Observations on Heat/Humidity Denaturation of Enzymes in Filter-Paper Blood Spots from Newborns

Freer, Dennis E.

doi:10.1373/clinchem.2005.049270

Cited by 15 publications

(13 citation statements)

References 3 publications

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“…GALT stability in DBSs stored at −20°C was only studied at low humidity because of difficulty in controlling humidity in the frozen state. The findings that GALT lost substantial activity during storage at elevated temperatures and low or high humidity are consistent with a previously reported controlled study of GALT stability [13] and with the observation that population means of GALT activities in newborn screening samples were more than 20% lower in summer than in winter [18]. …”

Section: Discussionsupporting

confidence: 91%

Galactose-1-phosphate uridyltransferase dried blood spot quality control materials for newborn screening tests

Adam

Flores

Hou

et al. 2015

Clinical Biochemistry

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Objectives We aimed to prepare dried-blood-spot (DBS) quality control (QC) materials for galactose-1-phosphate uridyltransferase (GALT), to evaluate their stability during storage and use, and to evaluate their performance in five DBS GALT test methods. Design and Methods We prepared and characterized GALT-normal and GALT-deficient DBS materials and compared GALT activities in DBSs after predetermined storage intervals at controlled temperatures and humidities. External evaluators documented the suitability of the DBS QC materials for use in five GALT test methods. Results GALT activity losses from DBSs stored in low (<30%) humidity for 14 days at 45°C, 35 days at 37°C, 91 days at room temperature, 182 days at 4°C, and 367 days at −20°C were 54%, 53%, 52% 23%, and 7% respectively. In paired DBSs stored in high humidity (>50%) for identical intervals, losses were: 45°C—68%; 37°C—79%; room temperature—72%, and 4°C—63%. GALT activities in DBSs stored at 4°C were stable throughout 19 excursions to room temperature. Twenty-five of 26 external evaluators, using five different GALT test methods, classified the GALT-deficient DBSs as “outside normal limits”. All evaluators classified the GALT-normal DBSs as “within normal limits”. Conclusions Most of the GALT activity loss from DBSs stored at elevated or room temperature was attributable to the effects of storage temperature. Most of the loss from DBSs stored at 4°C was attributable to the effects of elevated humidity. Loss from DBSs stored at −20°C was insignificant. The DBS materials were suitable for monitoring performance of all five GALT test methods.

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Section: Discussionsupporting

confidence: 91%

Galactose-1-phosphate uridyltransferase dried blood spot quality control materials for newborn screening tests

Adam

Flores

Hou

et al. 2015

Clinical Biochemistry

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“…Moreover, enzymes can also be sensitive to heat and humidity, with for consequence a drop of activity levels. For instance, after 1 day of storage at 35°C in a humid environment, activities of biotinidase, galactose‐1‐phosphate uridyltransferase, and glucose‐6‐phosphate dehydrogenase decrease by ≥60% (Freer, ). Such instabilities might explain seasonal changes in population‐scale mean activities, with losses from February to July of, respectively, 26%, 23%, and 38%.…”

Section: Blood Collectionmentioning

confidence: 99%

The use of mass spectrometry to analyze dried blood spots

Wagner

Tonoli

Varesio

et al. 2014

Mass Spectrometry Reviews

226

228

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Dried blood spots (DBS) typically consist in the deposition of small volumes of capillary blood onto dedicated paper cards. Comparatively to whole blood or plasma samples, their benefits rely in the fact that sample collection is easier and that logistic aspects related to sample storage and shipment can be relatively limited, respectively, without the need of a refrigerator or dry ice. Originally, this approach has been developed in the sixties to support the analysis of phenylalanine for the detection of phenylketonuria in newborns using bacterial inhibition test. In the nineties tandem mass spectrometry was established as the detection technique for phenylalanine and tyrosine. DBS became rapidly recognized for their clinical value: they were widely implemented in pediatric settings with mass spectrometric detection, and were closely associated to the debut of newborn screening (NBS) programs, as a part of public health policies. Since then, sample collection on paper cards has been explored with various analytical techniques in other areas more or less successfully regarding large‐scale applications. Moreover, in the last 5 years a regain of interest for DBS was observed and originated from the bioanalytical community to support drug development (e.g., PK studies) or therapeutic drug monitoring mainly. Those recent applications were essentially driven by improved sensitivity of triple quadrupole mass spectrometers. This review presents an overall view of all instrumental and methodological developments for DBS analysis with mass spectrometric detection, with and without separation techniques. A general introduction to DBS will describe their advantages and historical aspects of their emergence. A second section will focus on blood collection, with a strong emphasis on specific parameters that can impact quantitative analysis, including chromatographic effects, hematocrit effects, blood effects, and analyte stability. A third part of the review is dedicated to sample preparation and will consider off‐line and on‐line extractions; in particular, instrumental designs that have been developed so far for DBS extraction will be detailed. Flow injection analysis and applications will be discussed in section IV. The application of surface analysis mass spectrometry (DESI, paper spray, DART, APTDCI, MALDI, LDTD‐APCI, and ICP) to DBS is described in section V, while applications based on separation techniques (e.g., liquid or gas chromatography) are presented in section VI. To conclude this review, the current status of DBS analysis is summarized, and future perspectives are provided. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:361–438, 2016.

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“…Confirmation of analyte stability is of particular concern for measuring enzymatic activity from a dried spot because the drying process can denature the enzyme or release proteases. 10-11 …”

Section: Introductionmentioning

confidence: 99%

Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

Perez

Pantazides

Watson³

et al. 2015

Anal. Chem.

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Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intra-spot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intra-spot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10-times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intra-spot variability without the need to control for initial sample volume, and enhances analyte stability.

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Observations on Heat/Humidity Denaturation of Enzymes in Filter-Paper Blood Spots from Newborns

Cited by 15 publications

References 3 publications

Galactose-1-phosphate uridyltransferase dried blood spot quality control materials for newborn screening tests

Galactose-1-phosphate uridyltransferase dried blood spot quality control materials for newborn screening tests

The use of mass spectrometry to analyze dried blood spots

Enhanced Stability of Blood Matrices Using a Dried Sample Spot Assay To Measure Human Butyrylcholinesterase Activity and Nerve Agent Adducts

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