Monitorização dos níveis de fenilalanina em pacientes com fenilcetonúria por meio de sangue impregnado em papel filtro : comparação de dois métodos

Introdução: Os Erros Inatos do Metabolismo (EIM) são doenças genéticas causadas pela atividade deficiente de uma ou mais enzimas específicas ou por defeitos no transporte de proteínas. A fenilcetonúria (PKU) é um EIM causado pela atividade deficiente da fenilalanina-hidroxilase (PAH) que é responsável pela conversão de Fenilalanina (Phe) em Tirosina (Tyr). Esta doença pode ser detectada precocemente através da Triagem Neonatal e seu diagnóstico confirmado através da dosagem de aminoácidos por Cromatografia Líquida de Alta Performance (HPLC). O excesso de Phe e o consequente desbalanço de aminoácidos causam efeito tóxico no Sistema Nervoso Central (SNC) e por consequência perda de funções cognitiva e déficit cognitivo. O tratamento atual é feito principalmente através de dieta restrita em Phe, suplementação de aminoácidos essenciais e acompanhamento laboratorial e clínico. É importante lembrar que o diagnóstico e o tratamento precoce podem levar a excelentes resultados na qualidade de vida destes pacientes. Objetivo: O objetivo do presente trabalho foi avaliar dois métodos distintos: um método fluorimétrico desenvolvido In House (método A) e o Kit comercial de Fenilalanina Neonatal (Neonatal Phenylalanine Kit – Perkin Elmer®) (método B) para detecção dos níveis de Phe em amostras de sangue impregnado em papel filtro (SIPF) em pacientes com PKU que estão em acompanhamento no ambulatório do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre (SGM-HCPA) e comparar estes resultados com os resultados obtidos por HPLC, o qual é considerado o padrão ouro para o diagnóstico e monitoramento de pacientes com PKU. Metodologia: Estudo transversal, prospectivo e observacional no qual foi realizada a determinação dos níveis de Phe em amostras de SIPF de pacientes com PKU acompanhados no ambulatório do SGM-HCPA.Os resultados obtidos foram comparados com os níveis de Phe apresentados, na mesma amostra, pelo método de HPLC (padrão ouro). As amostras foram obtidas a partir de sangue total heparinizado. Após a impregnação do sangue total no papel filtro a amostra foi centrifugada e houve a separação do plasma para a análise pelo método de HPLC. O papel filtro ficou secando a temperatura ambiente por no mínimo 4 horas e no máximo 24 horas. Após secagem total a amostra foi armazenada em saco plástico com sílica em freezer -80ºC até o dia da análise. Resultados: Foram analisadas 89 amostras de SIPF de 47 pacientes que são acompanhados no SGM-HCPA. Dos 47 pacientes testados, 20 (42,5%) possuem a forma clássica de PKU, 16 (34,1%) a forma leve da doença e em 11 o fenótipo não está definido. A correlação entre o método In House (método A) e o HPLC, e entre o Kit comercial de Fenilalanina Neonatal (método B) e o HPLC feita através da correlação de Spearman, o coeficiente angular, interceptação em y e correlação linear foram de 0,990, 1,0365, 3,8125, 09873 e 0,974, 1,0795, 31,98 e 0,895 respectivamente. O nível de significância das duas análises foi de p<0,05. Todas as amostras analisadas por HPLC que eram alteradas ou normais, também se mostraram da mesma forma com ambos os métodos testados. A análise feita através da razão do diagrama de Bland Altman mostrou que quanto menor o valor do HPLC, maior é a dispersão dos valores nos métodos A e B. Conclusões: Nossos testes sugerem que amostras de SIPF nos métodos A e B são boas alternativas para a monitorização dos níveis de Phe em pacientes com PKU, sendo que o método A apresentou maior concordância em relação ao padrão ouro.Introduction: Inborn Errors of Metabolism (IEM) are genetic diseases caused by the absense of activity of one or more specific enzymes or by defects in the transport of proteins. Phenylketonuria (PKU) is an IEM caused by the deficiency of the phenylalanine hydroxylase (PAH), enzyme that is responsible for the conversion of Phenylalanine (Phe) into Tyrosine (Tyr). This disease can be detected early through Neonatal Screening and its diagnosis is confirmed through the measurement of amino acids by High Performance Liquid Chromatography (HPLC). The excess of Phe and the consequent amino acid imbalance cause toxic effect on the Central Nervous System (CNS) and consequently loss of cognitive function and cognitive deficit. The current treatment is mainly made through restricted diet in Phe, supplementation of essential amino acids and laboratory and clinical follow-up. It is important to remember that early diagnosis and treatment can lead to excellent results in the quality of life of these patients. Objective: The objective of the present study was to evaluate two different methods: a fluorimetric method developed In House (method A) and the commercial Neonatal Phenylalanine Kit (Perkin Elmer®) (method B) for the detection of Phe levels in dried blood spots samples (DBS) in patients with PKU who are being followed up at the ambulatory of the Medical Genetics Service of the Hospital de Clínicas of Porto Alegre (MGS-HCPA) and compare these results with the results obtained by HPLC, which is considered the gold standard for the diagnosis and monitoring of patients with PKU. Methodology: A cross-sectional, prospective and observational study in which the determination of Phe levels in DBS samples of patients with PKU was carried out in the MGS-HCPA outpatient clinic using two different methods: the In House (method A) and the commercial Neonatal Phenylalanine Kit (method B). The results obtained were compared with the Phe levels presented by the HPLC method (gold standard). Samples of DBS were obtained from heparinized whole blood. After impregnation of the whole blood on the filter paper the sample was centrifuged and the plasma separated for analysis by the HPLC method. The filter paper was dried at room temperature for at least 4 hours and at most 24 hours. After complete drying the sample was stored in plastic bag with silica in freezer -80ºC until the day of analysis. Results: A total of 89 DBS samples were analyzed from 47 patients who are followed up at GMS-HCPA. The correlation between the In House (method A) and HPLC, and between the commercial Neonatal Phenylalanine Kit (method B) and HPLC using Spearman's correlation, slope, y-intercept and linear correlation were 0.990, 1.0365, 3.8125, 09873 and 0.974, 1.0795, 31.98 and 0.895 respectively. The level of significance of the two analyzes was p <0.05. Of the 47 patients tested, 20 (42.5%) had the classic form of PKU, 16 (34.1%) the mild form of the disease and 11 (23.4%) were not yet genotyped. All samples analyzed by HPLC that were altered or normal, also showed the same way with both methods tested. The analysis done through the ratio of the Bland Altman diagram showed that the lower the HPLC value, the greater the dispersion of the values in methods A and B. Conclusions: Our tests suggest that DBS samples in methods A and B are good alternatives for the monitoring of Phe levels in patients with PKU, and method A showed greater agreement with the gold standard

Monitoring of phenylalanine levels in patients with phenylketonuria using dried blood spots : a comparison of two methods

Phenylketonuria (PKU) is caused by deficient activity of phenylalanine hydroxylase (PAH), responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr). Monitoring of patients with PKU requires the measurement of Phe in plasma using highperformance liquid chromatography (HPLC) or in dried blood spots (DBS) using different techniques to adjust treatment strategy. The objective of this study was to evaluate Phe levels in DBS measured by two different methods and compare them with Phe levels measured in plasma by HPLC. We analyzed 89 blood samples from 47 PKU patients by two different methods: fluorometric method developed in-house (method A) and the commercially available PerkinElmer® Neonatal Phenylalanine Kit (method B) and in plasma by HPLC. The mean Phe levels by method A, method B, and HPLC were 430.4±39.9μmol/L, 439.3±35.4μmol/L, and 442.2±41.6μmol/L, respectively. The correlation values between HPLC and methods A and B were 0.990 and 0.974, respectively (p<0.001 for both). Our data suggest that methods A and B are useful alternatives for monitoring Phe levels in patients with PKU, with method A being in closer agreement with the reference standard (HPLC)

Updated birth prevalence and relative frequency of mucopolysaccharidoses across Brazilian regions

The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by 11 enzyme deficiencies, classified into seven types. Data on the birth prevalence of each MPS type are available for only a few countries, and the totality of cases may be underestimated. To determine the epidemiological profile of MPS in each Brazilian region, we analyzed data collected between 1982 and 2019 by a national reference laboratory and identified 1,652 patients. Using data between 1994 and 2018, the birth prevalence (by 100,000 live births) for MPS was 1.57. MPS II was the most common type of MPS in Brazil, and its birth prevalence was 0.48 (0.94 considering only male births). Regarding the number of cases per region, MPS II was the most frequent in the North and Center-West (followed by MPS VI), and also in the Southeast (followed by MPS I); MPS I and MPS II were the most common types in the South; and MPS VI was the most common in the Northeast (followed by MPS II). The differences observed in the relative frequencies of MPS types across Brazilian regions are likely linked to founder effect, endogamy, and consanguinity, but other factors may be present and need further investigation

Follow-up of pre-motor symptoms of Parkinson’s disease in adult patients with Gaucher disease type 1 and analysis of their lysosomal enzyme profiles in the CSF

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide. Its classic motor symptoms may be preceded by non-motor symptoms (NMS). Population studies have identified GBA variants as risk factors for idiopathic PD. The increased risk of PD has also been suggested in other Lysosomal Storage Disorders (LSDs). Objective: To assess the evolution of the prevalence of NMS compatible with PD in a cohort of South Brazilian adult patients with Gaucher Disease (GD) type 1, already evaluated 3 years ago (2018). Cerebrospinal Fluid (CSF) was collected to assess the levels of LSD enzymes (beta-hexosaminidases, beta-glucuronidase) and biomarker of macrophage activation (chitotriosidase, ChT), compared to controls (metachromatic leukodystrophy, MLD). Cognition was evaluated by the Montreal Cognitive Assessment (MoCA) questionnaire, daytime sleepiness by the Epworth Sleepiness Scale (ESS), depression by Beck´s Inventory, constipation by the Unified Multiple System Atrophy Rating Scale (UMSARS) scale, and REM sleep behavior disorder by the single-question screen. Hyposmia was assessed with Sniffin’ Sticks (SST). Results Nineteen patients completed the follow-up (mean age of the sample was 44 years; range, 26–71). The patient with the highest number of NMS at the baseline (4 including the lowest SST score) was diagnosed with PD four years later. Apart from an improvement in the ESS score, no other statistical significance was found between the number of NMS between the first and second evaluation, nor between patients with one L444P variant (n = 5) and the rest of the cohort. CSF was collected in five patients (mean age of the sample was 40 years, range 30–53. A significant difference was found in the mean CSF activity levels of beta-hexosaminidases and beta-glucuronidase between GD1 and MLD patients. Mean ChT (CSF) was 62 nmol/h/mL in GD patients and 142 in MLD (n = 6) patients. Conclusions: The patient with the highest number of NMS in our 2018 cohort was the one that developed PD, corroborating with the importance of this longitudinal follow-up. CSF and plasma analysis might allow a better understanding of the neurodegenerative processes connecting PD and the lysosomal environment. Further analysis is needed to understand this relationshi

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Monitorização dos níveis de fenilalanina em pacientes com fenilcetonúria por meio de sangue impregnado em papel filtro : comparação de dois métodos

Introdução: Os Erros Inatos do Metabolismo (EIM) são doenças genéticas causadas pela atividade deficiente de uma ou mais enzimas específicas ou por defeitos no transporte de proteínas. A fenilcetonúria (PKU) é um EIM causado pela atividade deficiente da fenilalanina-hidroxilase (PAH) que é responsável pela conversão de Fenilalanina (Phe) em Tirosina (Tyr). Esta doença pode ser detectada precocemente através da Triagem Neonatal e seu diagnóstico confirmado através da dosagem de aminoácidos por Cromatografia Líquida de Alta Performance (HPLC). O excesso de Phe e o consequente desbalanço de aminoácidos causam efeito tóxico no Sistema Nervoso Central (SNC) e por consequência perda de funções cognitiva e déficit cognitivo. O tratamento atual é feito principalmente através de dieta restrita em Phe, suplementação de aminoácidos essenciais e acompanhamento laboratorial e clínico. É importante lembrar que o diagnóstico e o tratamento precoce podem levar a excelentes resultados na qualidade de vida destes pacientes. Objetivo: O objetivo do presente trabalho foi avaliar dois métodos distintos: um método fluorimétrico desenvolvido In House (método A) e o Kit comercial de Fenilalanina Neonatal (Neonatal Phenylalanine Kit – Perkin Elmer®) (método B) para detecção dos níveis de Phe em amostras de sangue impregnado em papel filtro (SIPF) em pacientes com PKU que estão em acompanhamento no ambulatório do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre (SGM-HCPA) e comparar estes resultados com os resultados obtidos por HPLC, o qual é considerado o padrão ouro para o diagnóstico e monitoramento de pacientes com PKU. Metodologia: Estudo transversal, prospectivo e observacional no qual foi realizada a determinação dos níveis de Phe em amostras de SIPF de pacientes com PKU acompanhados no ambulatório do SGM-HCPA.Os resultados obtidos foram comparados com os níveis de Phe apresentados, na mesma amostra, pelo método de HPLC (padrão ouro). As amostras foram obtidas a partir de sangue total heparinizado. Após a impregnação do sangue total no papel filtro a amostra foi centrifugada e houve a separação do plasma para a análise pelo método de HPLC. O papel filtro ficou secando a temperatura ambiente por no mínimo 4 horas e no máximo 24 horas. Após secagem total a amostra foi armazenada em saco plástico com sílica em freezer -80ºC até o dia da análise. Resultados: Foram analisadas 89 amostras de SIPF de 47 pacientes que são acompanhados no SGM-HCPA. Dos 47 pacientes testados, 20 (42,5%) possuem a forma clássica de PKU, 16 (34,1%) a forma leve da doença e em 11 o fenótipo não está definido. A correlação entre o método In House (método A) e o HPLC, e entre o Kit comercial de Fenilalanina Neonatal (método B) e o HPLC feita através da correlação de Spearman, o coeficiente angular, interceptação em y e correlação linear foram de 0,990, 1,0365, 3,8125, 09873 e 0,974, 1,0795, 31,98 e 0,895 respectivamente. O nível de significância das duas análises foi de p<0,05. Todas as amostras analisadas por HPLC que eram alteradas ou normais, também se mostraram da mesma forma com ambos os métodos testados. A análise feita através da razão do diagrama de Bland Altman mostrou que quanto menor o valor do HPLC, maior é a dispersão dos valores nos métodos A e B. Conclusões: Nossos testes sugerem que amostras de SIPF nos métodos A e B são boas alternativas para a monitorização dos níveis de Phe em pacientes com PKU, sendo que o método A apresentou maior concordância em relação ao padrão ouro.Introduction: Inborn Errors of Metabolism (IEM) are genetic diseases caused by the absense of activity of one or more specific enzymes or by defects in the transport of proteins. Phenylketonuria (PKU) is an IEM caused by the deficiency of the phenylalanine hydroxylase (PAH), enzyme that is responsible for the conversion of Phenylalanine (Phe) into Tyrosine (Tyr). This disease can be detected early through Neonatal Screening and its diagnosis is confirmed through the measurement of amino acids by High Performance Liquid Chromatography (HPLC). The excess of Phe and the consequent amino acid imbalance cause toxic effect on the Central Nervous System (CNS) and consequently loss of cognitive function and cognitive deficit. The current treatment is mainly made through restricted diet in Phe, supplementation of essential amino acids and laboratory and clinical follow-up. It is important to remember that early diagnosis and treatment can lead to excellent results in the quality of life of these patients. Objective: The objective of the present study was to evaluate two different methods: a fluorimetric method developed In House (method A) and the commercial Neonatal Phenylalanine Kit (Perkin Elmer®) (method B) for the detection of Phe levels in dried blood spots samples (DBS) in patients with PKU who are being followed up at the ambulatory of the Medical Genetics Service of the Hospital de Clínicas of Porto Alegre (MGS-HCPA) and compare these results with the results obtained by HPLC, which is considered the gold standard for the diagnosis and monitoring of patients with PKU. Methodology: A cross-sectional, prospective and observational study in which the determination of Phe levels in DBS samples of patients with PKU was carried out in the MGS-HCPA outpatient clinic using two different methods: the In House (method A) and the commercial Neonatal Phenylalanine Kit (method B). The results obtained were compared with the Phe levels presented by the HPLC method (gold standard). Samples of DBS were obtained from heparinized whole blood. After impregnation of the whole blood on the filter paper the sample was centrifuged and the plasma separated for analysis by the HPLC method. The filter paper was dried at room temperature for at least 4 hours and at most 24 hours. After complete drying the sample was stored in plastic bag with silica in freezer -80ºC until the day of analysis. Results: A total of 89 DBS samples were analyzed from 47 patients who are followed up at GMS-HCPA. The correlation between the In House (method A) and HPLC, and between the commercial Neonatal Phenylalanine Kit (method B) and HPLC using Spearman's correlation, slope, y-intercept and linear correlation were 0.990, 1.0365, 3.8125, 09873 and 0.974, 1.0795, 31.98 and 0.895 respectively. The level of significance of the two analyzes was p <0.05. Of the 47 patients tested, 20 (42.5%) had the classic form of PKU, 16 (34.1%) the mild form of the disease and 11 (23.4%) were not yet genotyped. All samples analyzed by HPLC that were altered or normal, also showed the same way with both methods tested. The analysis done through the ratio of the Bland Altman diagram showed that the lower the HPLC value, the greater the dispersion of the values in methods A and B. Conclusions: Our tests suggest that DBS samples in methods A and B are good alternatives for the monitoring of Phe levels in patients with PKU, and method A showed greater agreement with the gold standard