Academiejaar 2015 – 2016 Dual-wavelength recording, a simple algorithm to eliminate interferences due to UV-absorbing substances in capillary electrophoresis. Liesbeth SEAUX Promotor: Prof. dr. J. Delanghe Masterproef voorgedragen in de master in de specialistische geneeskunde ………………………………… Academiejaar 2015 – 2016 Dual-wavelength recording, a simple algorithm to eliminate interferences due to UV-absorbing substances in capillary electrophoresis. Liesbeth SEAUX Promotor: Prof. dr. J. Delanghe Masterproef voorgedragen in de master in de specialistische geneeskunde ………………………………… VOORWOORD Ik wens via deze weg Prof. Dr. J. Delanghe te bedanken voor mij de kans die hij mij bood om dit artikel tot stand te brengen. Naast het geven van deze gelegenheid en de steun bij het uitvoeren van de testen en het schrijven, heeft het enthousiasme van Prof. Dr. J. Delanghe samen met de MLT’s van labo speciale scheikunde mijn interesse in deze deeldiscipline nog vergroot. Ik hoop dan ook in de toekomst mij als klinisch bioloog hier nog verder in te kunnen specialiseren. Liesbeth Seaux 2248 Electrophoresis 2014, 35, 2248–2252 Liesbeth Seaux Research Article Sofie Van Houcke Els Dumoulin Tom Fiers Dual-wavelength recording, a simple Elke Lecocq Joris R. Delanghe algorithm to eliminate interferences due Department of Clinical to UV-absorbing substances in capillary Chemistry, Microbiology and Immunology, Ghent University electrophoresis Hospital, Ghent, Belgium Analytical interferences have been described due to the presence of various exogenous UV-absorbing substances in serum. Iodine-based X-ray contrast agents and various an- Received May 26, 2014 tibiotics have been reported to interfere with interpretation of serum protein pherograms, Accepted June 16, 2014 resulting in false diagnosis of paraproteinemia. In the present study, we have explored the possibility of measuring UV absorbance at two distinct wavelengths (210 and 246 nm) to distinguish between true and false paraproteins on a Helena V8 clinical electrophoresis instrument. This study demonstrates that most substances potentially interfering with serum protein electrophoresis show UV-absorption spectra that are distinct from those of serum proteins. Scanning at 246 nm allows detection of all described interfering agents. Comparing pherograms recorded at both wavelengths (210 and 246 nm) enables to dis- tinguish paraproteins from UV-absorbing substances. In case of a true paraprotein, the peak with an electrophoretic mobility in the gamma-region decreases, whereas the X-ray contrast media and antibiotics show an increased absorption when compared to the basic setting (210 nm). The finding of iodine-containing contrast media interfering with serum protein electrophoresis is not uncommon. In a clinical series, interference induced by con- trast media was reported in 54 cases (of 13 237 analyses), corresponding with a prevalence of 0.4%. In the same series, 1631 true paraproteins (12.3%) were detected. Implemen- tation of the proposed algorithm may significantly improve the interpretation of routine electrophoresis results. However, attention should still be paid to possible interference due to presence of atypical proteins fractions (e.g., tumor markers, C3). Keywords: Antibiotics / Capillary electrophoresis / Iodine-containing contrast media / Paraproteins DOI10.1002/elps.201400259 1Introduction ioxitalamic acid, meglumine iotroxate, iopromide, iobitridol, iodixanol) and antibiotics [4,5] (e.g., piperacillin–tazobactam, Current guidelines for diagnosis and monitoring of mono- sulfamethoxazole–trimetoprim, and ampicilline–sulbactam) clonal gammopathy of undetermined significance (MGUS) have been reported to interfere with interpretation of serum and smoldering (asymptomatic) multiple myeloma require protein pherograms, resulting in false diagnosis of parapro- quantification of paraproteins in serum [1]. CE of serum pro- teinemia (Table 1). teins has become a standard analysis for the detection and The Helena V8 is a clinical CE instrument (Helena quantification of paraproteins (M-proteins). Most commonly Biosciences Europe, Newcastle, UK), which is equipped the quantification of electrophoretic protein fractions is based with a monochromator and therefore allows to measure on absorbance of the peptide bonds (200–210 nm). Occa- absorbance at any wavelength in the range between 200 sionally, analytical interferences have been described due to and 600 nm [6, 7]. As the interfering substances show UV the presence of various exogenous UV-absorbing substances absorbance spectra that are distinct from those of proteins in serum. Iodine-based contrast agents [2, 3] (e.g., iohexol, (Table 1), analyzing serum protein pherograms at different wavelengths could allow detection of interfering agents and facilitate pherogram interpretation. Correspondence:ProfessorJorisR.Delanghe,Departmentof Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, In the present study we want to explore the possibility B-9000 Gent, Belgium of measuring absorbance at two distinct wavelengths (210 E-mail: [email protected] and 246 nm) in order to distinguish between true and false C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com ⃝ Electrophoresis 2014, 35,2248–2252 CE and CEC 2249 Ta b l e 1 . List of interfering substances lowing electrophoretic separation, polyclonal antisera against ␥, ␣, ␮ heavy Ig chains, and ␬ and ␭ light chains were used to Compound ␭ (nm) Expected max fix the specific Ig chains. The precipitated immune complexes concentration range in serum were then visualized with acid-violet stain [8]. Ig concentration (IgA, IgM, IgG) in serum was assayed Ioxitalamic acid 240 1.0–62.3 g/L immunonephelometrically on a BN II analyzer (Siemens, Iobitridol 242 2.6–84 g/L Marburg, Germany) [9] using Siemens polyclonal antibodies Iohexol 242 1.1–22 g/L (Siemens, Marburg, Germany). Iopromide 242 7.7–31 g/L Iodixanol 245 0.9–33 g/L Iomeprol 244 0.6–24 g/L 2.3 Subjects Sulfamethoxazole– 257 (sulfamethoxazole) 3.4 0.3 and ± trimetoprim 289 (trimethoprim) 46.3 2.7 mg/L ± A laboratory information-system query for all serum protein (after 1 h) electrophoresis requests between January 1, 2013, and March 31, 2014, was done. The comments of the 13 237 reports were paraproteins and to validate dual-wavelength reading of checked for presence of paraproteins, and remarks dealing serum protein electrophoresis in the clinical laboratory. with interferences due to contrast media and fibrinogen. Concomitantly, a series of serum samples containing paraproteins (n 31), IgG (n 15), IgA (n 9), IgM 2Materialsandmethods = = = (n 7) with M-protein concentrations ranging from 4.0 to = 32.7 g/L was analyzed in a similar way. 2.1 Electrophoresis In the Helena V8 instrument, 2 ␮Lofeachserumsample 2.4 UV spectra is 50-fold diluted and is automatically injected. Separation of proteins is obtained by applying a voltage of 9 kV in eight UV-absorption spectra (wavelength range: 200–300 nm) of fused-silica capillaries controlled by Peltier effect. A ninth sera containing paraproteins, sera spiked with contrast media capillary is used as reference (buffer only). In the standard (concentration: 52 mg/L), and aqueous solutions of contrast operating mode, direct detection of proteins is performed media (concentration range: 52 to 10 mg/L) were recorded in by measuring UV absorbance of the separated fractions at a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, 210 nm. Throughput is 90 samples/h. Helena V8 CE was used Japan). Due to high absorption of sera, sera samples were 40- in a standard and modified setting for serum electrophoresis. to 100-fold diluted in 0.9% NaCl. In the modified setting, the reading wavelength was changed from 210 to 246 nm, whereas all other settings of the instru- ment were kept identical. 3Results Pooled serum from a group of normal subjects (n = Figure 1 compares the UV-absorption spectra of a 1:100 20) was used as a standard matrix for studying ana- dilution in saline of a human serum pool (total protein lytical interferences. Pooled serum samples were spiked with iohexol (Omnipaque, final concentration in serum: 51.8 g/L), iomeprol (Iomeron, 71.44 g/L), iopromide (Ultra- vist, 76.9 g/L), ioxitalamic acid (Telebrix, 74.8 g/L), iodix- anol (Visipaque, 65.2 g/L), iobitidrol (Xenetix, 76.8 g/L), and sulfamethoxazole-trimetoprime (Eusaprim, 40 g/200 g/L), respectively, and analyzed at the two wavelengths. In order to test interference caused by fibrinogen, two plasma samples containing 0.36 and 0.47 g/L fibrinogen were subjected to electrophoresis and recorded at both wave- lengths. In order to investigate the detection range of the interfer- ing substances, a dilution series of iohexol in normal pooled serum was prepared. Final iohexol concentration ranged from 52 to 52 g/L. 2.2 Immunofixation—immunonephelometry Immunofixation was used for the identification of parapro- Figure 1. UV-absorption spectrum of human serum (total protein teins. Hereby, a commercial agarose gel based electrophoresis content: 73 g/L, (A) and an aqueous solution (51.8 mg/L) of iohexol system (Sebia Hydrasys, Sebia, Evry, France) was used. Fol- (B). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com ⃝ 2250 L. Seaux et al. Electrophoresis 2014, 35,2248–2252 Ta b l e 2 . UV absorption of plasma proteins and contrast media Compound a (210 nm, a (246 nm, Ratio 1 1 1 1 Lg− cm− ) Lg− cm− ) A210/A246 Plasma proteins 21 0.6 35 Iohexol 26.0 34.8 0.75 a, specific absorption coefficient; A210,absorbanceat210nm;