Interference by gelatin-based plasma substitutes in capillary zone electrophoresis.
Analysis of serum proteins by capillary zone electrophoresis (CZE) is used to detect dysproteinemias and monoclonal proteins [reviewed in (1)]. Most interferences in this method are caused by interference with ultraviolet detection [reviewed in (2)]. For example, radio-opaque contrast agents and, to a lesser extent, some antibiotics absorb at 200-214 nm and produce distinct peaks on the electropherogram. A gelatin-based plasma substitute has also been reported to produce an increase in the [gamma]-region in a polyclonal-like way (3). We describe the systematic evaluation of commercially available synthetic colloidal plasma substitutes for their potential to interfere with serum protein analysis by CZE.
WE used the Paragon 2000[R] (Beckman Coulter) and the Capillarys[R] (Sebia) CZE systems. The former uses 214 nm and the latter 200 nm as detection wavelength. The Hydrasys/Hyrys/Hydragel[R] (Sebia) agarose gel electrophoresis (AGE) system was used for comparison.
Synthetic colloidal plasma substitutes can be divided into products based on dextran, starch, or gelatin (4). We studied (a) dextran-based Rheomacrodex[R] (depolymerized dextran; Mr 40 000;100 g/L; Pharmalink), (b) starch-based Voluven[R] [poly(O-2-hydroxyethyl)amylum; [M.sub.r] 130 000; 60 go/ L; Fresenius Kabi] and HAES-steril[R] (hydroxyethyl amylum; [M.sub.r] 200 000; 60 g/L; Fresenius Kabi), and (c) gelatin-based Gelo-plasma[R] (modified gelatin; mean [M.sub.r] 30 000; 30 g/L; Fresenius Kabi) and Gelofusine[R] (modified gelatin; mean [M.sub.r] 30 000; 40 g/L; B. Braun).
We first evaluated the in vitro effects of the plasma substitutes by mixing equal volumes of the solutions (or phosphate-buffered saline for controls) with normal serum for CZE analysis. The resulting electropherograms are shown in Fig. 1, A-L; only the gelatin-based plasma substitutes showed clear interference in the electropherograms, although the dextran-based Rheomacrodex produced a small additional peak cathodal to the [gamma]-globulin zone on the Capillarys. Geloplasma induced a broad increase of the [gamma]-globulin fraction extending well into the [beta]-globulin fraction, and Gelofusine caused a similar increase of the [beta]-globulin fraction with less interference in the [beta]-globulin fraction. Quantification corroborated these findings (see Table 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem. org/content/vo150/issue8/). These interference patterns were confirmed by electrophoresis of the pure plasma substitute (not shown). Serial dilutions of the gelatin-based substitutes in serum indicated that interference became invisible/nonquantifiable at or below a concentration of 125 mL/L (12.5% by volume; tested on the Paragon 2000 system only).
After receiving approval by our Institutional Review Board and the patients' informed consent, we evaluated the in vivo effect of Gelofusine on CZE analysis of samples from patients (n = 10) who had received 500-mL infusions of Gelofusine over 1 h for hemodilution during orthopedic surgery by comparing the electropherograms for samples obtained before and immediately after the infusion. The interference was polyclonal-like in the [beta]-/[gamma]-regions and could not be mistaken for a M-peak (Fig. 1). The increases in the [beta]-/[gamma]-regions were obvious in all 10 patient samples tested. The median increase in the [beta]-region was 30% on the Paragon 2000 and 23% on the Capillarys CZE system, whereas there was a small but significant increase in the [[alpha].sub.2]-region on the Hydrasys AGE system (see Table 1 in the online Data Supplement). The [beta]-fraction (expressed as a percentage of total serum protein) after infusion exceeded our upper reference limit for this fraction in all patients.
[FIGURE 1 OMITTED]
Gelatin-based plasma substitutes are denatured collagens and thus contain complex mixtures of proteins (4). Differences in composition among products might explain the distinct migration patterns seen, as well as the different absorbances at the wavelengths used by the CZE instruments. The proteinaceous nature of these plasma substitutes produces both absorbance in the ultraviolet range and protein dye-binding, thus giving interference in CZE as well as in AGE (but to a much lesser extent, and even more anodal, including interference in the [[alpha].sub.2]-fraction; see Table 1 in the online Data Supplement). Synthetic plasma substitutes are given mainly at times when serum electrophoresis is rarely indicated, e.g., during emergency fluid resuscitation or for intraoperative hemodilution (4). Moreover, gelatin-based products are rapidly eliminated with an estimated half-life of 2.5 h (5).
In conclusion, gelatin-based plasma expanders may cause interference with serum protein electrophoresis (CZE and to a lesser extent AGE). With CZE these substances typically produce a (polyclonal-like) increase in the [beta]-/[gamma]-region. To avoid problems, instructions should be given not to collect samples for serum protein electrophoresis during the first hours after infusion of gelatin-based plasma substitutes.
We thank J. Vunckx (MCH, Leuven, Belgium) for use of the Capillarys CZE system.
(1.) Bossuyt X. Separation of serum proteins by automated capillary zone electrophoresis. Clin Chem Lab Med 2003;41:762-72.
(2.) Bossuyt X. Interferences in capillary zone electrophoresis of serum proteins. Electrophoresis 2004;25:1485-7.
(3.) Gay-Bellile C, Bengoufa D, Houze P, Le Carrer D, Benlakehal M, Bousquet B, et al. Automated multicapillary electrophoresis for analysis of human serum proteins. Clin Chem 2003;49: 1909-15.
(4.) Roberts JS, Bratton SL. Colloid volume expanders. Drugs 1998;55:621-30.
(5.) Mishler JM. Synthetic plasma volume expanders: their pharmacology, safety and clinical efficacy. Clin Haematol 1984;13:75-92.
Koenraad Gijbels 
Johan De Coster 
Xavier Bossuyt  *
 Laboratory Medicine, Immunology and  Anesthesia Leuven University Hospitals Katholieke Universiteit Leuven Leuven, Belgium
* Address correspondence to this author at: Leuven University Hospitals, Laboratory Medicine, Herestraat 49, B-3000 Leuven, Belgium. Fax 32-16-34-7931; e-mail email@example.com. ac.be.
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|Author:||Gijbels, Koenraad; De Coster, Johan; Bossuyt, Xavier|
|Article Type:||Letter to the editor|
|Date:||Aug 1, 2004|
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