Utility of Commercially Available Quantitative hCG Immunoassays as Tumor Markers in Trophoblastic and Non-Trophoblastic Disease.

BACKGROUND: The use of quantitative human chorionic gonadotropin (hCG) as a tumor marker is widely accepted despite lack of FDA-approval for oncology. Differences in iso- and glycoform recognition among hCG immunoassays is well established, exhibiting wide inter-method variability. Here, we assess the utility of 5 quantitative hCG immunoassays for use as tumor markers in trophoblastic and non-trophoblastic disease. METHODS: Remnant specimens were obtained from 150 patients with gestational trophoblastic disease (GTD), germ cell tumors (GCT), or other malignancies. Specimens were identified by review of results from physician-ordered hCG and tumor marker testing. Five analyzer platforms were used for split specimen analysis of hCG: Abbott Architect Total, Roche cobas STAT, Roche cobas Total, Siemens Dimension Vista Total, and Beckman Access Total. RESULTS: Frequency of elevated hCG concentrations (above reference cutoffs) was highest in GTD (100%), followed by GCT (55% to 57%), and other malignancies (8% to 23%). Overall, the Roche cobas Total detected elevated hCG in the greatest number of specimens (63/150). Detection of elevated hCG in trophoblastic disease was nearly equivalent among all immunoassays (range, 41 to 42/60). CONCLUSIONS: While no immunoassay is likely to be perfect in all clinical situations, results for the 5 hCG immunoassays evaluated suggest that all are adequate for use of hCG as a tumor marker in gestational trophoblastic disease and select germ cell tumors. Further harmonization of hCG methods is needed as serial testing for biochemical tumor monitoring must still be performed using a single method. Additional studies are needed to assess the utility of quantitative hCG as a tumor marker in other malignant disease.

A Case of Dubin-Johnson Syndrome Presenting as Neonatal Cholestasis With Paucity of Interlobular Bile Ducts.

Dubin-Johnson syndrome (DJS) is a rare autosomal recessive disorder that typically manifests in young adulthood as jaundice with conjugated hyperbilirubinemia. We report a case presenting as neonatal cholestasis with the unexpected histologic finding of paucity of interlobular bile ducts, a feature that is not typically seen in DJS. The diagnosis was confirmed by absent canalicular multidrug-resistance-associated protein 2 (MRP2) immunohistochemical staining on liver biopsy tissue and molecular genetic testing that demonstrated heterozygous mutations in the ATP-Binding Cassette Subfamily C Member 2 (ABCC2) gene, including a novel missense mutation. This report describes a case of DJS with atypical clinicopathologic findings and suggests that DJS should be considered in patients with neonatal cholestasis and bile duct paucity.

Elevated C5-hydroxy acylcarnitine in an infant girl as a result of holocarboxylase synthetase deficiency.

CONTEXT: Elevated 3-hydroxyisovaleryl-/2-methyl-3-hydroxybutyryl (C5-OH) acylcarnitine in blood can result from several genetic enzyme deficiencies: 3-methylcrotonyl CoA carboxylase deficiency, 3-hydroxy 3-methylglutaryl-CoA lyase deficiency, beta-ketothiolase deficiency, 2-methyl 3-hydroxybutyryl-CoA dehydrogenase deficiency, primary 3-methylglutaconic aciduria, multiple biotin-dependent carboxylase deficiencies and biotin metabolism disorders. Biochemical tests help differentiate these causes while molecular tests are usually required for definitive diagnosis. CASE DESCRIPTION: We reported an infant girl with newborn screen findings of elevated C5-OH acylcarnitine. She had further confirmational biochemical testing including plasma acylcarnitines, urine organic acids and urine acylglycines. Patient's urine organic acid profile showed markedly increased 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. Urine acylglycine test reported a large increase of 3-methylcrotonylglycine and plasma acylcarnitine test repeated the finding of elevated C5-OH acylcarnitine together with propionyl acylcarnitine elevation. These results point to multiple biotin-dependent carboxylase deficiency. Molecular tests revealed a homozygous mutation in the holocarboxylase synthetase gene that is consistent with her biochemical test findings. This case demonstrated the critical role of newborn screen in identifying inborn errors of metabolism that may otherwise be missed and lead to severe morbidity later in life. It also showcased that both biochemical and molecular tests are essential tools in the diagnosis.

Newborn Screening: Current Practice and Our Journey over the Last 60 Years.

BACKGROUND: Inborn errors of metabolism comprise a set of more than 2000 known disorders which can result in significant morbidity and may be rapidly fatal. Diagnosing these disorders at birth and treating immediately, however, may often result in a normal to near-normal life for the affected infant. Thus, newborn screening (NBS) has saved or improved the lives of countless individuals since its inception in the 1960s. CONTENT: This review covers NBS, from its early beginnings up to the current day practice. We follow the evolution of NBS, as well as describe the need and how disorders are added to NBS programs, the testing and how its performance is monitored, and the follow-up to the testing. We also briefly touch on NBS outside the United States. SUMMARY: Newborn screening in the United States is a major public health success story and it continues to grow and evolve to cover more disorders and utilize new technological advances.

Clinical applications of 3-hydroxy fatty acid analysis by gas chromatography-mass spectrometry.

BACKGROUND: L-3-Hydroxy fatty acids are unusual metabolites and rarely occur in significant quantities in normal human physiology. Genetic defects of both long-chain and medium-/short-chain mitochondrial L-3 hydroxyacyl coenzyme A dehydrogenases (LCHAD, M/SCHAD) have been identified as significant metabolic diseases in humans often with severe clinical phenotypes and pathophysiology that appears to differ from other defects of straight chain fatty acid oxidation. It is felt that accumulation of these atypical fatty acid species may play a role in this pathology. We have therefore developed an assay to measure these compounds in body fluids, and tissue culture medium to help in the diagnosis of these disorders and to better study the effects of 3-hydroxy fatty acid accumulation. METHODS: We have developed a stable isotope dilution, selected ion-monitoring gas chromatography-mass spectrometric assay for the measurement of all 3-hydroxy fatty acids from chain lengths C6 to C18 using 1,2 (13)C-labeled internal standards for all species. Authentic patient samples were utilized to develop reference intervals for control subjects, for those associated with patient samples confirmed at the molecular level to have either LCHAD or M/SCHAD deficiency and for patients who did not have disease but were fasting or on diets high in medium-chain fatty acids. Likewise, skin fibroblasts were obtained from patients with confirmed disease for additional study. Samples were also obtained from the hadh (M/SCHAD) knockout mouse. RESULTS: The measurement of 3-hydroxy fatty acids in patient plasma is a valuable tool in the identification of defects of both enzymes. Severe starvation, prolonged fasting and increased medium-chain triglycerides in the diet produce a profile that is similar to that seen in M/SCHAD deficiency, making this a more difficult condition to diagnose but these biomarkers provide an important clue to the diagnosis, particularly in non-fasted, diet-controlled patients. Fibroblast studies in LCHAD deficiency demonstrate that long-chain 3-hydroxy fatty acid accumulation can be observed in cultured tissues. Incubation of cultured fibroblasts from LCHAD deficient patients with labeled fatty acids demonstrated a process of chain lengthening that has not previously been recognized. CONCLUSIONS: The measurement of body fluid and cultured cell 3-hydroxy fatty acids provides both diagnostic and pathogenic information regarding these genetic diseases of fatty acid oxidation in the mitochondrion. Presently, the measurement of medium- and short-chain species provides a major metabolic biomarker for the recognition of M/SCHAD deficiency.

Treatment of vinasse from tequila production using polyglutamic acid.

Vinasse, the wastewater from ethanol distillation, is characterised by high levels of organic and inorganic matter, high exit process temperature (ca. 90°C) and low pH (3.0-4.5). In this study, the treatment of tequila vinasse was achieved by a flocculation-coagulation process using poly-γ-glutamic acid (PGA). Results showed that the use of PGA (250-300 ppm) combined with sodium hypochlorite and sand filtration managed to remove about 70% of the turbidity and reduced chemical oxygen demand (COD) by 79.5% with the extra benefit of colour removal. PGA showed its best flocculating activity at pH 2.5-3.5 and a temperature of 30-55°C. Such a treatment may be a solution for small tequila companies for which other solutions to deal with their vinasse may not be economically affordable.

Current State of Pediatric Reference Intervals and the Importance of Correctly Describing the Biochemistry of Child Development: A Review.

Importance: Appropriately established pediatric reference intervals are critical to the clinical decision-making process and should reflect the physiologic changes that occur during healthy child development. Reference intervals used in pediatric care today remain highly inconsistent across a broad range of common clinical biomarkers. Observations: This narrative review assesses biomarker-specific pediatric reference intervals and their clinical utility with respect to the underlying biological changes occurring during development. Pediatric reference intervals from PubMed-indexed articles published from January 2015 to April 2021, commercial laboratory websites, study cohorts, and pediatric reference interval books were all examined. Although large numbers of pediatric reference intervals are published for some biomarkers, very few are used by clinical and commercial laboratories. The patterns, extent, and timing of biomarker changes are highly variable, particularly during developmental stages with rapid physiologic changes. However, many pediatric reference intervals do not capture these changes and thus do not accurately reflect the underlying biochemistry of development, resulting in significant inconsistencies between reference intervals. Conclusions and Relevance: There is a need to correctly describe the biochemistry of child development as well as to identify strategies to develop accurate and consistent pediatric reference intervals for improved pediatric care.

Urine Organic Acid Analysis: Key Diagnostic Test for Fumaric Aciduria in a Sri Lankan Child.

Fumaric aciduria resulting from fumarate hydratase deficiency is a rare inherited disorder of the Krebs tricarboxylic acid cycle that is characterized by neurologic manifestations, a spectrum of brain abnormalities, and the excretion of fumaric acid in urine. We describe a 3 year old Sri Lankan boy who was referred at age 10 months with poor weight gain and hypotonia for further laboratory investigations. In addition to global developmental delay, there were noticeable dysmorphic features with a prominent forehead, low-set ears, micrognathia, and hypertelorism with persistent neutropenia. Urine organic acid assay revealed a massive elevation of fumaric acid on 2 occasions. Molecular analysis revealed a homozygous likely pathogenic missense variant, NM000143.3:c.1048C>T p. (Arg350Trp), in the FH gene, confirming the biochemical diagnosis. Our patient was the first patient in Sri Lanka molecularly diagnosed with fumaric aciduria. This case study highlights the importance of performing organic acid assays in children presenting with neurologic manifestations especially when these are suspected to have a metabolic basis.

Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

Taxus globosa S. cell lines: initiation, selection and characterization in terms of growth, and of baccatin III and paclitaxel production.

Of the initial six cell lines originating from explants of Taxus globosa, or Mexican yew (stem internode, leaves and meristematic tissue), three were selected for their microbial and oxidation resistance, two from leaves and the other from stem internode. A study of their behavior, both in terms of cell growth, and of baccatin III and paclitaxel production, was developed in suspension cultures with an initially standardized biomass (fresh weight 0.23 g/L) using modified Gamborg's B5 medium, and an elicitor (methyl jasmonate), on either the first or seventh day of culture, at several levels (0, 0.1, 1, 10, 100 microM). In most of the conditions used, the three cell lines showed growth associated baccatin III production. The cell line from stem internode was the highest producer of baccatin III using 1 microM elicitor, sampling at 10 days (p < or = 0.01, 6.45 mg/L). This same line also had the highest biomass production (6.85 g/L, p < or = 0.01) at 10 days of culture but at the higher elicitor concentration of 10 microM. All three cell lines did not produce paclitaxel under experimental conditions used.

Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatization and an automated liquid handler.

Amino acid analysis is central to newborn screening and the investigation of inborn errors of metabolism. Ion-exchange chromatography with ninhydrin derivatization remains the reference method for quantitative amino acid analysis but offers slow chromatography and is susceptible to interference from other co-eluting compounds. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) provides a rapid and highly specific alternative, but sample preparation is frequently laborious and sometimes cost prohibitive. To address these limitations, we validated an LC-MS/MS method using the aTRAQ Reagents Application Kit with a modified protocol consuming only half reagents. Adequate performance for clinical specimen measurement of 26 amino acids with high clinical relevance was achieved. An automated liquid handler and modified calibration and normalization approaches were used to ensure reproducible assay performance. Linear measurement between 5 and 2000 µM was achieved for most analytes despite use of a small, 10 µl sample size. Overall the method achieved near substantially improved throughput and enabled use of smaller samples volumes for batched analyses of clinical samples.

Interest of locally weighted regression to overcome nonlinear effects during in situ NIR monitoring of CHO cell culture parameters and antibody glycosylation.

Animal cell culture processes have become the standard platform to produce therapeutic proteins such as recombinant monoclonal antibodies (mAb). Since the mAb quality could be subject to significant changes depending on manufacturing process conditions, real time monitoring and control systems are required to ensure mAb specifications mainly glycosylation and patient safety. Up to now, real time monitoring glycosylation of proteins has received scarce attention. In this article, the use of near infrared (NIR) to monitor mAb glycosylation has been reported for the first time. Whereas monitoring models are mainly constructed using linear partial least squares regressions (PLSR), evidences presented in this study indicate nonlinearity relationship between in situ captured spectra and compound concentrations, compromising the PLSR performances. A novel and simple approach was proposed to fit nonlinearity using the locally weighted regression (LWR). The LWR models were found to be more appropriate for handling information contained in spectra so that real time monitoring of cultures were accurately performed. Moreover, for the first time, the LWR calibration models allowed mAb glycosylation to be monitored, in a real time manner, by using in situ NIR spectroscopy. These results represent a further step toward developing active-control feedback of animal cell processes, particularly for ensuring properties of biologics.

Measurement of Oncometabolites D-2-Hydroxyglutaric Acid and L-2-Hydroxyglutaric Acid.

We describe a liquid chromatography-tandem mass spectrometry assay for measurement of D-2-hydroxyglutaric acid and L-2-hydroxyglutaric acid. These metabolites are increased in specific inborn errors of metabolism and are now recognized as oncometabolites. The measurement of D-2-hydroxyglutarate in peripheral blood may be used as a biomarker for screening and follow-up of patients with IDH-mutated acute myeloid leukemia.