Elsevier

Clinical Biochemistry

Volume 44, Issues 17–18, December 2011, Pages 1445-1450
Clinical Biochemistry

The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States

https://doi.org/10.1016/j.clinbiochem.2011.09.010Get rights and content

Abstract

Objective

We aimed to measure separately the contributions of heat and humidity to changes in levels of 34 markers of inborn disorders in dried-blood-spot (DBS) samples.

Design and methods

We stored paired sets of DBSs at 37 °C for predetermined intervals in low-humidity and high-humidity environments. Marker levels of all samples in each complete sample set were measured in a single analytic run.

Results

During the 30 ± 5 day studies, galactose-1-phosphate uridyltransferase and biotinidase lost almost 65% of initial activities in low-humidity storage; most of the degradation in 27 other markers was attributable to adverse effects of high-humidity storage; seven markers in DBSs stored at high humidity lost more than 90% of initial levels by the end of the study and 4 of the 7 lost more than 50% of initial levels within the first week of storage.

Conclusions

Minimizing both humidity and temperature in DBS transportation and storage environments is essential to maintaining sample integrity.

Highlights

►We stored paired sets of blood-spots at 37 °C for 30 ± 5 days at low- and high-humidity. ►We measured marker levels of all samples in each set in a single analytic run. ►High blood-spot storage humidity caused most of the loss of 27 disorder markers. ►High (37 °C) blood-spot storage temperature caused most of the loss of 4 markers. ►Little of the marker losses were recovered by extending blood-spot elution times.

Introduction

Blood spot samples–collected from blood obtained by heel pricks, applied to filter paper and dried–are used to screen newborns for more than 50 treatable inborn disorders [1], [2], [3]. For more than 30 years, investigators have studied the relationship between temperature and the stabilities of markers for many of these disorders in dried-blood-spot (DBS) samples [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. These fragmentary retrospective and prospective studies of DBSs illustrate the adverse effects of uncontrolled or elevated temperatures and/or humidities on marker stabilities. However, differences in markers studied, study durations, DBS storage conditions, and data analysis methods make comparison of results among studies difficult. Published guidelines [15] for optimal long-term storage of residual DBSs after newborn screening analysis only address some of the markers presently recommended.

The United States' recommended uniform newborn screening panel consists of 30 core disorders recommended for screening and 26 secondary targeted disorders that are detectable in the course of screening for the 30 core disorders [1], [2], [3]. Examination of the stabilities of markers of the core and targeted disorders has not occurred in a single normalized study that included temperature, humidity and elution variables. Understanding and controlling for these variables are critical in sustaining effective operations of newborn screening and associated external quality assurance programs that store and transport DBS samples in a wide range of environments.

We performed accelerated degradation studies of 34 markers as part of routine evaluations of DBS materials prepared by the Newborn Screening Quality Assurance Program (NSQAP) of the Centers for Disease Control and Prevention (CDC) to assist laboratories with monitoring the performance of their newborn screening tests [16]. This report summarizes results from those studies and covers markers for 25 recommended core disorders and 22 targeted disorders. Hemoglobin S (the marker for the 3 recommended sickle cell diseases) was excluded from the accelerated degradation studies because it is usually measured in a qualitative manner (no calibration), and hearing loss was excluded because accelerated degradation studies are not applicable. Targeted panel markers for galactokinase and galactoepimerase deficiencies and markers for disorders measured by DNA testing–the core panel severe combined immune deficiency [3] and targeted panel T-cell-related lymphocyte deficiencies–were also excluded from our studies. These studies were initiated to compare the stabilities of the markers in DBSs stored at an elevated temperature (37 °C) and at low (below 30%) or high (above 50%) relative humidity for predetermined intervals. The objectives of the studies were to measure separately, under normalized conditions for all markers tested, the contributions of heat and humidity to the degradation of markers of inborn disorders in DBSs and to evaluate the effectiveness of extending DBS elution times to improve marker recoveries from the treated samples.

Section snippets

Preparation of DBS materials

Sets of DBS materials for 34 inborn disorder markers were prepared from human blood adjusted to a hematocrit of 50 ± 1% to simulate the hematocrit of newborns and dispensed in 75 μL aliquots onto Whatman Grade 903 filter paper. The blood enriched with amino acids and some acylcarnitines was prepared from saline-washed packed red cells and clarified serum to create a normalized base matrix for these markers. Other hematocrit adjustments were made by plasma removal, except for the

Accelerated degradation study outcomes

The degradations of 33 of the 34 disorder markers during storage are summarized in Table 1. Recovery of C0 increased during storage because C0 is liberated when acylcarnitines degrade. The increase in C0 concentration of paired sets of DBSs enriched with an array of acylcarnitines and stored for 35 days at 37 °C was 6-fold greater in DBSs stored at high humidity than in paired DBSs stored at low humidity.

Results from the comparison of marker levels on the initial and final days of the 37 °C

Discussion

The accelerated degradation studies were carried out at 37 °C at low and high relative humidities for 30 ± 5 days for study convenience. (Elevated storage temperature [4], [9], [11] or elevated humidity [7], [8], [10] accelerates the degradation process, thus enabling measurement of changes in marker levels in a short study period.) Additionally, the study conditions can mimic those encountered during some transportation environments, such as summer transit in hot courier trucks, in which DBSs may

Acknowledgments

We thank Hien Nguyen for performing the tandem mass spectrometry amino acid analyses for the studies reported here. Ms. Nguyen and author D. Simms were funded by the Research Participation Program at the Centers for Disease Control and Prevention, an interagency agreement with the U.S. Department of Energy administered by the Oak Ridge Institute for Science and Education. Authors S.R. Flores, E.M. Hall, and S. O'Brien are employees of Battelle, Atlanta, GA who are under contract to CDC.

References (22)

  • T.H. Zytkovicz et al.

    Instability of enzymes, antibodies, and other analytes in dried blood spots — is the major problem heat or humidity

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