Elevated fluid and glycosaminoglycan content in the Achilles tendon contribute to higher intratendinous pressure: Implications for Achilles tendinopathy.

BACKGROUND: Tendinopathy alters the compositional properties of the Achilles tendon by increasing fluid and glycosaminoglycan content. It has been speculated that these changes may affect intratendinous pressure, but the extent of this relationship remains unclear. Therefore, we aimed to investigate the impact of elevated fluid and glycosaminoglycan content on Achilles tendon intratendinous pressure and to determine whether hyaluronidase (HYAL) therapy can intervene in this potential relationship. METHODS: Twenty paired fresh-frozen cadaveric Achilles tendons were mounted in a tensile-testing machine and loaded up to 5% strain. Intratendinous resting (at 0% strain) and dynamic pressure (at 5% strain) were assessed using the microcapillary infusion technique. First, intratendinous pressure was measured under native conditions before and after infusion of 2 mL physiological saline. Next, 80 mg of glycosaminoglycans were administered bilaterally to the paired tendons. The right tendons were additionally treated with 1500 units of HYAL. Finally, both groups were retested, and the glycosaminoglycan content was analyzed. RESULTS: It was found that both elevated fluid and glycosaminoglycan content resulted in higher intratendinous resting and dynamic pressures (p < 0.001). HYAL treatment induced a 2.3-fold reduction in glycosaminoglycan content (p = 0.002) and restored intratendinous pressures. CONCLUSION: The results of this study demonstrated that elevated fluid and glycosaminoglycan content in Achilles tendinopathy contribute to increased intratendinous resting and dynamic pressures, which can be explained by the associated increased volume and reduced permeability of the tendon matrix, respectively. HYAL degrades glycosaminoglycans sufficiently to lower intratendinous pressures and may, therefore, serve as a promising treatment.

Clinical Heterogeneity in MT-ATP6 Pathogenic Variants: Same Genotype-Different Onset.

Human mitochondrial disease exhibits large variation of clinical phenotypes, even in patients with the same causative gene defect. We illustrate this heterogeneity by confronting clinical and biochemical data of two patients with the uncommon pathogenic homoplasmic NC_012920.1(MT-ATP6):m.9035T>C variant in MT-ATP6. Patient 1 presented as a toddler with severe motor and speech delay and spastic ataxia without extra-neurologic involvement. Patient 2 presented in adolescence with ataxia and ophthalmoplegia without cognitive or motor impairment. Respiratory chain complex activities were normal in cultured skin fibroblasts from both patients when calculated as ratios over citrate synthase activity. Native gels found presence of subcomplexes of complex V in fibroblast and/or skeletal muscle. Bioenergetic measurements in fibroblasts from both patients detected reduced spare respiratory capacities and altered extracellular acidification rates, revealing a switch from mitochondrial respiration to glycolysis to uphold ATP production. Thus, in contrast to the differing disease presentation, biochemical evidence of mitochondrial deficiency turned out quite similar. We conclude that biochemical analysis remains a valuable tool to confirm the genetic diagnosis of mitochondrial disease, especially in patients with new gene variants or atypical clinical presentation.

Belgian rare diseases plan in clinical pathology: identification of key biochemical diagnostic tests and establishment of reference laboratories and financing conditions.

BACKGROUND: One objective of the Belgian Rare Diseases plan is to improve patients' management using phenotypic tests and, more specifically, the access to those tests by identifying the biochemical analyses used for rare diseases, developing new financing conditions and establishing reference laboratories. METHODS: A feasibility study was performed from May 2015 until August 2016 in order to select the financeable biochemical analyses, and, among them, those that should be performed by reference laboratories. This selection was based on an inventory of analyses used for rare diseases and a survey addressed to the Belgian laboratories of clinical pathology (investigating the annual analytical costs, volumes, turnaround times and the tests unavailable in Belgium and outsourced abroad). A proposal of financeable analyses, financing modalities, reference laboratories' scope and budget estimation was developed and submitted to the Belgian healthcare authorities. After its approval in December 2016, the implementation phase took place from January 2017 until December 2019. RESULTS: In 2019, new reimbursement conditions have been published for 46 analyses and eighteen reference laboratories have been recognized. Collaborations have also been developed with 5 foreign laboratories in order to organize the outsourcing and financing of 9 analyses unavailable in Belgium. CONCLUSIONS: In the context of clinical pathology and rare diseases, this initiative enabled to identify unreimbursed analyses and to meet the most crucial financial needs. It also contributed to improve patients' management by establishing Belgian reference laboratories and foreign referral laboratories for highly-specific analyses and a permanent surveillance, quality and financing framework for those tests.

Longitudinal study of Pex1-G844D NMRI mouse model: A robust pre-clinical model for mild Zellweger spectrum disorder.

Zellweger spectrum disorders (ZSD) are inborn errors of metabolism caused by mutations in PEX genes that lead to peroxisomal biogenesis disorder (PBD). No validated treatment is able to modify the dismal progression of the disease. ZSD mouse models used to develop therapeutic approaches are limited by poor survival and breeding restrictions. To overcome these limitations, we backcrossed the hypomorphic Pex1 p.G844D allele to NMRI background. NMRI mouse breeding restored an autosomal recessive Mendelian inheritance pattern and delivered twice larger litters. Mice were longitudinally phenotyped up to 6 months of age to make this model suitable for therapeutic interventions. ZSD mice exhibited growth retardation and relative hepatomegaly associated to progressive hepatocyte hypertrophy. Biochemical studies associated with RNA sequencing deciphered ZSD liver glycogen metabolism alterations. Affected fibroblasts displayed classical immunofluorescence pattern and biochemical alterations associated with PBD. Plasma and liver showed very long-chain fatty acids, specific oxysterols and C27 bile acids intermediates elevation in ZSD mice along with a specific urine organic acid profile. With ageing, C26 fatty acid and phytanic acid levels tended to normalize in ZSD mice, as described in patients reaching adulthood. In conclusion, our mouse model recapitulates a mild ZSD phenotype and is suitable for liver-targeted therapies evaluation.

Cystathionine beta synthase deficiency and brain edema associated with methionine excess under betaine supplementation: Four new cases and a review of the evidence.

CBS deficient individuals undergoing betaine supplementation without sufficient dietary methionine restriction can develop severe hypermethioninemia and brain edema. Brain edema has also been observed in individuals with severe hypermethioninemia without concomitant betaine supplementation. We systematically evaluated reports from 11 published and 4 unpublished patients with CBS deficiency and from additional four cases of encephalopathy in association with elevated methionine. We conclude that, while betaine supplementation does greatly exacerbate methionine accumulation, the primary agent causing brain edema is methionine rather than betaine. Clinical signs of increased intracranial pressure have not been seen in patients with plasma methionine levels below 559 µmol/L but occurred in one patient whose levels did not knowingly exceed 972 µmol/L at the time of manifestation. While levels below 500 µmol/L can be deemed safe it appears that brain edema can develop with plasma methionine levels close to 1000 µmol/L. Patients with CBS deficiency on betaine supplementation need to be regularly monitored for concordance with their dietary plan and for plasma methionine concentrations. Recurrent methionine levels above 500 µmol/L should alert clinicians to check for clinical signs and symptoms of brain edema and review dietary methionine intake. Levels approaching 1000 µmol/L do increase the risk of complications and levels exceeding 1000 µmol/L, despite best dietetic efforts, should be acutely addressed by reducing the prescribed betaine dose.

Probiotics in cystic fibrosis patients: A double blind crossover placebo controlled study: Pilot study from the ESPGHAN Working Group on Pancreas/CF.

BACKGROUND: A potential positive effect of probiotics in cystic fibrosis (CF) on fecal calprotectin (FCP), pulmonary exacerbations and weight has been described in small controlled trials. METHODS: A double-blind multicenter cross-over study (2 × 4 m) was performed looking at abdominal pain, nutritional status, pulmonary function, pulmonary exacerbation, FCP and lactulose/mannitol gut permeability test. Patients kept a diary with daily scoring of abdominal pain, stool frequency and consistency as well as treatment changes. RESULTS: 31 CF patients entered the study of which 25 finished it. At start patients aged 9.3yrs (6.9-12.2), had a median BMI z-score of -0.5 (-1.5-0.08), height z-score of -0.4 (-1.1-0.05) and FEV1% of 100% (87.2-106.6). Median FCP at start was 61 µg/g (17-108) and gut permeability 0.079 (0.051-0.122). No significant changes were observed in the clinical parameters (BMI, FEV1%, abdominal pain, exacerbations). Despite being frequently abnormal (17/28 (61%) >50 mg/kg), FCP did not change significantly with probiotics. The proportion of patients with normal permeability was 8% during placebo and 32% during probiotic treatment (p = 0.031). FCP correlated to BMI z-score (p = 0.043) and gut permeability to abdominal pain (p = 0.015). The microbiome revealed a high predominance of Actinobacteria and Proteobacteriae. Probiotic supplementation did not result in a shift at the phylum nor at phylogenetic level. CONCLUSION: Normalization of gut permeability was observed in 13% of patients during probiotic treatment. However, none of the previously described effects could be confirmed.

Severe hepatopathy and neurological deterioration after start of valproate treatment in a 6-year-old child with mitochondrial tryptophanyl-tRNA synthetase deficiency.

BACKGROUND: The first subjects with deficiency of mitochondrial tryptophanyl-tRNA synthetase (WARS2) were reported in 2017. Their clinical characteristics can be subdivided into three phenotypes (neonatal phenotype, severe infantile onset phenotype, Parkinson-like phenotype). RESULTS: Here, we report on a subject who presented with early developmental delay, motor weakness and intellectual disability and who was considered during several years as having a non-progressive encephalopathy. At the age of six years, she had an epileptic seizure which was treated with sodium valproate. In the months after treatment was started, she developed acute liver failure and severe progressive encephalopathy. Although valproate was discontinued, she died six months later. Spectrophotometric analysis of the oxidative phosphorylation complexes in liver revealed a deficient activity of complex III and low normal activities of the complexes I and IV. Activity staining in the BN-PAGE gel confirmed the low activities of complex I, III and IV and, in addition, showed the presence of a subcomplex of complex V. Histochemically, a mosaic pattern was seen in hepatocytes after cytochrome c oxidase staining. Using Whole Exome Sequencing two known pathogenic variants were detected in WARS2 (c.797delC, p.Pro266ArgfsTer10/ c.938 A > T, p.Lys313Met). CONCLUSION: This is the first report of severe hepatopathy in a subject with WARS2 deficiency. The hepatopathy occurred soon after start of sodium valproate treatment. In the literature, valproate-induced hepatotoxicity was reported in the subjects with pathogenic mutations in POLG and TWNK. This case report illustrates that the course of the disease in the subjects with a mitochondrial defect can be non-progressive during several years. The subject reported here was first diagnosed as having cerebral palsy. Only after a mitochondriotoxic medication was started, the disease became progressive, and the diagnosis of a mitochondrial defect was made.

Measurements for 8 common analytes in native sera identify inadequate standardization among 6 routine laboratory assays.

BACKGROUND: External quality assessment (EQA) with commutable samples is essential for assessing the quality of assays performed by laboratories, particularly when the emphasis is on their standardization status and interchangeability of results. METHODS: We used a panel of 20 fresh-frozen single-donation serum samples to assess assays for the measurement of creatinine, glucose, phosphate, uric acid, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. The commercial random access platforms included: Abbott Architect, Beckman Coulter AU, Ortho Vitros, Roche Cobas, Siemens Advia, and Thermo Scientific Konelab. The assessment was done at the peer group level and by comparison against the all-method trimmed mean or reference method values, where available. The considered quality indicators were intraassay imprecision, combined imprecision (including sample-matrix interference), bias, and total error. Fail/pass decisions were based on limits reflecting state-of-the-art performance, but also limits related to biological variation. RESULTS: Most assays showed excellent peer performance attributes, except for HDL- and LDL cholesterol. Cases in which individual assays had biases exceeding the used limits were the Siemens Advia creatinine (-4.2%), Ortho Vitros phosphate (8.9%), Beckman Coulter AU triglycerides (5.4%), and Thermo Scientific Konelab uric acid (6.4%), which lead to considerable interassay discrepancies. Additionally, large laboratory effects were observed that caused interlaboratory differences of >30%. CONCLUSIONS: The design of the EQA study was well suited for monitoring different quality attributes of assays performed in daily laboratory practice. There is a need for improvement, even for simple clinical chemistry analytes. In particular, the interchangeability of results remains jeopardized both by assay standardization issues and individual laboratory effects.

Long-term stability of laboratory tests and practical implications for quality management.

BACKGROUND: Long-term stability of analytical performance is required for adequate patient management. We investigated the use of patient data to document test stability, and the relevance of observed instabilities on a surrogate medical outcome. We used multiyear patient and internal quality control (IQC) data from two laboratories for tests to monitor chronic kidney and thyroid disease. METHODS: We plotted moving means of the 50th percentiles of stratified patient data and of the daily IQC means. We evaluated observed instabilities based on goals inferred from the analytes' biological variation and investigated their effect on classification of results against reference intervals. RESULTS: Patient and IQC data generally matched well, except for analytes, for which other than analytical variation sources prevailed. Analytical instabilities were predominantly due to reagent/calibrator lot changes, however, for immunoassays also to within-lot instabilities, urging frequent recalibrations. The relevance of biased results on medical decisions ranged from negligible to very pronounced, indicating the need for assessment of analytical performance in relation to quality goals inferred from biological variation. CONCLUSIONS: Patient percentiles offer great potential to assess/monitor the medium- to long-term analytical stability of a test within certain constraints. Differences in analytical quality between assays can significantly affect medical outcome.

Standardization of measurements of 25-hydroxyvitamin D3 and D2.

The vitamin D status is increasingly assessed/monitored in different populations, research cohorts and individual patients. This is done by measuring the liver metabolites 25-hydroxyvitamin D3 and D2 as biomarkers. Recommendations for using specific serum concentrations of these biomarkers to assess a person's vitamin D status were done. This requires current vitamin D assays to be sufficiently accurate over time, location and laboratory procedures. In view of the fact that several studies demonstrated that current 25(OH)D measurement methods do not meet this prerequisite, standardization is needed. This paper rehearses the basic concept of standardization, in particular applied to measurements of 25-hydroxyvitamin D. Progress has been made by establishing a reference measurement system consisting of reference methods and reference materials. Coordinated efforts to improve the accuracy and standardize measurements are being performed by organizations such as the U.S. NIH, the CDC and Prevention, the NIST together with their national and international partners. Beyond describing the available reference measurement system and its use as calibration hierarchy to establish traceability of measurements with routine laboratory methods to the SI-unit, this report will also focus on other aspects considered essential for a successful and sustainable standardization, such as analytical issues related to the definition of the measurand and analytical performance goals.

Status of serum-calcium and -albumin measurement in Argentina assessed in 300 representative laboratories with 20 fresh frozen single donation sera.

BACKGROUND: The Fundación Bioquímica Argentina (FBA) performs external quality assessment (EQA) of >3200 laboratories. However, FBA realizes that sample non-commutability and predominant use of heterogeneous systems may bias the estimated performance and standardization status. To eliminate these confounding factors, a study using frozen single donation sera was undertaken with the focus on serum-calcium and -albumin measurement. METHODS: Target values were established from the results produced with homogeneous systems. In groups of n=7, system effects were investigated. Laboratory performance was evaluated from the correlation coefficient r between the measurement results for all sera and the target values. This allowed ranking of the laboratories and judgment of the deviation for individual samples (total error) against a 10% limit. The total error specification was a deviation for ≥ 5 samples exceeding 10% and/or causing a result outside the laboratory's reference interval. RESULTS: For calcium (n=303) (range: 2.06-2.42 mmol/L), 81 laboratories had an r-value <0.6, 43 even <0.4; the total error was relevant for 97 (10% limit) and 111 (reference interval) laboratories. For albumin (n=311) (range: 34.7-45.7 g/L) r was <0.7 (<0.4) in 44 (16) laboratories; 83 and 36 laboratories exceeded the total error criteria. Laboratories using homogeneous systems were generally ranked higher by correlation. System effects were moderate for calcium, but significant for albumin. CONCLUSIONS: The study demonstrated the need to improve the quality and harmonization of calcium and albumin testing in the investigated laboratories. To achieve this objective, we promote co-operation between laboratories, EQA provider and manufacturers.

Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: 25-hydroxyvitamin D [25(OH)D] assays are characterized by poor between-assay comparability. This result emphasizes the need for reference measurement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on the basis of isotope- dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3. METHODS: Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimension, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Reference Material (SRM) 2972. Validation comprised assessment of interference and limit of quantification/detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV<5% and bias<1.7%). The expanded uncertainty for quadruplicate measurements was estimated. RESULTS: Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was resolved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D(2). Mean total CVs and differences from the SRM 972 target (±1-sided 95% CI) were 2.1% and 1.1%±1.5% [25(OH)D3] and 3% and 1.3%±0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%. CONCLUSIONS: From the validation data, we conclude that we achieved our objective of 2 state-of-the-art candidate RMPs for serum 25(OH)D3 and 25(OH)D2.

Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D3 in serum of infants and adults.

BACKGROUND: Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D3 (25OHD3) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD3 sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD3 in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults. METHODS: Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD3 from 25OHD3. Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H](+) precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison. RESULTS: In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD3 and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD3 in infants ranged from 15% to 41%, and in adults from 2.5% to 17%. CONCLUSIONS: This preliminary finding suggests that the prevalence of 3-epi-25OHD3 in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.