Improving Harmonization and Standardization of Expanded Newborn Screening Results by Optimization of the Legacy Flow Injection Analysis Tandem Mass Spectrometry Methods and Application of a Standardized Calibration Approach.

BACKGROUND: Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors. METHODS: Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches. RESULTS: Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 µL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer's stated values of ≥98%. CONCLUSIONS: For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.

Olive ingestion causing a false suspicion of relapsed neuroblastoma: A case of "oliveblastoma?"

Measurement of the urine catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) are the standard method for detecting disease recurrence in neuroblastoma. We present a case of abnormal concentrations of catecholamine metabolites that prompted investigations for relapsed neuroblastoma. However, further study revealed that the abnormal biochemistry was likely due to ingestion of olives. Olive ingestion should be considered when interpreting urine HVA and VMA results, and excluded if concentrations are unexpectedly abnormal.

A computer vision approach to the assessment of dried blood spot size and quality in newborn screening.

BACKGROUND: Dried blood spot (DBS) size and quality affect newborn screening (NBS) test results. Visual assessment of DBS quality is subjective. METHODS: We developed and validated a computer vision (CV) algorithm to measure DBS diameter and identify incorrectly applied blood in images from the Panthera DBS puncher. We used CV to assess historical trends in DBS quality and correlate DBS diameter to NBS analyte concentrations in 130,620 specimens. RESULTS: CV estimates of DBS diameter were precise (percentage coefficient of variation < 1.3%) and demonstrated excellent agreement with digital calipers with a mean (standard deviation) difference of 0.23 mm (0.18 mm). An optimised logistic regression model showed a sensitivity of 94.3% and specificity of 96.8% for detecting incorrectly applied blood. In a validation set of images (n = 40), CV agreed with an expert panel in all acceptable specimens and identified all specimens rejected by the expert panel due to incorrect blood application or DBS diameter > 14 mm. CV identified a reduction in unsuitable NBS specimens from 25.5% in 2015 to 2% in 2021. Each mm decrease in DBS diameter decreased analyte concentrations by up to 4.3%. CONCLUSIONS: CV can aid assessment of DBS size and quality to harmonize specimen rejection both within and between laboratories.

Paracetamol toxicity in classic homocystinuria: Effect of N-acetylcysteine on total homocysteine.

Classical homocystinuria (HCU) is caused by cystathionine ß-synthase deficiency leading to impaired homocysteine transsulfuration and accumulation of homocysteine and methionine. Patients present with a wide spectrum of manifestations including ocular, skeletal, neuropsychiatric, and vascular manifestations. We report a 48-year-old female with pyridoxine-unresponsive HCU treated with betaine, cyanocobalamin, and folate. Her diet was non-restricted due to intolerance of low-methionine diet. She was admitted to hospital following a fall, with multiple fractures and subsequently developed acute liver failure with encephalopathy. Shock, sepsis, and liver ischaemia/thrombosis were excluded. In the context of glutathione depletion expected in HCU, hepatic dysfunction was presumed to be due to iatrogenic paracetamol toxicity, despite paracetamol intake at conventional therapeutic dose, with role of hypermethioninemia as a contributing factor being uncertain. Betaine was discontinued on hospital admission. N-Acetylcysteine (NAC) infusion was initiated. Plasma total homocysteine (tHcy) was 3.4 µmol/L 9 days following initiation of NAC treatment with a markedly elevated plasma methionine of 1278 µmol/L. tHcy concentration returned to pre-admission baseline after NAC was discontinued. Recovery following this episode was slow with a prolonged cholestatic phase and gradual improvement in jaundice and coagulopathy. We recommend that paracetamol should be administered cautiously in HCU patients due to underlying glutathione depletion and risk of toxicity even at therapeutic doses. NAC is clearly effective in lowering tHcy in classical HCU in the short-term however further research is required to assess clinical efficacy and use as a potential therapy in classical HCU.

Investigation of the relationship between phenylalanine in venous plasma and capillary blood using volumetric blood collection devices.

Measurement of plasma and dried blood spot (DBS) phenylalanine (Phe) is key to monitoring patients with phenylketonuria (PKU). The relationship between plasma and capillary DBS Phe concentrations has been investigated previously, however, differences in methodology, calibration approach and assumptions about the volume of blood in a DBS sub-punch has complicated this. Volumetric blood collection devices (VBCDs) provide an opportunity to re-evaluate this relationship. Paired venous and capillary samples were collected from patients with PKU (n = 51). Capillary blood was collected onto both conventional newborn screening (NBS) cards and VBCDs. Specimens were analysed by liquid-chromatography tandem mass-spectrometry (LC-MS/MS) using a common calibrator. Use of VBCDs was evaluated qualitatively by patients. Mean bias between plasma and volumetrically collected capillary DBS Phe was -13%. Mean recovery (SD) of Phe from DBS was 89.4% (4.6). VBCDs confirmed that the volume of blood typically assumed to be present in a 3.2 mm sub-punch is over-estimated by 9.7%. Determination of the relationship between plasma and capillary DBS Phe, using a single analytical method, common calibration and VBCDs, demonstrated that once the under-recovery of Phe from DBS has been taken into account, there is no significant difference in the concentration of Phe in plasma and capillary blood. Conversely, comparison of plasma Phe with capillary DBS Phe collected on a NBS card highlighted the limitations of this approach. Introducing VBCDs for the routine monitoring of patients with PKU would provide a simple, acceptable specimen collection technique that ensures consistent sample quality and produces accurate and precise blood Phe results which are interchangeable with plasma Phe.

Cross-sectional audit assessing the quality of dried bloodspot specimens received by UK metabolic biochemistry laboratories for the biochemical monitoring of individuals with Phenylketonuria.

BACKGROUND: Sapropterin has been approved as a treatment option for individuals with Phenylketonuria in the United Kingdom. Individuals are assessed as responsive to Sapropterin by a ≥30% reduction in Phenylalanine (Phe) concentrations using dried blood spot (DBS) specimens. DBS quality is critical for accurate and precise measurement of Phe. Currently, UK national guidelines for DBS specimen acceptance do not exist for patient-collected DBS specimens. We adopted evidence-based guidelines for specimen acceptance criteria and retrospectively assessed the impact of introducing these guidelines on specimen rejection rates. Methods: Laboratories were invited to audit the quality of DBS specimens routinely received for Phe monitoring using: (1) existing acceptance/rejection criteria and (2) proposed national guidelines. RESULTS: Ten laboratories audited 2111 specimens from 1094 individuals. Using existing local guidelines, the median rejection rate was 4.0% (IQR 1.5-7.2%). This increased to 21.9% (IQR 10.0-33.0%) using the proposed guidelines. Where reason(s) for rejection were provided (n = 299); 211/299 (70.6%) of DBS specimens were too small or multi-spotted. 380 individuals had more than one sample evaluated; 231/380 (60.8%) provided specimens of acceptable quality, 37/380 (9.7%) consistently provided poor-quality DBS specimens. CONCLUSIONS: There is significant variability in the quality of patient-collected DBS specimens. If unacceptable specimens are not rejected, imprecise/inaccurate results will be used in clinical decision making. Using annual workload data for England, this equates to 12,410 incorrect results. Individuals and parents/carers should receive ongoing training in blood collection technique to ensure use of evidence-based acceptability criteria does not cause undue distress from increased sample rejection rates.

Incidental Detection of Classical Galactosemia through Newborn Screening for Phenylketonuria: A 10-Year Retrospective Audit to Determine the Efficacy of This Approach.

In the UK, Classical Galactosaemia (CG) is identified incidentally from the Newborn Screening (NBS) for phenylketonuria (PKU) using an "Other disorder suspected" (ODS) pathway when phenylalanine (Phe) and tyrosine (Tyr) concentrations are increased. We aimed to determine the efficacy of CG detection via NBS and estimate the incidence of CG in live births in the UK. A survey was sent to all UK NBS laboratories to collate CG cases diagnosed in the UK from 2010 to 2020. Cases of CG diagnosed were determined if detected clinically, NBS, or by family screening, as well as age at diagnosis. Cases referred via the ODS pathway were also collated, including the final diagnosis made. Responses were obtained from 13/16 laboratories. Between 2010 and 2020, a total of 6,642,787 babies were screened, and 172 cases of CG were identified. It should be noted that 85/172 presented clinically, 52/172 were identified by NBS, and 17/172 came from family screening. A total of 117 referrals were made via the ODS pathway, and 45/117 were subsequently diagnosed with CG. Median (interquartile range) age at diagnosis by NBS and clinically was 8 days (7-11) and 10 days (7-16), respectively (Mann-Whitney U test, U = 836.5, p-value = 0.082). The incidence of CG is 1:38,621 live births. The incidence of CG in the UK is comparable with that of other European/western countries. No statistical difference was seen in the timing of diagnosis between NBS and clinical presentation based on the current practice of sampling on day 5. Bringing forward the day of NBS sampling to day 3 would increase the proportion diagnosed with CG by NBS from 52/172 (30.2%) to 66/172 (38.4%).

Thirty-years of screening for cystic fibrosis in East Anglia.

BACKGROUND: Newborn screening for cystic fibrosis (CF) relies on the measurement of immunoreactive trypsinogen (IRT) originating from the pancreas. The Norfolk, Suffolk and Cambridgeshire screening programme initially exploited the persistent increase in IRT seen in CF (IRT-IRT protocol) and later changed to include mutation analysis as a second tier test (IRT-DNA-IRT protocol). RESULTS: During a 30 year period 582 966 babies have been screened by IRT-IRT and 147 764 by IRT-DNA-IRT (total 730730), resulting in 296 screen positive cases of CF and 29 false negatives (including 10 false negatives with meconium ileus). Ten missed CF cases were pancreatic insufficient, however all were diagnosed before their first birthday, suggesting that a false negative result did not forestall appropriate clinical investigation. The IRT-DNA-IRT protocol had a much improved positive predictive value (PPV) of 85.9% compared to 67.3% for IRT-IRT, excluding CF babies with meconium ileus. The PPVs increased to 82.2% and 98.2% respectively if only well, term babies were considered. The main factor to account for this improvement in PPV has probably been the incorporation of DNA analysis in the second tier testing. CONCLUSIONS: The diagnosis of screen-positive babies proved difficult in a minority of cases with the classification of some patients changing with evolving phenotype. Our results illustrate the importance of collecting outcome data over a long time period for accurate assessment of the screening programme. This study provides evidence that newborn screening for CF is a valid undertaking that detects 95% of unsuspected CF cases presenting before 3 years of age.

Attention in musicians is more bilateral than in non-musicians.

Attention in neurologically intact adults normally errs towards the left side of space, as documented in studies involving tasks of visual attention (i.e., line bisection). The aim of this study was to further investigate lateralisation of attention in musicians and non-musicians. Reaction times and accuracy were recorded to stimuli presented to the left and right of a vertical line in 20 right-handed musicians and 20 matched non-musician controls. While both groups performed more accurately to left-sided stimuli, performance by the musician group was significantly more accurate than the non-musician group for the right-sided stimuli. Musicians also had faster reaction times overall. Consistent with previous research, the results indicate a more balanced attentional capacity in musicians, as well as enhanced visuomotor ability, and are interpreted with reference to extended musical training.