Extended use of dried-leukocytes impregnated in filter paper samples for detection of Pompe, Gaucher, and Morquio A diseases.

BACKGROUND: Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. METHODS: We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), ß-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. RESULTS: We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. CONCLUSIONS: We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.

Galactocerebrosidase assay on dried-leukocytes impregnated in filter paper for the detection of Krabbe disease.

BACKGROUND: Krabbe disease (KD) is an inherited lysosomal storage disease (LSD) caused by the deficiency of galactocerebrosidase (GALC) and is characterized by a severe and progressive leukodystrophy with death frequently before one year of life in the classical early-onset form. As a consequence of the enzyme defect, globoid cells containing undigested galactosylceramide are observed and are characteristic of the disease. Hematopoietic stem cell transplantation is the current treatment for this disease, with some success in the classical cases if performed very early in life. Definitive diagnosis of KD is generally accessed by determination of GALC in leukocytes or fibroblasts. For the last few years, dried-blood filter paper (DBFP) samples have been increasingly used for lysosomal enzyme assays. Originally, some lysosomal enzymes could not be tested in DBFP samples using fluorometric assays, including GALC, heparan-sulfamidase and a few others. Recently, we reported successful results using dried-leukocytes filter paper (DLFP) samples for heparan sulfamidase and ß-galactosidase. Extending these studies, we present now a new GALC assay on these type of samples. METHODS: Adapted leukocyte fluorometric assay was used for the evaluation of GALC in DLFP samples. RESULTS: Our results using this method showed a clear discrimination between GALC levels observed in KD patients and healthy controls. CONCLUSIONS: The assay is robust and reliable and could be adopted by reference laboratories for diagnosis of LSDs. It is expected that the use of DLPF would make it possible to diagnose patients living in isolated areas, where liquid samples usually have to be transported over several days and sometimes across country borders before reaching reference laboratories.

Extended use of a selective inhibitor of acid lipase for the diagnosis of Wolman disease and cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) deficiency produces two well defined inborn disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe, early-onset condition involving massive storage of triglycerides and cholesteryl esters in the liver, with death usually occurring before one year of life. CESD is a more attenuated, later-onset disease that leads to a progressive and variable liver dysfunction. Diagnosis of LAL deficiency is mainly based on the enzyme assay of LAL activity in fibroblasts. Recently, a selective acid lipase inhibitor was used for the determination of enzyme activity in dried-blood filter paper (DBFP) samples. To extend and to validate these studies, we tested LAL activity with selective inhibition on DBFP samples, leukocytes and fibroblasts. Our results showed a clear discrimination between patients with LAL deficiency and healthy controls when using DBFP, leukocytes or fibroblasts (p<0.001). Deficiency of LAL was also demonstrated in individuals referred to our laboratory with suspected clinical diagnosis of WD, CESD, and Niemann-Pick type B. We conclude that the assay of LAL using selective inhibitor is a reliable and useful method for the identification of LAL deficiency, not only in DBFP samples but also in leukocytes and fibroblasts. This is important as enzyme replacement therapy for LAL deficiency is currently being developed, making the correct diagnosis a critical issue.

Assay of heparan-N-sulfamidase in dried leukocytes impregnated in filter paper: a new tool for the identification of mucopolisaccharidosis IIIA and potentially other lysosomal disorders.

Diagnosis of lysosomal storage disorders (LSDs) is mainly based on specific enzyme assays in leucocytes. Dried blood spots have also been used as sample for the enzyme assays. However, some lysosomal enzymes such as heparan-N-sulfamidase (HNS) and others cannot be assayed by this material. We developed an assay for HNS using dried leukocytes impregnated in filter paper (DLFP) as source of enzyme, and the results allowed the correct identification of Mucopolisaccharidosis IIIA. From this proof of concept we predict that the assay of lysosomal enzymes in DLFP samples, which still needs further development, could be a useful tool for the diagnosis of LSDs, especially in regions where transportation of liquid blood samples in appropriate conditions for long distances and/or across country borders is challenging.

Reliable detection of mucopolysacchariduria in dried-urine filter paper samples.

BACKGROUND: The mucopolysaccharidoses (MPS) are inherited metabolic disorders with bone, joint, and visceral abnormalities, leading to multi-organ dysfunction and, sometimes, neurological manifestations. These diseases are caused by storage of glycosaminoglycans (GAGs) and other complex molecules in tissues, among other pathogenic mechanisms. Definitive diagnosis of the affected individual is mainly based on the identification of the specific enzyme deficiency. New therapies are available or are in development for these pathologies, and early diagnosis seems to be important for the therapy outcomes. Almost all MPS patients have increased levels of GAGs in urine being their evaluation usually the first step in the screening of these conditions. Test on urine may be challenging as transportation of liquid urine samples in appropriate conditions for long distances, especially across international borders, could be difficult. METHODS: With the aim of overcoming the difficulties related to the use of liquid samples, we extended and validated previous studies about colorimetric determination of GAGs in dried-urine filter paper (DUFP) samples. RESULTS: In the conditions we described, there are no differences in the concentration of GAGs between urine and DUFP samples. Untreated patients with MPS and normal controls were well discriminated using any of the samples. CONCLUSIONS: Dried-urine filter paper is a suitable sample for the colorimetric quantitation of GAGs, and that its incorporation as an additional tool for screening of MPS should be considered by reference laboratories.

Persistent tyrosinemia detected by thin-layer chromatography.

Screening for tyrosinemia is not routinely performed worldwide. Using a low expense thin-layer chromatography (TLC) for amino acids we detected a high frequency of transient tyrosinemia with secondary hyperphenylalaninemia in some newborns. Serum follow up showed the need to introduce adequate therapy in these babies.

A comprehensive screening program in South Brazil.

We present the experience and figures about a screening program in South Brazil carried on in Porto Alegre, capital of the Southern Brazilian State. We present the tests performed routinely in our laboratory, the prevalence of some diseases and tests for infectious diseases to be added in the most comprehensive regional program in our country.

Validity study of the Brazilian version of the Calgary Depression Scale for Schizophrenia.

INTRODUCTION: Although depression is a well-established feature of schizophrenia, it is difficult to measure, because it overlaps with negative symptoms and extrapyramidal symptoms (EPS). Routinely adopted depression scales were not designed to be used in--cases of schizophrenia, and are known to perform poorly when trying to distinguish depression from other symptoms. OBJECTIVE: The aim of this study was to evaluate the validity of the Brazilian version of the Calgary Depression Rating Scale for Schizophrenia (CDSS). METHOD: Outpatients from four mental health units in the city of São Paulo, diagnosed as having schizophrenia by DSM-IV criteria, were evaluated by two independent raters who applied the DSM-IV depression criteria. All patients were assessed by means of the CDSS, the Positive and Negative Syndrome Scale (PANSS), and the Extrapyramidal Symptom Rating Scale (ESRS). RESULTS: Eighty patients were recruited for the study. The analysis was carried out by comparing the DSM-IV criteria of depression with the CDSS scores, by means of the receiver operating characteristic (ROC) curves. The area under the ROC curve for major depression was 0.95 (SD = 0.02), and at a cut-off point of 6/7 the validity coefficients were as follows: sensibility 77%, specificity 92%, positive predictive value 67% and negative predictive value 95%. The area under the ROC curve for minor depression was 0.95 (SD = 0.02), and at a cut-off point of 4/5 the validity coefficients were as follows: sensibility 95%, specificity 88%, positive predictive value 75% and negative predictive value 98%. The correlation coefficients between the CDSS scores, the PANSS negative and positive subscale scores, and the ESRS scores were all below 0.50. CONCLUSION: It can be concluded that the Brazilian version of the CDSS is a valid research tool to assess depressive episodes for stabilized patients with schizophrenia.

[Transient neonatal tyrosinemia: a frequent abnormality] / Tirosinemia neonatal transitória: uma alteração freqüente.

OBJECTIVE: To evaluate the frequency of transient neonatal tyrosinemia, with or without secondary hyperphenylalaninemia,observed through neonatal screening for metabolic disorders, andthe need of monitoration and intervention with drugs and/or specialdiet in selected cases. METHODS: 457.870 dried blood samples obtained by heel stickfrom 3 to 20 days old babies were qualitatively evaluated by aminoacid thin-layer chromatography. Positive cases were quantitativelyconfirmed in serum samples by fluorimetric measurement of tyrosineand phenylalanine. RESULTS: 1.231 dried blood samples displayed positive resultsfor tyrosine in the cromatographic evaluation. The fluorimetricserum analysis disclosed normal levels of tyrosine and phenylalaninein 822 patients. The remained 409 patients showed hightyrosine levels and were placed in three groups according to thetyrosine concentration. In 118 of these cases serum phenylalaninewas also increased. CONCLUCIONS: Transient neonatal tyrosinemia is a very frequentdisorder in neonates (1/372); in some cases very high levels werefound, not only of tyrosine but also phenylalanine. As this findingis not yet accepted as absolutely harmless, the monitoration of thissituation and the use of measures to lower the tyrosine and phenylalaninelevels should be considered by the pediatrician.