Analytical Method Validation for Estimation of Neurotransmitters (Biogenic Monoamines) from Cerebrospinal Fluid Using High Performance Liquid Chromatography.

Biogenic amine neurotransmitters such as serotonin and dopamine are essential for signaling in both central and peripheral nervous system. Their metabolism is a multistep pathway and any defect in this results in alteration in metabolites of serotonin 5-Hydroxyindole acetic acid (5HIAA) and dopamine homovanillic acid (HVA) and 3-O-Methyl Dopa (3-OMD). Estimation of these metabolites in cerebrospinal fluid (CSF) assists in diagnosis of neurotransmitter defects. Their estimation is technically demanding and is currently available only in referral centers. We aimed to optimize a method for analysis of 5HIAA, HVA and 3-OMD. A high performance liquid chromatography (HPLC) method with electro chemical detector (ECD) was standardized for estimation. Analysis for method validation, reference range verification and clinical correlation was performed. Linearity obtained for 5-HIAA, HVA and 3-OMD was 65.35-2615.0 nmoles/l, 68.62-2745.0 nmoles/l and 236.5-4730.0 nmoles/l respectively. The coefficient of variation for internal quality controls ranged from 5 to 14% and the external proficiency testing samples (n = 16) were within peer group range. CSF metabolite levels of samples for reference range analysis overlapped with age matched ranges reported in literature. Among the 40 suspected patients analyzed for clinical testing four were found to have a neurotransmitter defect. These patients were then confirmed with molecular testing and clinical correlation. The method is validated and can be adapted in a clinical laboratory with analytical competence in HPLC.

Paroxysmal Nonepileptic Events in a Pediatric Epilepsy Clinic.

AIMS: We aimed to study the frequency, age, and gender distribution of paroxysmal nonepileptic events (PNEs) in children referred to epilepsy clinic with the diagnosis of epilepsy. We also evaluated the therapeutic implications of correct diagnosis and co-existence of true epilepsy in this population. SETTINGS AND DESIGN: All new patients below 18 years attending the Pediatric epilepsy out-patient clinic of PD Hinduja hospital over 6 months were evaluated. MATERIALS AND METHODS: Patients with history of paroxysmal events characterized by abrupt changes in consciousness or behavior or movement were included. They were assessed on description of events aided by recorded videos. If the diagnosis was not confirmed by this preliminary evaluation, further investigations were advised. STATISTICAL ANALYSIS USED: Chi-square/Fisher's exact test was used to analyze differences between categorical variables and Kruskal-Wallis test between continuous variables. The data were analyzed by SAS University Edition. All significance tests were two-tailed with α <0.05. RESULTS: Two hundred new patients presenting with paroxysmal events were enrolled over 6 months. After diagnoses, 19% of these children had PNEs, 80% had epileptic events, and 1% remained undiagnosed. Common nonepileptic events seen were physiological in patients below 5 years and psychogenic in older children. Thirty-four percent of patients with PNEs were on anti-epileptic drugs (AEDs). After confirming nonepileptic attacks, only 2.6% patients needed AEDs for coexisting epilepsy which was statistically significant (P < 0.001) change in treatment. CONCLUSIONS: Epilepsy mimics are common in children and are often misdiagnosed causing undue stress. Correct diagnosis leads to a drastic change in management like withdrawal of drugs, commencing new treatment if needed, and appropriate referrals.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

BACKGROUND: Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. METHODS: The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. RESULTS: Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. CONCLUSION: The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.

GPR56-Related Polymicrogyria: Clinicoradiologic Profile of 4 Patients.

Bilateral frontoparietal polymicrogyria is an autosomal recessive cortical malformation associated with abnormalities of neuronal migration, white matter changes, and mild brainstem and cerebellar abnormalities. Affected patients present with delayed milestones, intellectual disability, epilepsy, ataxia, and eye movement abnormalities. The clinicoradiologic profile resembles congenital muscular dystrophy. However, no muscle disease or characteristic eye abnormalities of congenial muscular dystrophy are detected in these children. GPR56 is the only confirmed gene associated with bilateral frontoparietal polymicrogyria. Antenatal diagnosis is possible if the index case is genetically confirmed. Four patients from different Indian families with a distinct clinicoradiologic profile resembling congenital muscular dystrophy with mutations in the GPR56 gene are described.