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Evaluation of a commercially available carbohydrate deficient transferrin kit to detect beta-2-transferrin in cerebrospinal fluid using capillary electrophoresis
Clinical Biochemistry 46 (2013) 1770–1773
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Evaluation of a commercially available carbohydrate deficient transferrin kit to detect beta-2-transferrin in cerebrospinal fluid using capillary electrophoresis Karina Rodríguez-Capote a,b,⁎,1, Joceline Turner c, Joseph Macri a,b a b c
Department of Pathology and Laboratory Medicine, McMaster University, Ontario, Canada Hamilton Regional Medicine Program, Ontario, Canada Department of Special Chemistry Laboratory, Hamilton Regional Medicine Program, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 6 May 2013 Received in revised form 29 June 2013 Accepted 2 July 2013 Available online 12 July 2013 Keywords: β-2-transferrin Capillary electrophoresis Cerebrospinal fluid leakage CSF rhinorrhea Desialylated transferrin Tau-protein
a b s t r a c t Objective: The aim of this study was to evaluate the CEofix™ CDT Capillary Electrophoresis (CE) kit for the detection of β-2-tranferrin (β-2-Tf) in cerebrospinal fluid (CSF). Design and method: Evaluation was performed according to CLSI EP5-A and EP12-A guidelines. Results: The method resolved β-2-Tf from other Tf isoforms. The C50 for β-Tf was determined to be 0.46 mg/L. Neither hemoglobin nor bilirubin co-migrated with β-2-Tf. Conclusions: The CDT kit can be used for detecting β-2-Tf in CSF by CE. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Leakage of cerebrospinal fluid (CSF) is a critical condition with a high risk of meningitis and potentially fatal outcome [1]. Βeta-2-transferrin (β-2-Tf) is the asialo transferrin isoform found only in CSF, ocular fluids and perilymph and is an accepted marker of CSF leakage [1,2]. Our objective was to evaluate a commercially available kit, Analis CEofix™ CDT Capillary Electrophoresis [3], for detecting β-2-Tf in fluids suspected to be CSF. This kit is intended to detect serum carbohydrate deficient transferrin (CDT) by capillary electrophoresis (CE) in patients suspected of chronic alcoholism [4,5]. Methodology Samples Residual CSF samples were obtained from the clinical laboratory after physician-ordered testing was complete. CSF pools were prepared representing sample condition (clear, hemolyzed and xanthochromic) and centrifuged to remove cells and debris. Total protein and transferrin ⁎ Corresponding author at: Clinical Chemist, DynaLIFEDx, #200, 10150-102 Street, Edmonton, Alberta, Canada, T5J 5E2. Fax: + 1 780 454 2845. E-mail address: [email protected] (K. Rodríguez-Capote). 1 Present address: DynaLIFEDx, Edmonton, Alberta, Canada.
concentrations were determined using the Roche Modular and the Dade Behring BNII Protein Analyzer, respectively and aliquots frozen at −20 °C until use. For method comparison, individual patient samples submitted and referred out for β-2-Tf testing with sufficient volume were split and recoded prior to use. All but one sample were nasal fluids; this specimen consisted of a cotton swab soaked with fluid collected after lumbar spine surgery. This sample was eluted with a known volume of sterile saline, as per referral laboratory protocol. Sample preparation and analysis by capillary electrophoresis Sialo-Tf isoforms were separated using the CEofix™ CDT kit [3] and the P/ACE™ MDQ Capillary Electrophoresis System from Beckman Coulter according to manufacturer's instructions. Running buffer (TRIS/Borate, pH 8.5), initiator buffer (Tris/phosphate, pH 2.0) as well as serum based quality controls are provided with the kit. These controls (normal and CDT) were run prior to sample analysis. Since Tf concentrations in CSF are 100-fold lower than in blood (estimated concentrations between 12 and 78 mg/l [1,6]), CSF samples were concentrated using Amicon® Ultra-4 centrifugal filtration devices (Millipore Ltd). Before CE analysis, samples (25 μl) were saturated with iron by incubation with 10 mM FeCl3 provided with the kit. Results were recorded on an electropherogram and peaks were integrated using the instrument software. Tetra-sialo-Tf was used as the reference peak with its migration time (MT) set at 6.08 min.
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.07.001
K. Rodríguez-Capote et al. / Clinical Biochemistry 46 (2013) 1770–1773
Method evaluation Analytical precision Analytical precision was calculated following CLSI guideline EP5-A [7], by repeatedly measuring CSF pools of low (2.0 mg/L) and high (23 mg/L) Tf concentrations. Analytical sensitivity Serial dilutions of CSF were performed to determine linearity. The C50 of the assay (analyte concentration at which 50% of the results are classified either as positive or negative) and the 95% interval (concentrations above and below the cutoff point at which repeated results are 95% positive or 95% negative) were calculated according to CLSI guideline EP12-A2 [8]. Analytical specificity Analytical specificity was verified by immunosubtraction of all Tf sialoforms using an anti-human Tf antibody (Sigma-Aldrich) prior to CE analysis. Specificity of the MT for β-2-Tf and 0-sialo-Tf was verified by treating CSF and serum with neuraminidase (Acetyl-neuraminyl hydrolase, New England BioLabs, USA) to digest the Tf sialic acid side chains. Interference studies were performed using hemolyzed and xanthochromic CSF pools. In addition, we examined the potential influence of CDT due to blood contamination, by mixing CSF with serum control material.
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the qualitative detection of its presence in fluid, and so the C50 was estimated according to the CLSI guideline EP12-A2 and determined to be 0.46 mg/L (95% interval: 0.25 to 0.68). Specificity and interference Specificity for transferrin was verified by immunosubstraction (Fig. 1A) and the MT for β-2-Tf was verified by neuraminidase treatment (Fig. 1B). We further examined the potential influence of blood contamination by mixing the serum control from the kit, containing 0-sialo-Tf, with CSF (Fig. 1C). This experiment confirmed that MTs of β-2-Tf and 0-sialo Tf can be discriminated in the same sample. Xanthochromia (CSF bilirubin = 9.7 μmol/L, Fig. 1D) produced an extra peak at 5.78 min whereas hemolysis (CSF hemoglobin = 2.4 g/L, Fig. 1E) suppressed the 1-and 3-sialo-Tf peaks but the MT of β-Tf was unaffected. Stability β-2-Tf was detected in all samples without significant decrease of the β-2-Tf/4-sialo-Tf area ratio, independent of storage temperature. All values were within 30% of baseline measurements. The analyte is stable for up to 6 months at − 20 °C, and up to 7 days refrigerated or at room temperature. Concordance of results with comparative method
Stability Aliquots of CSF (β-2-Tf = 2.0 mg/L) stored at room temperature and 4 °C were tested every second day for a week and aliquots stored at − 20 °C were tested weekly for six months. Adequate analyte stability was defined as a β-2-Tf/4-sialo-Tf peak area ratio relative to the baseline measurement being within or close to the total imprecision of the test. Diagnostic performance Patient specimens with sufficient volume, proficiency testing samples and diluted CSF of known β-2-Tf concentrations were analyzed and compared to reference laboratory results and to clinical outcome of the patient, where available. The comparative method is a qualitative immunofixation electrophoresis (IFE) method developed in-house by the reference laboratory (Scarborough General Hospital, Toronto). The reference laboratory is the B-2-Tf testing facility used by Hospitals In-Common Laboratory Inc. with a published turnaround time of 10 days. Clinical evidence of CSF leak was determined by retrospective chart review and included development of meningitis or observation of fistulas during physical exam, surgery or imaging studies. The degree of agreement between the two methods was evaluated using Kappa statistics. Statistical analysis was performed using MedCalc v12.4.0 (MedCalc Software, Mariakerke, Belgium). Results Beta-2-Tf was clearly resolved from 0-sialo Tf (MT ± SD = 5.52 ± 0.01 min and 5.59 ± 0.02 min respectively) as well as from the other sialo Tf forms (Figure C). Peak area ratios (AR, β-2-Tf to 4-sialo-Tf) were highly reproducible. Intra-assay CVs and total imprecision were 21.8% (n = 20) and 26.9% (n = 27) for the low concentration (2.0 mg/L, AR = 0.11) and 2.7% (n = 22) and 8.3% (n = 27) for the high concentration (23 mg/L, AR = 0.25). Analytical sensitivity Analytical sensitivity was calculated by serially diluting pooled CSF (total Tf concentration = 0.23 g/L; β-2-Tf = 32 mg/L). The test is linear between 0.25 and 23 mg/L β-2-Tf (y = 1.21 × − 0.0046, R2 = 0.99). The clinical utility of β-2-Tf measurement, however, is
Of the 20 samples analyzed by both the test method and the comparative method (Table 1), eleven results were positive, five were negative, and four were discordant (positive by CE and negative by IFE) representing a moderate degree of agreement (weighted kappa = 0.58; 95% CI 0.24 to 0.91%). Not significant difference between paired proportions was found by the McNemar test (p = 0.13). Diagnostic performance Diagnostic performance was calculated using patient specimens and proficiency samples analyzed by both laboratories (Table 1). Using clinical outcome assessed by retrospective chart review as the gold standard, diagnostic sensitivities were 91.67% (61.52% to 99.79%) and 66.7% (34.89% to 90.08%) for CE and IFE respectively, with corresponding specificities of 100% (2.5% to 100%). Discussion and limitations The present evaluation presents an adapted method for off-label use of the Analis CEofix™ CDT kit in conjunction with capillary electrophoresis for β-2-Tf detection in body fluids. Analytically, MTs were highly reproducible and β-2-Tf was resolved from serum 0-sialoTf. Neither hemoglobin nor bilirubin co-migrated with β-2-Tf. Since current IFE methods of detecting β-2-Tf are hindered when samples are contaminated with blood [9] (a common occurrence in this type of specimen), this presents a major improvement over existing β-2-Tf methods. Current IFE methods can also be limited by allelic variants of transferrin that resemble the CSF isoform, as reported in cases of hypoglycosylation secondary to cystic fibrosis, Epstein–Barr virus infection, galactosemia or liver failure [10], suggesting that additional patient populations may benefit from CE analysis. In addition, the CE method presents an opportunity to improve result turn-around-times, with the whole procedure of sample concentration, instrument calibration and sample analysis taking no more than 60 min. CE appears to be more sensitive than IFE for the detection of CSF leakage (higher positive rate). However, difficulty in obtaining patient specimens of sufficient volume resulted in the inclusion of only one true negative nasal sample, preventing an accurate estimated of the
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Fig. 1. Specificity of the migration time (MT) for beta transferrin (β-Tf). 1A. Electropherograms of untreated and transferrin immunoprecipitated CSF: sample was incubated with Tf antiserum prior to CE analysis. 1B. Neuraminidase was used to digest sialic acid side chains from transferrin in CSF and serum, revealing their asialo-isoforms. 1C. Electropherograms of clear CSF, serum control (CDT-rich) and CSF mixed 1:1 with the CDT-rich serum control. 1D. Electropherograms of clear CSF, icteric CSF (bilirubin = 9.7 μmol/L) and a 1:1 clear: icteric CSF. 1E. Electropherogram of hemolytic CSF showing suppression of 1- and 3-sialo Tf.
Table 1 Diagnostic performance and concordance of results with comparative method. CE, capillary electrophoresis; CSF, cerebrospinal fluid; IFE, immunofixation electrophoresis; Ref. lab, reference laboratory; Tf, transferrin. Proficiency samples consisted of true patient samples. CE: positive the peaks for β-2-Tf and 4-sialo-Tf are present in the correct migration times; traces, when in the presence of 4-sialo-Tf a small peak is visible in the correct migration time for β-2-Tf, but the software cannot integrate it. IFE: positive if beta-1 and beta-2 transferrin are detected; traces, if beta-1 is present and a very faint band is visible in beta 2. Sample type
Our lab (CE)
Ref. lab (IFE)
Clinical outcome
1
Nasal fluid
Positive
Positive
2 3
Nasal fluid Cotton swab
Positive Positive
Positive Traces
4 5 6 7 8 9 10 11
Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Proficiency B2T 2008-1 Proficiency B2T 2008-2 Proficiency B2T 2008-4 Serum control CSF filtrate CSF CSF CSF CSF CSF
Negative Positive Positive Positive Positive Negative Positive Positive
Negative Negative Positive Negative Negative Negative Positive Positive
Endoscopic transphenoidal resection of pituitary tumor. Confirmed positive Confirmed positive Postoperative CSF leak, compressive epidural hematoma. Lumbar spine surgery. Confirmed positive. Confirmed positive. Confirmed positive. Confirmed positive. Confirmed positive in surgery. Laryngectomy, confirmed fistulae. Confirmed positive. Head trauma. Confirmed negative. Confirmed positive. Fluid from anterior neck Hemovac. Confirmed positive.
Traces
Traces
Positive
Positive
Negative Negative Negative Positive Positive Positive Positive
Negative Negative Negative Negative Traces Positive Positive
12 13 14 15 16 17 18 19 20
Confirmed CSF leak by MR cisternogram as well as CT scan. Confirmed positive. Leak identified in skull base, fluid and fluorescein dye actively pouring out of nasal cavity. Confirmed positive. Tf =0, β-2-T = 0 mg/L Tf =0, β-2-T = 0 mg/L Tf = 2.3 mg/L, β-2-T = 0.3 mg/L Tf = 3.5 mg/L, β-2-T = 0.46 mg/L Tf = 4.6 mg/L, β-2-T = 0.6 mg/L Tf = 5.8 mg/L, β-2-T = 0.7 mg/L Tf = 22.5 mg/L, β-2-T = 2.8 mg/L
K. Rodríguez-Capote et al. / Clinical Biochemistry 46 (2013) 1770–1773
false positive rate using CE. Therefore, analysis of a suitable number of truly negative nasal, oral, and ear fluids should be undertaken by laboratories who consider performing this testing. The finding that β-2-Tf in CSF samples is stable for at least a week indicates that a negative result is unlikely to be caused by protein degradation if tested within this period. A positive result in an old specimen, however, should be interpreted with caution since false positives by IFE have been reported due to colonization of samples by neuraminidase secreting bacteria [1,11]. Whether this will affect detection by CE remains to be tested. Regardless, all results positive or negative should be critically correlated with clinical history and with results from other diagnostic procedures. Conclusions The method effectively resolves β-2-Tf from other Tf sialoforms. The Analis CEofix™ CDT kit can be used in conjunction with CE for the detection of β-2-Tf. The procedure is fast, specific and sensitive. Acknowledgment The authors express their gratitude to Dr. Barakauskas for her insightful comments and critical review of the manuscript. We want to extend our thanks to Dr. Samsoondar (Scarborough General Hospital) for his collaboration with the method comparison.
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References [1] Warnecke A, Averbeck T, Wurster U, Harmening M, Lenarz T, Stover T. Diagnostic relevance of beta2-transferrin for the detection of cerebrospinal fluid fistulas. Arch Otolaryngol Head Neck Surg 2004;130:1178–84. [2] Reisinger PW, Hochstrasser K. The diagnosis of CSF fistulae on the basis of detection of beta 2-transferrin by polyacrylamide gel electrophoresis and immunoblotting. J Clin Chem Clin Biochem 1989;27:169–72. [3] ANALIS SA. Ceofix application note 040605. Belgium: ANALIS S.A. [4] Legros FJ, Nuyens V, Baudoux M, Zouaoui Boudjeltia K, Ruelle JL, Colicis J, et al. Use of capillary zone electrophoresis for differentiating excessive from moderate alcohol consumption. Clin Chem 2003;49:440–9. [5] Wuyts B, Delanghe JR. The analysis of carbohydrate-deficient transferrin, marker of chronic alcoholism, using capillary electrophoresis. Clin Chem Lab Med 2003;41:739–46. [6] Zaret DL, Morrison N, Gulbranson R, Keren DF. Immunofixation to quantify beta 2-transferrin in cerebrospinal fluid to detect leakage of cerebrospinal fluid from skull injury. Clin Chem 1992;38:1908–12. [7] Clinical Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline. CLSI Document EP05-A2. Wayne, PA, US: CLSI; 2004 . [8] Clinical Laboratory Standards Institute. User protocol for evaluation of qualitative test performance; approved guideline. CLSI Document EP12-A. Wayne, PA, US: CLSI; 2008 . [9] Gorogh T, Rudolph P, Meyer JE, Werner JA, Lippert BM, Maune S. Separation of beta2-transferrin by denaturing gel electrophoresis to detect cerebrospinal fluid in ear and nasal fluids. Clin Chem 2005;51:1704–10. [10] Perez-Cerda C, Quelhas D, Vega AI, Ecay J, Vilarinho L, Ugarte M. Screening using serum percentage of carbohydrate-deficient transferrin for congenital disorders of glycosylation in children with suspected metabolic disease. Clin Chem 2008;54:93–100. [11] Boscato L, Edwards G, Bayoun R, Brady L. Spurious elevation of b2 transferrin in bacterially contaminated specimens. Clin Biochem Rev 1994;15.
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Evaluation of a commercially available carbohydrate deficient transferrin kit to detect beta-2-transferrin in cerebrospinal fluid using capillary electrophoresis Karina Rodríguez-Capote a,b,⁎,1, Joceline Turner c, Joseph Macri a,b a b c
Department of Pathology and Laboratory Medicine, McMaster University, Ontario, Canada Hamilton Regional Medicine Program, Ontario, Canada Department of Special Chemistry Laboratory, Hamilton Regional Medicine Program, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 6 May 2013 Received in revised form 29 June 2013 Accepted 2 July 2013 Available online 12 July 2013 Keywords: β-2-transferrin Capillary electrophoresis Cerebrospinal fluid leakage CSF rhinorrhea Desialylated transferrin Tau-protein
a b s t r a c t Objective: The aim of this study was to evaluate the CEofix™ CDT Capillary Electrophoresis (CE) kit for the detection of β-2-tranferrin (β-2-Tf) in cerebrospinal fluid (CSF). Design and method: Evaluation was performed according to CLSI EP5-A and EP12-A guidelines. Results: The method resolved β-2-Tf from other Tf isoforms. The C50 for β-Tf was determined to be 0.46 mg/L. Neither hemoglobin nor bilirubin co-migrated with β-2-Tf. Conclusions: The CDT kit can be used for detecting β-2-Tf in CSF by CE. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Leakage of cerebrospinal fluid (CSF) is a critical condition with a high risk of meningitis and potentially fatal outcome [1]. Βeta-2-transferrin (β-2-Tf) is the asialo transferrin isoform found only in CSF, ocular fluids and perilymph and is an accepted marker of CSF leakage [1,2]. Our objective was to evaluate a commercially available kit, Analis CEofix™ CDT Capillary Electrophoresis [3], for detecting β-2-Tf in fluids suspected to be CSF. This kit is intended to detect serum carbohydrate deficient transferrin (CDT) by capillary electrophoresis (CE) in patients suspected of chronic alcoholism [4,5]. Methodology Samples Residual CSF samples were obtained from the clinical laboratory after physician-ordered testing was complete. CSF pools were prepared representing sample condition (clear, hemolyzed and xanthochromic) and centrifuged to remove cells and debris. Total protein and transferrin ⁎ Corresponding author at: Clinical Chemist, DynaLIFEDx, #200, 10150-102 Street, Edmonton, Alberta, Canada, T5J 5E2. Fax: + 1 780 454 2845. E-mail address: [email protected] (K. Rodríguez-Capote). 1 Present address: DynaLIFEDx, Edmonton, Alberta, Canada.
concentrations were determined using the Roche Modular and the Dade Behring BNII Protein Analyzer, respectively and aliquots frozen at −20 °C until use. For method comparison, individual patient samples submitted and referred out for β-2-Tf testing with sufficient volume were split and recoded prior to use. All but one sample were nasal fluids; this specimen consisted of a cotton swab soaked with fluid collected after lumbar spine surgery. This sample was eluted with a known volume of sterile saline, as per referral laboratory protocol. Sample preparation and analysis by capillary electrophoresis Sialo-Tf isoforms were separated using the CEofix™ CDT kit [3] and the P/ACE™ MDQ Capillary Electrophoresis System from Beckman Coulter according to manufacturer's instructions. Running buffer (TRIS/Borate, pH 8.5), initiator buffer (Tris/phosphate, pH 2.0) as well as serum based quality controls are provided with the kit. These controls (normal and CDT) were run prior to sample analysis. Since Tf concentrations in CSF are 100-fold lower than in blood (estimated concentrations between 12 and 78 mg/l [1,6]), CSF samples were concentrated using Amicon® Ultra-4 centrifugal filtration devices (Millipore Ltd). Before CE analysis, samples (25 μl) were saturated with iron by incubation with 10 mM FeCl3 provided with the kit. Results were recorded on an electropherogram and peaks were integrated using the instrument software. Tetra-sialo-Tf was used as the reference peak with its migration time (MT) set at 6.08 min.
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.07.001
K. Rodríguez-Capote et al. / Clinical Biochemistry 46 (2013) 1770–1773
Method evaluation Analytical precision Analytical precision was calculated following CLSI guideline EP5-A [7], by repeatedly measuring CSF pools of low (2.0 mg/L) and high (23 mg/L) Tf concentrations. Analytical sensitivity Serial dilutions of CSF were performed to determine linearity. The C50 of the assay (analyte concentration at which 50% of the results are classified either as positive or negative) and the 95% interval (concentrations above and below the cutoff point at which repeated results are 95% positive or 95% negative) were calculated according to CLSI guideline EP12-A2 [8]. Analytical specificity Analytical specificity was verified by immunosubtraction of all Tf sialoforms using an anti-human Tf antibody (Sigma-Aldrich) prior to CE analysis. Specificity of the MT for β-2-Tf and 0-sialo-Tf was verified by treating CSF and serum with neuraminidase (Acetyl-neuraminyl hydrolase, New England BioLabs, USA) to digest the Tf sialic acid side chains. Interference studies were performed using hemolyzed and xanthochromic CSF pools. In addition, we examined the potential influence of CDT due to blood contamination, by mixing CSF with serum control material.
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the qualitative detection of its presence in fluid, and so the C50 was estimated according to the CLSI guideline EP12-A2 and determined to be 0.46 mg/L (95% interval: 0.25 to 0.68). Specificity and interference Specificity for transferrin was verified by immunosubstraction (Fig. 1A) and the MT for β-2-Tf was verified by neuraminidase treatment (Fig. 1B). We further examined the potential influence of blood contamination by mixing the serum control from the kit, containing 0-sialo-Tf, with CSF (Fig. 1C). This experiment confirmed that MTs of β-2-Tf and 0-sialo Tf can be discriminated in the same sample. Xanthochromia (CSF bilirubin = 9.7 μmol/L, Fig. 1D) produced an extra peak at 5.78 min whereas hemolysis (CSF hemoglobin = 2.4 g/L, Fig. 1E) suppressed the 1-and 3-sialo-Tf peaks but the MT of β-Tf was unaffected. Stability β-2-Tf was detected in all samples without significant decrease of the β-2-Tf/4-sialo-Tf area ratio, independent of storage temperature. All values were within 30% of baseline measurements. The analyte is stable for up to 6 months at − 20 °C, and up to 7 days refrigerated or at room temperature. Concordance of results with comparative method
Stability Aliquots of CSF (β-2-Tf = 2.0 mg/L) stored at room temperature and 4 °C were tested every second day for a week and aliquots stored at − 20 °C were tested weekly for six months. Adequate analyte stability was defined as a β-2-Tf/4-sialo-Tf peak area ratio relative to the baseline measurement being within or close to the total imprecision of the test. Diagnostic performance Patient specimens with sufficient volume, proficiency testing samples and diluted CSF of known β-2-Tf concentrations were analyzed and compared to reference laboratory results and to clinical outcome of the patient, where available. The comparative method is a qualitative immunofixation electrophoresis (IFE) method developed in-house by the reference laboratory (Scarborough General Hospital, Toronto). The reference laboratory is the B-2-Tf testing facility used by Hospitals In-Common Laboratory Inc. with a published turnaround time of 10 days. Clinical evidence of CSF leak was determined by retrospective chart review and included development of meningitis or observation of fistulas during physical exam, surgery or imaging studies. The degree of agreement between the two methods was evaluated using Kappa statistics. Statistical analysis was performed using MedCalc v12.4.0 (MedCalc Software, Mariakerke, Belgium). Results Beta-2-Tf was clearly resolved from 0-sialo Tf (MT ± SD = 5.52 ± 0.01 min and 5.59 ± 0.02 min respectively) as well as from the other sialo Tf forms (Figure C). Peak area ratios (AR, β-2-Tf to 4-sialo-Tf) were highly reproducible. Intra-assay CVs and total imprecision were 21.8% (n = 20) and 26.9% (n = 27) for the low concentration (2.0 mg/L, AR = 0.11) and 2.7% (n = 22) and 8.3% (n = 27) for the high concentration (23 mg/L, AR = 0.25). Analytical sensitivity Analytical sensitivity was calculated by serially diluting pooled CSF (total Tf concentration = 0.23 g/L; β-2-Tf = 32 mg/L). The test is linear between 0.25 and 23 mg/L β-2-Tf (y = 1.21 × − 0.0046, R2 = 0.99). The clinical utility of β-2-Tf measurement, however, is
Of the 20 samples analyzed by both the test method and the comparative method (Table 1), eleven results were positive, five were negative, and four were discordant (positive by CE and negative by IFE) representing a moderate degree of agreement (weighted kappa = 0.58; 95% CI 0.24 to 0.91%). Not significant difference between paired proportions was found by the McNemar test (p = 0.13). Diagnostic performance Diagnostic performance was calculated using patient specimens and proficiency samples analyzed by both laboratories (Table 1). Using clinical outcome assessed by retrospective chart review as the gold standard, diagnostic sensitivities were 91.67% (61.52% to 99.79%) and 66.7% (34.89% to 90.08%) for CE and IFE respectively, with corresponding specificities of 100% (2.5% to 100%). Discussion and limitations The present evaluation presents an adapted method for off-label use of the Analis CEofix™ CDT kit in conjunction with capillary electrophoresis for β-2-Tf detection in body fluids. Analytically, MTs were highly reproducible and β-2-Tf was resolved from serum 0-sialoTf. Neither hemoglobin nor bilirubin co-migrated with β-2-Tf. Since current IFE methods of detecting β-2-Tf are hindered when samples are contaminated with blood [9] (a common occurrence in this type of specimen), this presents a major improvement over existing β-2-Tf methods. Current IFE methods can also be limited by allelic variants of transferrin that resemble the CSF isoform, as reported in cases of hypoglycosylation secondary to cystic fibrosis, Epstein–Barr virus infection, galactosemia or liver failure [10], suggesting that additional patient populations may benefit from CE analysis. In addition, the CE method presents an opportunity to improve result turn-around-times, with the whole procedure of sample concentration, instrument calibration and sample analysis taking no more than 60 min. CE appears to be more sensitive than IFE for the detection of CSF leakage (higher positive rate). However, difficulty in obtaining patient specimens of sufficient volume resulted in the inclusion of only one true negative nasal sample, preventing an accurate estimated of the
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K. Rodríguez-Capote et al. / Clinical Biochemistry 46 (2013) 1770–1773
Fig. 1. Specificity of the migration time (MT) for beta transferrin (β-Tf). 1A. Electropherograms of untreated and transferrin immunoprecipitated CSF: sample was incubated with Tf antiserum prior to CE analysis. 1B. Neuraminidase was used to digest sialic acid side chains from transferrin in CSF and serum, revealing their asialo-isoforms. 1C. Electropherograms of clear CSF, serum control (CDT-rich) and CSF mixed 1:1 with the CDT-rich serum control. 1D. Electropherograms of clear CSF, icteric CSF (bilirubin = 9.7 μmol/L) and a 1:1 clear: icteric CSF. 1E. Electropherogram of hemolytic CSF showing suppression of 1- and 3-sialo Tf.
Table 1 Diagnostic performance and concordance of results with comparative method. CE, capillary electrophoresis; CSF, cerebrospinal fluid; IFE, immunofixation electrophoresis; Ref. lab, reference laboratory; Tf, transferrin. Proficiency samples consisted of true patient samples. CE: positive the peaks for β-2-Tf and 4-sialo-Tf are present in the correct migration times; traces, when in the presence of 4-sialo-Tf a small peak is visible in the correct migration time for β-2-Tf, but the software cannot integrate it. IFE: positive if beta-1 and beta-2 transferrin are detected; traces, if beta-1 is present and a very faint band is visible in beta 2. Sample type
Our lab (CE)
Ref. lab (IFE)
Clinical outcome
1
Nasal fluid
Positive
Positive
2 3
Nasal fluid Cotton swab
Positive Positive
Positive Traces
4 5 6 7 8 9 10 11
Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Nasal fluid Proficiency B2T 2008-1 Proficiency B2T 2008-2 Proficiency B2T 2008-4 Serum control CSF filtrate CSF CSF CSF CSF CSF
Negative Positive Positive Positive Positive Negative Positive Positive
Negative Negative Positive Negative Negative Negative Positive Positive
Endoscopic transphenoidal resection of pituitary tumor. Confirmed positive Confirmed positive Postoperative CSF leak, compressive epidural hematoma. Lumbar spine surgery. Confirmed positive. Confirmed positive. Confirmed positive. Confirmed positive. Confirmed positive in surgery. Laryngectomy, confirmed fistulae. Confirmed positive. Head trauma. Confirmed negative. Confirmed positive. Fluid from anterior neck Hemovac. Confirmed positive.
Traces
Traces
Positive
Positive
Negative Negative Negative Positive Positive Positive Positive
Negative Negative Negative Negative Traces Positive Positive
12 13 14 15 16 17 18 19 20
Confirmed CSF leak by MR cisternogram as well as CT scan. Confirmed positive. Leak identified in skull base, fluid and fluorescein dye actively pouring out of nasal cavity. Confirmed positive. Tf =0, β-2-T = 0 mg/L Tf =0, β-2-T = 0 mg/L Tf = 2.3 mg/L, β-2-T = 0.3 mg/L Tf = 3.5 mg/L, β-2-T = 0.46 mg/L Tf = 4.6 mg/L, β-2-T = 0.6 mg/L Tf = 5.8 mg/L, β-2-T = 0.7 mg/L Tf = 22.5 mg/L, β-2-T = 2.8 mg/L
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false positive rate using CE. Therefore, analysis of a suitable number of truly negative nasal, oral, and ear fluids should be undertaken by laboratories who consider performing this testing. The finding that β-2-Tf in CSF samples is stable for at least a week indicates that a negative result is unlikely to be caused by protein degradation if tested within this period. A positive result in an old specimen, however, should be interpreted with caution since false positives by IFE have been reported due to colonization of samples by neuraminidase secreting bacteria [1,11]. Whether this will affect detection by CE remains to be tested. Regardless, all results positive or negative should be critically correlated with clinical history and with results from other diagnostic procedures. Conclusions The method effectively resolves β-2-Tf from other Tf sialoforms. The Analis CEofix™ CDT kit can be used in conjunction with CE for the detection of β-2-Tf. The procedure is fast, specific and sensitive. Acknowledgment The authors express their gratitude to Dr. Barakauskas for her insightful comments and critical review of the manuscript. We want to extend our thanks to Dr. Samsoondar (Scarborough General Hospital) for his collaboration with the method comparison.
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