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Technical Briefs |
-Hydroxyprogesterone in Dried Blood Spots after Autoclaving and Prolonged Storage
Departments of
1
Pediatrics and
4
Medical Computer Sciences, University of Vienna, A-1090 Vienna, Austria
2
2nd Department of Pediatrics, Semmelweis University, H-1094 Budapest, Hungary
3
Department of Pediatrics, Charles University, CZ-10081 Prague, Czech Republic
aaddress correspondence to this author at: Department of Pediatrics, University of Vienna, AKH-Wien, Währinger Gürtel 18-20, A-1090 Vienna, Austria; fax 43-1-40-400-3238, e-mail franz.waldhauser{at}akh-wien.ac.at
In the last few decades, management of patients with inborn errors of metabolism or congenital endocrine disorders has been greatly improved by the introduction of neonatal mass screening programs in many parts of the world. For this purpose, blood from newborns, ideally 4–7 days of age, is collected on filter-paper cards by means of a heel prick. A standard set of up to 20 tests is usually carried out at a centralized laboratory within a few days. Until recently, either when the tests were being performed or during the final step, the entire filter-paper card was autoclaved and then stored (1). Thus, leftover blood spots on filter paper from the entire young population in many countries are available in native or autoclaved form, although autoclaving has now become outdated because of a change in analytical methods (1). The stored cards provide a potentially valuable source for population studies or retrospective examinations. Documentation of the long-term stability of the analytes to be examined is a prerequisite for such studies.
In many countries, the latest addition to the neonatal screening program is the examination for congenital adrenal hyperplasia (CAH), by measurement of 17
-hydroxyprogesterone (17-OHP) in a dried blood spot (2)(3)(4). The technique, first described by Pang et al. in 1977 (5), is based on a direct immunoassay and is used as the standard procedure in programs screening for CAH (2). In several trials, 17-OHP in dried blood spots on filter-paper cards was resistant to freezing, high ambient temperatures, humidity, and short- or intermediate-term (up to 1 year) storage (6)(7)(8)(9)(10). In the present study, we examined the impact of autoclaving (i.e., extremely high temperature, high humidity and pressure) and prolonged (more than a decade) storage under typical room conditions on the 17-OHP concentrations in dried blood spots on filter-paper cards.
In Austria and the Czech Republic, nationwide neonatal mass screening programs have been in use for more than two decades. Several blood drops are collected from each newborn baby on a filter-paper card, dried, and mailed to a centralized laboratory, where various tests for congenital metabolic and endocrine disorders are performed. Until 2001 in Austria, several tests were done on blood spots on native cards as a first step; the cards were then autoclaved and screened for phenylketonuria, and the leftovers were stored at room temperature. In the Czech Republic, the cards are usually cut into two parts: one half is assayed native, whereas the other is autoclaved; leftovers from the native half are stored at room temperature. Since 2001, Austrian cards have been treated similarly. Accordingly, autoclaved cards are available for 1991–2000 from Austria, along with native cards, whereas from the Czech Republic, native cards are available dating back to 1988.
To evaluate the effect of autoclaving on 17-OHP concentrations in dried blood spots (experiment 1a), native filter-paper cards from 25 term and 25 premature neonates were retrieved from the Austrian neonatal screening bank. The cards were collected in 2001. The 17-OHP concentration in a blood spot on each native card was first assayed (native 17-OHP). Subsequently, the cards were autoclaved and then reassayed for 17-OHP (autoclaved 17-OHP).
To evaluate the effect of autoclaving on 17-OHP concentrations in a fluid environment (experiment 1b), an aqueous solution was prepared that contained 29.59 nmol/L 17-OHP (solution A). One-half of this solution was autoclaved in an open glass flask, and the evaporated fluid was substituted (solution B). Solutions A and B were dripped onto filter-paper cards, and 17-OHP was determined in six spots on each. Solution A was also dripped onto an additional filter-paper card, which was dried and autoclaved. The 17-OHP concentration in the autoclaved card was then determined (six replicates).
To evaluate the impact of long-term storage at room temperature on 17-OHP in autoclaved filter-paper cards (experiment 2a), we retrieved the first 40 cards from neonates fulfilling certain birth weight criteria (25 neonates weighing >2500 g, 5 weighing 2000–2500 g, 5 weighing 1500–2000 g, and 5 weighing <1500 g) from each year between 1991 and 2000 from the Austrian screening bank. A total of 400 cards were examined altogether.
To evaluate the impact of long-term storage at room temperature on 17-OHP in native filter-paper cards (experiment 2b), 520 cards from neonates born between 1988 and 2000 were retrieved from the Czech screening bank, according to the selection criteria described above (Fig. 1
).
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In the year of sampling, Austrian cards were autoclaved in a Varioklav Dampfsterilisator, Type 400 (H+P Labortechnik GmbH) for 5 min at a temperature of 121 °C, a humidity of 100%, and pressure set automatically.
All 17-OHP measurements were carried out using a time-resolved fluoroimmunoassay (DELFIA neonatal 17-OH-progesterone reagent set; Wallac Oy) according to the manufacturer’s instructions (11). When we applied the overnight incubation protocol (3-mm filter-paper discs with dried calibrators, controls, or unknowns are incubated for 18–22 h), the lower detection limit of the assay was <0.2 nmol/L of blood, and the upper limit of detection was 260 nmol/L of blood. However, the useful part of the calibration curve (80–20% inhibition) was 7–110 nmol 17-OHP/L of blood. The intraassay (within-run) imprecision (CV) of the method was 7.0% (n = 5 x 24) at 21.9 nmol/L on cards that had not been autoclaved (native 17-OHP) and 6.8% (n = 5 x 24) at 12.3 nmol/L on autoclaved cards (autoclaved 17-OHP); at corresponding concentrations, the interassay (between-run) CV was 7.9% (n = 11 x 12) and 8.0% (n = 11 x 12) for native and autoclaved 17-OHP, respectively. A Victor 1420 Multilabel Counter (Wallac Oy) was used for automatic measurement of the fluorescence and for result calculation.
The 17-OHP values are presented as median and quartiles (25th and 75th percentiles) because data were not normally distributed. Concentrations before and after autoclaving were compared by a paired t-test and correlated by means of the Pearson linear correlation coefficient. Linear regression analysis was performed to adjust the 17-OHP concentrations for the systematic effect of autoclaving.
The influence of storage time on 17-OHP concentrations was assessed by linear regression of the log-transformed 17-OHP values vs time. P <0.05 was considered as indicating statistical significance. The software products SigmaStat, SigmaPlot (both from SPSS Inc.), and SAS (SAS Institute Inc.) were used for statistical analysis.
17-OHP concentrations in newborn dried blood spots collected on filter-paper cards (experiment 1a) were significantly lower after autoclaving (autoclaved 17-OHP) than before (native 17-OHP): median (25th–75th percentiles), 11.9 (5.6–21.5) nmol/L vs 18.8 (13.6–33.2) nmol/L; P = 0.001. However, there was a strong correlation between autoclaved 17-OHP and native 17-OHP (r = 0.952; P <0.001; y = -1.177 + 0.68x). Autoclaving decreased the measurable amount of 17-OHP by 
32%.
In experiment 1b, 17-OHP concentrations were similar when measured before (solution A) and after autoclaving in a glass flask (solution B) or after autoclaving on filter paper (28.9 ± 3.7 nmol/L vs 27.7 ± 3.5 nmol/L vs 28.6 ± 3.6 nmol/L, mean ± SE). The reason for the different behaviors compared with experiment 1a is not known.
In experiment 2a, in which we compared 17-OHP concentrations in dried blood spots from autoclaved cards, median year-group 17-OHP concentrations were 4.6–6.3 nmol/L (Fig. 1
). There was a small (2.1% per year), insignificant decrease in 17-OHP on storage (r = 0.06; P = 0.23; y = e-40.5 + 0.021x, where y is the 17-OHP concentration in nmol/L and x is the year of collection).
In experiment 2b, in which we compared 17-OHP concentrations in dried blood spots from native cards, median year-group 17-OHP concentrations were 7.0–9.1 nmol/L (Fig. 1
). There was also a small (3.2% per year), significant decrease in 17-OHP on storage (r = 0.12; P < 0.001; y = e-62.6 + 0.032x, where y is the 17-OHP concentration in nmol/L and x is the year of collection).
In the autoclaved cards, 17-OHP concentrations were 27.3% lower than in the native cards: median (25th–75th percentiles), 5.6 (3.6–8.9) nmol/L vs 7.7 (5.3–11.1) nmol/L; P = 0.001.
Thus, 17-OHP in dried blood spots on autoclaved or native filter-paper cards decreased 2–3% annually, and the decrease was significant on native cards but not on autoclaved cards. However, given an interassay CV of 8%, this small decrease may be unimportant for interpretation of samples stored for a few years. If samples are kept for a decade or more, however, it may be wise to correct for the loss, certainly at the expense of an increase in the error of estimate. Thus, in a moderate climate such as in Middle Europe, 17-OHP is basically stable in native or autoclaved filter-paper cards stored at room temperature over a prolonged period. The stability of 17-OHP on filter paper for up to 1 year has been ascertained in previous studies (5)(12). Similarly, progesterone has been shown to be stable in filter paper for 15 weeks at 25 °C and for 9 weeks at 37 °C (13). Steroids are known to be stable in serum stored at -25 °C for up to a decade (14).
We conclude that 17-OHP estimates in dried blood on native or autoclaved filter papers stored for more than a decade can reliably be used for retrospective examinations and population studies.
Acknowledgments
This study was initiated by the "MEWPE–CAH" study group and supported by a grant from the Ministry of Health of the Czech Republic (6133-3) and the Central European Exchange Program for University Studies (CEEPUS SLO-013). Drs. Dóra Török and Felix Votava were recipients of program fellowships.
References
-hydroxyprogesterone RIA: its application for rapid screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1977;45:1003-1008.[ISI][Medline]
[Order article via Infotrieve]
-hydroxyprogesterone in whole-blood samples from newborns. Clin Chem 1985;31:1127-1130.The following articles in journals at HighWire Press have cited this article:
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  V. S Lando, M. C Batista, I. T Nakamura, C. R Mazi, B. B Mendonca, and V. N Brito Effects of long-term storage of filter paper blood samples on neonatal thyroid stimulating hormone, thyroxin and 17-alpha-hydroxyprogesterone measurements J Med Screen, September 1, 2008; 15(3): 109 - 111. [Abstract] [Full Text] [PDF]
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 K. A. Strnadova, M. Holub, A. Muhl, G. Heinze, R. Ratschmann, H. Mascher, S. Stockler-Ipsiroglu, F. Waldhauser, F. Votava, J. Lebl, et al. Long-Term Stability of Amino Acids and Acylcarnitines in Dried Blood Spots Clin. Chem., April 1, 2007; 53(4): 717 - 722. [Abstract] [Full Text] [PDF]
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 F. Votava, D. Torok, J. Kovacs, D. Moslinger, S. M Baumgartner-Parzer, J. Solyom, Z. Pribilincova, T. Battelino, J. Lebl, H. Frisch, et al. Estimation of the false-negative rate in newborn screening for congenital adrenal hyperplasia Eur. J. Endocrinol., June 1, 2005; 152(6): 869 - 874. [Abstract] [Full Text] [PDF]
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