Dried-blood Filter Paper Samples Research Articles

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

BackgroundLysosomal 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. MethodsWe 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), β-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. ResultsWe show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. ConclusionsWe 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

BackgroundKrabbe 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 β-galactosidase. Extending these studies, we present now a new GALC assay on these type of samples. MethodsAdapted leukocyte fluorometric assay was used for the evaluation of GALC in DLFP samples. ResultsOur results using this method showed a clear discrimination between GALC levels observed in KD patients and healthy controls. ConclusionsThe 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.

Impact of Inborn Errors of Metabolism on Admission in a Neonatal Intensive Care Unit—A Prospective Cohort Study

To estimate the incidence of Inborn errors of metobolism (IEM) in Neonatal intensive care unit (NICU) using tandem mass spectrometry and to determine the impact that these disorders have on NICU resources. During the period of study, 724 (81% eligible cases) dried blood filter-paper samples were collected from a NICU. The samples were analysed using tandem mass spectrometry. The diagnosis was further confirmed through clinical symptoms and by gas chromatography-mass spectrometry. The results were also confirmed by clinical follow-up of all positive patients in an overall interval of 1 year. The mean observation period was 11 months per neonate. In total, 22 cases were screen positive and 8 cases of inborn errors of metabolism were detected. The incidence of IEM in the population of patients admitted to the authors' NICU was 1.1%. The most common inborn error found was methylmalonic acidemia (3 cases, 37.5%), and all of the cases needed aggressive treatment and invasive mechanical ventilation. There were two cases of Tyrosinemia type 1, one case each of Maple Syrup Urine Disease, Propionic Acidemia, and Multiple Acyl-CoA dehydrogenase deficiency (MADD). Five of the eight patients required invasive mechanical ventilation. The median length of NICU stay was 3 days (1~7 days) and early therapeutic intervention was effective for four of them and other four patients (50%) died. The incidence of IEM in NICU was 1.1%, indicating an underestimation of the incidence of metabolic disorders prior to implementing screening. Most patients with IEM in the NICU required invasive mechanical ventilation and the mortality was increased due to underlying IEM.

Screening for inborn errors of metabolism using automated electrospray tandem mass spectrometry: Study in high-risk Indian population

Objectives: Tandem mass spectrometry is a major technological advance in the screening for inborn errors of metabolism. It has the advantage of sensitive and simultaneous multiple disease screening with minimal sample requirement. The diseases detected include aminoacidemias, fatty acid oxidation disorders, and organic acidemias. Design and methods: Using automated electrospray tandem mass spectrometry we screened 3550, clinically selected, symptomatic children for inborn errors of metabolism by analyzing amino acids and acylcarnitines in dried blood filter-paper samples. Results: Among these, 113 (3.2%) children were identified with a metabolic disorder: 61 (54%) patients had amino acid disorders, 47 (41.6%) had organic acidemias, and 5 (4.4%) children had disorders of fatty acid oxidation. The diagnoses were further confirmed through clinical symptoms, and other biochemical studies. Conclusions: These results show that inherited metabolic disorders are not rare in India, a rapidly developing country with a high birth rate and relatively frequent occurrence of consanguineous marriages.

Selective screening for inborn errors of metabolism on clinical patients using tandem mass spectrometry in China: A four‐year report

We have initiated clinical selective screening for inborn errors of metabolism in China by analysing amino acids and acylcarnitines in a dried blood filter-paper samples using tandem mass spectrometry. Samples from a total of 3070 children suspected of inborn errors of metabolism were collected through a study network which covered most provinces of China. The diagnoses were further confirmed through clinical symptoms, by gas chromatography-mass spectrometry and other biochemistry studies, and in a few cases by DNA analysis. In all, 212 cases were diagnosed (6.6%) including 92 (43.4%) with amino acids disorders (48 with phenylketonuria, 12 with ornithine carbamoyltransferase deficiency, 7 with tyrosinaemia type I, 9 with maple syrup urine disease, 5 with citrullinaemia type I, 8 with citrullinaemia type II, 2 with homocystinuria, and 1 with argininaemia); 107 (50.5%) with organic acid disorders (including 58 with methylmalonic acidaemia, 13 with propionic acidaemia, 6 with isovaleric acidaemia, 7 with glutaric acidaemia type I, 6 with 3-methylcrotonyl-CoA carboxylase deficiency, 2 with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, 10 with multiple carboxylase deficiency, and 5 with beta-ketothiolase deficiency); and 13 (6.1%) with fatty acid oxidation disorders (including 1 with carnitine palmitoyltransferase deficiency type I, 1 with carnitine palmitoyltransferase deficiency type II, 1 with short-chain acyl-CoA dehydrogenase deficiency, 5 with medium-chain acyl-CoA dehydrogenase deficiency, 3 with very long-chain acyl-CoA dehydrogenase deficiency, and 2 with multiple acyl-CoA dehydrogenase deficiency). It is suggested that tandem mass spectrometry is useful for selective screening of clinically suspected patients. The majority of diseases (94%) in this study were amino acid disorders and organic acid disorders. Fatty acid oxidation disorders are relatively rare in the Chinese, but medium-chain acyl-CoA dehydrogenase deficiency should be further investigated.

Usefulness of thyroxine and free thyroxine filter paper measurements in neonatal screening for congenital hypothyroidism of preterm babies.

Low thyroxine (T4) with normal thyroid stimulating hormone (TSH) is a well known condition in preterm (PT) infants. The establishment of T4 and freeT4 (FT4) values in filter paper dried blood spots in PT could provide useful information in the neonatal period. To study T4 and FT4 levels in dried blood filter paper samples of PT and full term (FT) babies. We measured T4 by fluoroimmunoassay (FIA) DELFIA and TSH by IFMA DELFIA (Wallac Inc Turku, Finland) in 193 PT (26 to 37 weeks of gestational age (GA)) in samples from the first and second week of life and in 153 FT babies in the first week of life. In 131 PT and 31 FT we determined FT4 in filter paper blood spots using FIA (Alonso Fernandez J). Infants were grouped according to GA. There was a significant difference in T4 between PT and FT (p < 0.001). The lowest T4 levels were at 26 to 29 weeks GA. T4 values were lower in the second week. FT4 in PT up to 35 weeks GA, during the first week, was significantly different with FT infants (G1 to G3 p < 0.01, G4 p < 0.05). FT4 values in the first and second weeks of life did not vary. T4 values were significantly lower in PT than in FT neonates, increasing with GA. PT infants had low T4 with normal FT4 values. This could suggest a decreased thyroxine binding globulin (TBG) or decreased protein binding and/or an adaptative mechanism that would not require therapeutical intervention.

The Detection of Hemoglobin Variants by Isoelectrofocusing Using EDTA-Collected and Filter Paper-Dried Cord Blood Specimens

A comparative study was conducted aimed at the detection of abnormal hemoglobin conditions (mainly AS, SS, AC, CC, SC, AE, AD, Hb Bart's or gamma 4) by isoelectrofocusing of cord blood samples stored as liquid blood and as dried hemoglobin on filter paper. Analyses were made within four to six days after the collection of the samples; storage conditions mimicked those of testing programs using liquid blood samples (as in Georgia) or dried blood filter paper samples (as in several other states). During analysis of hemoglobin solutions extracted from dried blood samples, considerable difficulties were encountered in detecting significant hemoglobinopathies such as SS and SC, whereas even simple abnormalities such as AS, AC, AD, and AE were also often not diagnosed. Detection of the fast-moving variant Hb Bart's or gamma 4 was not possible. These results again cause doubt regarding the general use of dried blood filter paper samples in newborn hemoglobin testing programs. Perhaps special precautions--such as speed in analyzing the samples, storage at -20 degrees C (or perhaps 4 degrees C) instead of at room temperature, and removal of unstable hemoglobin from the filter paper extract by centrifugation--might eliminate some of the problems that were observed.