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Letters to the Editor |
1 Department of Pediatrics Center for Metabolic Disease Katholieke Universiteit Leuven Leuven, Belgium
2 Department of Pediatrics University of Kinshasa Kinshasa, Congo
aAddress correspondence to this author at: Center for Metabolic Disease, University Hospital Gasthuisberg, Herestraat 49, Box 817, B-3000 Leuven, Belgium. Fax 32-1634-7284; e-mail hubert.carchon{at}med.kuleuven.be.
To the Editor:
Serum transferrin (Tf) isoform profiling by isoelectric focusing (IEF) (1) is the preferred procedure to screen for congenital disorders of glycosylation (CDG). HPLC (2) and capillary electrophoresis (CE) (3) are recently introduced techniques that offer mainly the advantage of automation. The analyses are usually performed in specialized laboratories, and serum samples are sent primarily via courier. Stibler and Cederberg (4) showed that blood samples spotted on Guthrie cards could be used instead of serum for investigating carbohydrate-deficient Tf. We had the same experience with Guthrie cards sent for Tf isoform profiling. In fact, a substantial proportion of the samples we have investigated (886 of 9654 samples) were transported this way (our unpublished data).
Tf obtained by extraction from Guthrie cards may show on IEF an isoform profile that corresponds to a known CDG pattern (Fig. 1A
, lane 2). The Tf profile on CE is unusable because of the high degree of hemolysis. However, when the Tf concentration in serum is low (Fig. 1A
, lane 3) or when only a small amount of blood has been spotted on the Guthrie card, the Tf bands are mostly too faint to exclude a CDG. This is particularly true for type II CDG (CDG-II) profiles because of the smaller increases in asialo- and monosialo-Tf. Increasing the amount of sample on the agarose gel is not useful because it increases the background (Fig. 1A
, lane 4).
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We obtained 393 abnormal IEF profiles suggestive of CDG among 8768 serum samples (4.5%) and 9 abnormal profiles among 886 blood samples on Guthrie cards (1%; our unpublished data), and investigated whether the application of serum instead of whole blood could combine the usefulness of Guthrie cards with the accuracy of serum analysis.
The protein concentration in each sample was determined by the BIO-RAD assay. IEF and CE analyses of sialo-Tf were performed as described previously (3).
Sera spotted on the Guthrie cards originated from serum pools that were obtained from control samples (permanently stored at 20 °C) and from patients with CDG-I or CDG-II. After 250 µL of serum was spotted on a Guthrie card, the card was enclosed in a plastic bag and sealed after air removal. After 2 weeks at room temperature or –20 °C, spots were cut and snipped and dispersed in a volume (in microliters) of physiologic saline–2 mL/L Triton X-100 equal to 10 times the weight of the paper (in milligrams); this surfactant solution is used for homogenization of intestinal mucosal biopsies (5). Protein was extracted overnight at room temperature under constant stirring. After centrifugation for 10 min at 13 000g, the supernatant was lyophilized and the residue was dissolved in 50 µL of physiologic saline. Tf isoform profiling was performed by IEF and by CE.
The mean (SD) protein recovery with this extraction procedure was 10 (0.3)% (n = 10). Thus, 
300 µL of serum should be applied on the Guthrie cards for IEF and CE analysis to provide the equivalent to 30 µL of serum in the extract. As this would correspond to at least 0.5 mL of blood, it is clear that in many cases the amount applied on Guthrie cards is too low for Tf analysis.
The intensities of the Tf isoforms in the samples stored at –20 °C were slightly higher than the intensities in samples stored at room temperature (Fig. 1B
). The CE profiles (Fig. 1C
) corresponded to the expected Tf patterns for the same control (trace 1), CDG-I (trace 2), and CDG-II (trace 3) sera on the Guthrie cards after 2 weeks of storage at –20 °C.
We have observed neither analytical differences, in both IEF and CE, between serum and serum spotted on Guthrie cards nor differences between the Tf profiles on CE. We therefore expect that the rate of positive outcomes suggesting CDG will be similar.
In conclusion, analysis of a sufficient amount of serum spotted on a Guthrie card, stored at –20 °C and transported by air, gives information about the Tf profile similar to that obtained by analysis of serum. Although serum samples are preferable, we recommend the present procedure if Guthrie cards are used.
Acknowledgments
This work was supported by the Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (Grant G.0371.02N), the Körber European Science Award 2004, and EUROGLYCANET.
References
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