Beta-Glucosidase analysis in collected dried blood spots, report of a new method applied to the control population and patients with suspicion of Gaucher disease

Análisis de beta-glucosidasa en sangre seca recolectada en papel filtro (DBS), reporte de un nuevo método aplicado a población control y pacientes con sospecha de enfermedad de Gaucher;
Análise de β-glucosidase em sangue seco coletado em papel filtro (DBS), relatório de um novo método aplicado à população controle e a pacientes com suspeita de doença de Gaucher

dc.creatorVásquez-Salazar, Ana Camila
dc.creatorUribe-Ardila, Alfredo
dc.date2021-06-04
dc.date2023-03-22T19:09:45Z
dc.date2023-03-22T19:09:45Z
dc.date.accessioned2023-09-06T17:42:09Z
dc.date.available2023-09-06T17:42:09Z
dc.identifierhttps://revistas.unimilitar.edu.co/index.php/rmed/article/view/4857
dc.identifier10.18359/rmed.4857
dc.identifierhttp://hdl.handle.net/10654/43511
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8692327
dc.descriptionGaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency in the enzymatic activity of β-glucosidase (BGLU), resulting in the accumulation of glucosylceramide in cells. Its diagnosis is aimed at checking the enzyme in the affected leukocytes. Studies have been conducted on dried blood spots (DBS) for BGLU activity to monitor high-risk populations; however, they exhibit interferences related to severe leukopenias or increased expression of the neutral BGLU isoform, a molecule not related to GD. This study intends to standardize a screening method on DBS (punch: 5 mm) using 4-methylumbelliferyl-β-D-glucopyranoside and chonduritol-β-epoxide. DBS samples from 395 individuals clinically suspected of GD (high-risk or HR population), 151 controls, and 16 affected patients were analyzed using the elution of 5 mm punches (≈10 μl of blood) in 300 μl of Triton X-100/ (0.5 %). As a result, the following ranges were obtained; hr: 0.84-26.92 nmol/ml/h, controls: 3.56-8.92 nmol/ml/h (M = 5.56, sd = 1.15), and patients with confirmed gd: 0.82-2.88 nmol/ml/h (M = 1.64, sd = 0.57). The cut-off point between patients with GD and controls was 3.22 nmol/ml/h, obtained from ROC analysis (99 % ci, 100 % sensitivity, and 100 % specificity). The protocol revealed a deficiency in all GD cases, confirmed by parallel BGLU analysis in isolated leukocytes. The use of CBE and the elution of 5 mm punches are recommended for enzymatic titration with a higher approximate volume of blood in the absence of neutral isoform activity.
dc.descriptionLa enfermedad de Gaucher (GD) es el trastorno de almacenamiento lisosomal que se caracteriza por la deficiencia en la actividad enzimática de la β-glucosidasa (BGLU), lo que produce la acumulación de glucosilceramida en las células. Su diagnóstico se orienta a la valoración de la enzima en los leucocitos afectados. Se han realizado estudios en DBS para la actividad de BGLU en el seguimiento de poblaciones de alto riesgo; sin embargo, presentan interferencias relacionadas a leucopenias severas o expresión aumentada de la isoforma neutra de la enzima BGLU, molécula no relacionada con GD. El objetivo de este estudio fue la estandarización de un método de tamizaje en DBS (punch: 5 mm) con el uso de 4-metilumbeliferil-β-D-glucósido y conduritol-β-epóxido. Se analizaron muestras de dbs de 395 individuos con sospecha clínica (población de alto riesgo o AR), 151 controles y 16 pacientes afectados, usando la elución de un corte de 5 mm (≈10 μl de sangre) en 300 μl de Tritón X-100/(0,5 %). Como resultados, se obtuvieron los rangos, AR: 0,84-26,92 nmol/ml/h, controles: 3,56- 8,92 nmol/ml/h (M = 5,56, ds = 1,15) y pacientes confirmados con GD: 0,82- 2,88 nmol/ml/h (M = 1,64, ds = 0,57). El punto de corte entre deficientes y controles fue 3,22 nmol/ml/h, obtenido a partir de análisis ROC (99 % confianza, 100 % sensibilidad y 100 % especificidad). El protocolo permitió evidenciar la deficiencia en todos los casos de GD, confirmados mediante el análisis en paralelo de la enzima en aislamiento leucocitario. Se recomienda el uso del CBE y realizar la elución del corte a 5 mm, a fin de llevar a cabo la valoración enzimática con un volumen mayor aproximado de sangre y en ausencia de la actividad generada por la isoforma neutra.
dc.descriptionA doença de Gaucher (GD) é o trastorno de armazenamento lisosomal caracterizado pela deficiência na atividade enzimática da β-glucosidase (BGLU), o que produz a acumulação de glucossilceramida nas células. Seu diagnóstico está orientado à avaliação da enzima nos leucócitos afetados. Foram realizados estudos em DBS para a atividade de BGLU no seguimento de populações de alto risco; contudo, são apresentadas interferências relacionadas a leucopenias graves ou a expressão aumentada da isoforma neutra da enzima BGLU, molécula não relacionada com GD. O objetivo deste estudo foi a padronização de um método de tamisação em DBS (punch: 5 mm) com o uso de 4-metilumbeliferil-β-D- glicosídeo e conduritol-β-epóxido. Foram analisadas amostras de dbs de 395 indivíduos com suspeita clínica (população de alto risco ou AR), 151 controles e 16 pacientes afetados, usando a eluição de um corte de 5 mm (≈10 μl de sangue) em 300 μl de Tritão X-100/(0,5 %). Como resultados, foram obtidos os intervalos: AR: 0,84-26,92 nmol/ml/h, controles: 3,56-8,92 nmol/ml/h (M = 5,56, ds = 1,15) e pacientes confirmados com GD: 0,82- 2,88 nmol/ml/h (M = 1,64, ds = 0,57). O ponto de corte entre deficientes e controles foi 3,22 nmol/ml/h, obtido a partir de análise ROC (99 % confiança, 100 % sensibilidade e 100 % especificidade). O protocolo permitiu evidenciar a deficiência em todos os casos de GD, confirmados mediante a análise em paralelo da enzima em isolamento leucocitário. É recomendado o uso do CBE e a realização da eluição do corte a 5 mm, a fim de implementar a avaliação enzimática com um volume maior aproximado de sangue e em ausência da atividade gerada pela isoforma neutra.
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dc.publisherUniversidad Militar Nueva Granada
dc.relationhttps://revistas.unimilitar.edu.co/index.php/rmed/article/view/4857/4644
dc.relationhttps://revistas.unimilitar.edu.co/index.php/rmed/article/view/4857/4782
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dc.rightsDerechos de autor 2021 Revista Med
dc.sourceRevista Med; Vol. 28 No. 2 (2020): july - december; 35-48
dc.sourceRevista Med; Vol. 28 Núm. 2 (2020): julio - diciembre; 35-48
dc.source1909-7700
dc.source0121-5256
dc.titleBeta-Glucosidase analysis in collected dried blood spots, report of a new method applied to the control population and patients with suspicion of Gaucher disease
dc.titleAnálisis de beta-glucosidasa en sangre seca recolectada en papel filtro (DBS), reporte de un nuevo método aplicado a población control y pacientes con sospecha de enfermedad de Gaucher
dc.titleAnálise de β-glucosidase em sangue seco coletado em papel filtro (DBS), relatório de um novo método aplicado à população controle e a pacientes com suspeita de doença de Gaucher
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