV) fluids are common in clinical laboratories. Current methods for detecting contamination rely on insensitive and workflow-disrupting delta checks or manual technologist review. Herein, we assessed the utility of large language models for detecting contamination by IV crystalloids and compared its performance to multiple, but variably trained healthcare personnel (HCP). METHODS: Contamination of basic metabolic panels was simulated using 0.9% normal saline (NS), with (n = 30) and without (n = 30) 5% dextrose (D5NS), at mixture ratios of 0.10 and 0.25. A multimodal language model (GPT-4) and a diverse panel of 8 HCP were asked to adjudicate between real and contaminated results. Classification performance, mixture quantification, and confidence was compared by Wilcoxon rank sum. RESULTS: The 95% CIs for accuracy were 0.57-0.71 vs 0.73-0.80 for GPT-4 and HCP, respectively, on the NS set and 0.57-0.57 vs 0.73-0.80 on the D5NS set. HCP overestimated severity of contamination in the 0.10 mixture group (95% CI of estimate error, 0.05-0.20) for both fluids, while GPT-4 markedly overestimated the D5NS mixture at both ratios (0.16-0.33 for NS, 0.11-0.35 for D5NS). There was no correlation between reported confidence and likelihood of a correct classification. CONCLUSIONS: GPT-4 is less accurate than trained HCP for detecting IV fluid contamination of basic metabolic panel results. However, trained individuals were imperfect at identifying contaminated specimens implying the need for novel, automated tools for its detection.

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products.

Comprehensive Determination of Amino Acids for Diagnosis of Inborn Errors of Metabolism.

Analysis of clinically relevant amino acids using ion exchange chromatography coupled to photometric/fluorescent detection has been an indispensable component in the detection of inborn errors of metabolism for six decades. Detection of amino acids using mass spectrometry offers advantages in speed and analytic specificity. Employing methanol extraction and controlled butylation, C8 reversed-phase chromatography, and MS/MS detection, 32 amino acids are quantified in 20 min with clinically appropriate imprecision in plasma, urine, and cerebrospinal fluid (CSF). Quantitation is linear to 2500 µM, and limits of detection are at least 1.0 µM. Important isobaric amino acids are distinguished by chromatography or by unique patterns of fragmentation following collision-induced dissociation (CID). The technique employs commercially available reagents and may be expanded and customized for specific clinical or research settings.

Comparison of the Friedewald equation with Martin and Sampson equations for estimating LDL cholesterol in hypertriglyceridemic adults.

Low density lipoprotein cholesterol (LDL-C) is traditionally calculated using the Friedewald (LDL-F) equation. New equations by Martin (LDL-M) and Sampson (LDL-S) have improved accuracy relative to LDL-F for samples with high triglycerides (TG) or low LDL-C. However, most labs still rely on LDL-F and few studies have examined the accuracy and impact of contemporary LDL-C equations applied to a retrospective dataset. 934 lipid panels with a concurrent direct enzymatic LDL-C (dLDL-C) result were extracted from the laboratory information system. LDL-F, LDL-M, and LDL-S were calculated and the accuracy of each equation determined in a predominantly hypertriglyceridemic population. The impact of implementing each equation was compared by analyzing the LDL-C treatment group miscategorization rate relative to dLDL-C. The slope for the LDL-F, LDL-M and LDL-S were 0.59, 0.78, and 0.94, relative to dLDL-C. The three equations performed comparably for samples with TG <4.52 mmol/L (<400 mg/dL). The LDL-C treatment group miscategorization rate was 48.6 % for LDL-F, 28.8 % for LDL-M and 37.2 % for LDL-S in specimens with TG ≥4.52 mmol/L (≥400 mg/dL) (n = 817). LDL-S underestimated treatment group category (31.3 %, 95 % CI 17.2-22.4) relative to LDL-M (9.0 %, 4.39-7.41, P < 0.001). 5.9 % of samples were overestimated for treatment group category by LDL-S vs 19.8 % for LDL-M (P = 0.1883). LDL-M and LDL-S demonstrate reduced bias with a dLDL-C method compared to LDL-F in samples with TG ≥4.52 mmol/L (≥400 mg/dL). LDL-M reduces LDL-C treatment group miscategorization rate leading to fewer underestimations of risk overall compared to LDL-S; however, neither may be sufficiently accurate to report LDL-C in patients with TG ≥4.52 mmol/L (≥400 mg/dL).

Pediatric ionized calcium reference intervals from archived radiometer data.

BACKGROUND: Measurement of ionized calcium (iCa) reflects bioavailable calcium and has significant utility in children. However, robust pediatric iCa reference intervals (RI) have not been well-established. In this study, we retrospectively calculated RI for iCa in a pediatric population by accessing archived data acquired on Radiometer instruments and applying stringent exclusion criteria. METHODS: Data saved on 4 Radiometer ABL800 FLEX blood gas analyzers were queried. Exclusion criteria were applied based on information available from these instruments. iCa results were plotted and inflection points were visually identified. Following outlier removal and partition verification, age-specific RI were calculated using a nonparametric rank order approach from > 5,000 individuals. Finally, the stringency of the exclusion criteria was assessed by calculating RI from additional results in the dataset and comparing to existing in-house ranges. RESULTS: Six age-specific iCa partitions were established from 0 to 19 years. Relative to adults, wider ranges for the central 95th percentile were observed early in life that progressively narrowed with increasing age and approached adult concentrations by 2.5 years. Analysis of concurrent data for sodium and creatinine in the dataset suggest the applied exclusion criteria reduced the likelihood of including results from acutely-ill children. CONCLUSIONS: Normal concentrations of iCa in children are more variable than adults. Observed differences may reflect the transition from maternally supplied calcium to nutritional sources, the maturation of calcium homeostatic mechanisms, and/or the need for calcium for growth/development. This study also demonstrates the feasibility and advantages of using data archived on Radiometer analyzers to establish pediatric RI. This approach enables rapid, cost-effective evaluation of large datasets and may be a feasible option when prospective or detailed retrospective analyses are not possible.

Development and Implementation of One-Step, Broad-Spectrum, High-Sensitivity Drug Screening by Tandem Mass Spectrometry in a Pediatric Population.

BACKGROUND: Drug screening by immunoassay is common in pediatric populations. However, false-positive and -negative results due to antibody cross-reactivity and dilute urine are frequent and underappreciated. Accurate ascertainment of drug exposure in children has significant clinical and medico-legal consequences. DESIGN AND METHODS: We developed and characterized an LC-MS/MS drug screening assay to supplant immunoassay and detect 38 compounds at the lowest concentrations distinguishable from analytic noise. Once implemented, we conducted a retrospective analysis of 3985 pediatric urine drug screens performed a year before (n = 1663) and after (n = 2322) implementation to examine the frequency and breadth of drug detection in our pediatric population. RESULTS: Using immunoassay, 23% (293/1269) of samples from the general pediatric and 37% (147/394) of nursery populations had presumptively positive results. Of the presumptive positive compounds, 85% (288/338) from the general pediatric population and 40% (65/162) from the nursery cohort were confirmed by mass spectrometry. After LC-MS/MS implementation, 31% (628/2052) of general pediatric, and 18% (48/270) of the nursery samples were positive for 1 or more compounds. In the nursery population, immunoassays over-detected the presence of THC but under-detected exposure to cocaine. CONCLUSION: A broadly targeted, analytically sensitive LC-MS/MS drug screening assay detects a larger number and variety of compounds in a single step compared to a screen-then-confirm approach initiated by immunoassay in our pediatric population. Rapid delivery of accurate results enables timely, appropriate disposition of patients in a variety of settings including the emergency department and labor/delivery.

Yield of Serum Dexamethasone Measurement for Reducing False-Positive Results of Low-Dose Dexamethasone Suppression Testing.

BACKGROUND: The low-dose dexamethasone suppression test (DST) using a cortisol cutoff of 1.8 µg/dL has approximate sensitivity of 95% and specificity of 80% for detecting Cushing syndrome. False-positive DST results can be caused by a variety of conditions, by low dexamethasone bioavailability, or by failure to take dexamethasone as instructed. In an effort to reduce false positives caused by low bioavailability or medication noncompliance, we evaluated the yield of serum dexamethasone measurement for identifying invalid results. METHODS: Data were queried for orders requesting concurrent measurement of serum cortisol and dexamethasone over a 41-month period. Inclusion criteria were serum cortisol and dexamethasone measured from the same specimen, specimen collection before 9 AM after 1 mg dexamethasone administration, and results for both analytes documented in the electronic medical record. Seventy paired measurements were identified with these criteria. Results were categorized into 4 groups based on observed cortisol and dexamethasone concentrations: (a) suppressed cortisol, low dexamethasone; (b) suppressed cortisol, therapeutic dexamethasone; (c) unsuppressed cortisol, low dexamethasone; or (d) unsuppressed cortisol, therapeutic dexamethasone. RESULTS: Overall, 35 (50%) results demonstrated suppressed cortisol and therapeutic dexamethasone levels. The next largest group was unsuppressed cortisol and therapeutic dexamethasone, representing approximately 32% (n = 22) of the study population. Ten result sets (14%) fell into the unsuppressed cortisol and low dexamethasone category, and 3 paired measurements (4%) fit the criteria for suppressed cortisol and low dexamethasone. CONCLUSIONS: The measurement of serum dexamethasone following DST reduces the false-positive rate associated with subtherapeutic dexamethasone levels.

Detecting Fentanyl Analogs in Urine Using Precursor Ion Scan Mode.

The opioid crisis has led many providers to inquire about the capabilities of urine drug testing to detect contemporary compounds such as fentanyl and fentanyl analogs. However, current methods for clinical urine drug testing, including immunoassays and targeted liquid chromatography tandem mass spectrometry, are not designed to broadly screen for the variety of fentanyl analogs that may be encountered. In this proof-of-principle study we developed a precursor ion scan method to enable semi-targeted data acquisition for structurally related fentanyl analogs. Based on the knowledge that many analogs fragment to m/z=188 and m/z=105, data was acquired on all precursor ions 250-400 Da that fragmented to these product ions. Using a tandem mass spectrometer we analyzed 102 residual urine specimens, in which we identified fentanyl, acetylfentanyl and acrylfentanyl. In 30 contrived urine samples, the precursor ion scan was also able to identify furanylfentanyl, butryrlfentanyl, 4-fluroisobutrylfentanyl, and despropionylfentanyl with accuracy ranging from 83-100%.

Progression of SARS-CoV-2 Seroprevalence in St. Louis, Missouri, through January 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seropositivity was assessed for 3,066 individuals visiting hospitals in St. Louis, Missouri, during July 2020, November 2020, or January 2021. Seropositivity in children increased from 5.22% in July to 21.16% in January. In the same time frame, seropositivity among adults increased from 4.52% to 19.03%, prior to initiation of mass vaccination. IMPORTANCE This study determined the percentage of children and adult samples from the St. Louis metropolitan area in Missouri with SARS-CoV-2 antibodies during three collection periods spanning July 2020 to January 2021. By January 2021, 20.68% of the tested individuals had antibodies. These results show the evolution of the SARS-CoV-2 pandemic in St. Louis, Missouri, and provide a snapshot of the extent of infection just prior to the start of mass vaccination.

Comparison of Two Commercially Available Fentanyl Screening Immunoassays for Clinical Use.

BACKGROUND: Fentanyl is a synthetic opioid associated with illicit drug use and overdose deaths. The SEFRIA Immunalysis (IAL) and ARK fentanyl assays are both FDA-cleared, open channel immunoassays for fentanyl detection in urine. However, limited data are available in the literature comparing these assays. The objective of this study was to perform a direct comparison of these two fentanyl immunoassays. METHODS: IAL and ARK fentanyl immunoassays were performed on a Roche Cobas e602 automated chemistry analyzer. Repeatability and total imprecision were compared by diluting fentanyl into urine at concentrations above, below, and at the manufacturers' cutoffs of 1.0 ng/mL. Cross-reactivity was assessed for norfentanyl and the fentanyl analogs acetylfentanyl, acrylfentanyl, and furanylfentanyl. Concordance was assessed in 90 patient samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard. RESULTS: Repeatability varied from 11.4%-17.8% on the IAL assay and 2.8%-5.5% on the ARK assay. Total imprecision was 18.9%-40.7% on the IAL assay and 2.9%-6.4% on the ARK assay. Both assays cross-reacted with acetylfentanyl (∼100%), acrylfentanyl (∼100%), and furanylfentanyl (∼20%), but only the ARK assay cross-reacted with norfentanyl (∼3%). An admixture of 0.5 ng/mL fentanyl and 6 ng/mL norfentanyl produced a positive result on the ARK assay. Total concordance between IAL and ARK for 90 tested patient samples was 93% (kappa = 0.85). Relative to LC-MS/MS, the IAL assay had a concordance of 90% (kappa = 0.79) and the ARK assay had a concordance of 94% (kappa = 0.88). Including norfentanyl in the LC-MS/MS confirmation increased the concordance of the ARK to 96% (kappa = 0.90). CONCLUSIONS: The ARK assay recognized the metabolite norfentanyl, demonstrated superior precision, and had better concordance with LC-MS/MS compared to the IAL assay.

Evaluation of a multiplex liquid chromatography-tandem mass spectrometry method for congenital adrenal hyperplasia in pediatric patients.

Multiplexed adrenal steroid measurement provides critical diagnostic information for patients with congenital adrenal hyperplasia (CAH) as confirmation of newborn screening (NBS) or as initial diagnosis. This study reports the implementation of an adrenal steroid profiling method with a turnaround time (TAT) of less than 24 h using liquid chromatography and tandem-mass spectrometry (LC-MS/MS). A lab-developed multiplexed LC-MS/MS assay was used to quantify levels of 11-deoxycortisol, cortisol, 17-hydroxy-progesterone (17-OHP), androstenedione, and testosterone. Intra and interassay imprecision were found to be <10%. Comparison with a reference laboratory revealed <20% bias for all 5 analytes and Deming correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit calibrator concentrations with 100- to 800-fold maximum dilution. Run to run carryover was <0.1%, and acceptable matrix effect was observed (i.e., ion suppression enhancement <15%). Compared to serum samples, ethylenediaminetetraacetic acid (EDTA) and heparin plasma had large positive bias in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and androstenedione (43.8% and 33.2%, respectively), while cortisol, 17-OHP and testosterone showed less than 20% bias between sample types. Hemoglobin, bilirubin, or triglyceride interference decreased 11-deoxycortisol measurement in EDTA plasma (-19.3%, -25.6%, and -25.0%, respectively). Lipemia increased the measurement of testosterone by 28.9%. In summary, our multiplexed LC-MS/MS method provided highly sensitive and specific measurement of adrenal steroids. EDTA, heparin, hemolysis, icterus and/or lipemia may significantly impact assay results and should be avoided. This method provides an effective strategy for improving TAT in CAH testing and confirmation of NBS results.