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Evaluation of a commercially available carbohydrate deficient transferrin kit to detect β2-transferrin in cerebrospinal fluid using capillary electrophoresis
ABSTRACTS / Clinical Biochemistry 41 (2008) 1263–1287
results, the difference between which represents error for not adjusting for albumin concentrations. Results:
1283
contamination, not all medications containing EtOH also contain PG and vice versa. doi:10.1016/j.clinbiochem.2008.08.054
Clinical impact of single-value protein compensation of Jaffe creatinine assay results on eGFR values Patient group
Patient records
Age/sex
Total
1 19–70 y, F 6201 2 19–70 y, M 5689 Total: 11890
Error in eGFR in patients with creatinine ≥ 88.4 μmol/L (1 mg/dL) Creatinine Total ≥88.4 range % μmol/L 1018 2242 3260
25th Median 75th Percentage Percentage percentile % percentile N 5% N 10% % %
−7.7 to 18.2 0.8 −8.7 to 16.7 − 0.2
2.1 1.4
4 19.0 3.1 14.2 Average: 16.6
4.3 3.5 3.9
Conclusion: For creatinine concentrations ≥ 88.4 μmol/L, 3.9% of the eGFR values have N 10% error and 16.6% have N 5% error due exclusively to not compensating for actual albumin concentrations. This undermines achieving reliable clinical interpretation of eGFR. doi:10.1016/j.clinbiochem.2008.08.053
158 Ethanol and propylene glycol contamination of blood specimens drawn from indwelling lines following intravenous medication administration Perkins S. L. 1,2, Forsythe J. E.1, Bookalam S. N.1 1 The Ottawa Hospital, Canada 2 University of Ottawa, Ottawa, Canada Case history: A 34-year-old male with known seizure disorder was brought to Emergency after two seizures. Blood samples analyzed by GC revealed 86 mmol/L propylene glycol (PG), 27.3 mmol/L ethanol (EtOH), and no other volatiles. Analysis of a second sample (same draw) and a fresh specimen detected no EtOH, PG or other volatiles. Investigation: We reviewed the intravenous medications and found PG and EtOH to be preservatives and solvents in two formulations: lorazepam (benzyl alcohol, polyethylene glycol, PG), phenytoin (40% PG, 10% EtOH). The patient had one indwelling line for administration of IV medications. It is our hospital's practice to use this saline lock to procure blood samples, however it must be thoroughly flushed and one “discard” tube drawn prior to sample procurement. We hypothesize that in the busy ER the saline lock may not be adequately flushed. We calculated that the phenytoin solution contained 5200 mmol/L PG and 2173 mmol/L EtOH. Small contamination of the blood sample drawn through the saline lock could have led to PG and EtOH detection in the first specimen. Conclusions: We believe the first specimen was contaminated with PG and EtOH during procurement through the indwelling line. The presence of small concentrations of PG is of little clinical consequence, however EtOH contamination could result in erroneous medical management and medical/ legal implications for impaired driving cases. Although the presence of PG in a sample may be a surrogate marker of sample
159 Are vitamin B12 results too low? Khajuria A.1, Higgins T.N.1, Prosser C.I.2, Janzen P.1 1 DynaLIFEDX, Edmonton, Alberta, Canada 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada Objectives: To explore the validity of low vitamin B12 results assayed by the Siemens' method after referring physicians expressed concern that results reported were too low and were not reflected in hematological changes. Method: Samples were separated into three groups based on Siemens' total vitamin B12 and the MCV:. 1) vitamin B12 (N150 pmol/L) and MCV (80–100 fL), 2) vitamin B12 (b 150 pmol/L) and MCV (80–100 fL), 3) vitamin B12 (b 150 pmol/L) and MCV (N 100 fL). All specimens were also analyzed for total vitamin B12 by Abbott Architect and Beckman Coulter DxI methods. In addition holo-cobalamin (Abbott), homocysteine (Siemens), intrinsic factor antibody and gastrin assays were performed on all samples. Results: Total B12 concentrations by the Siemens' method were higher than Abbott and Beckman Coulter methods (Bias − 18% and − 5% respectively). Patients with low holocobalamin (b 35 pmol/L) had increased homocysteine (N13.5 μmol/L). Total vitamin B12 compared reasonably well with holo-cobalamin and homocysteine independent of MCV. There was little association between low total vitamin B12 and either gastrin or anti-intrinsic factor results. Conclusions: The low B12 results seen on the Siemens method were confirmed by other methods. In patients with low total B12 and normal MCV, holo-cobalamin results were decreased and homocysteine results increased suggesting that low total B12 results are reasonable from a physiological perspective. Because MCV was elevated only at levels of vitamin B12 less then 70 pmol/L, decreased B12 results may precede hematological changes. doi:10.1016/j.clinbiochem.2008.08.055
160 Evaluation of a commercially available carbohydrate deficient transferrin kit to detect β2-transferrin in cerebrospinal fluid using capillary electrophoresis Rodríguez-Capote K.1, Turner J.2, Macri J.1 1 Department of Laboratory Medicine, HRLMP and McMaster University, Canada 2 Special Chemistry, HRLMP, Canada Objective: To evaluate a carbohydrate deficient transferrin (CDT) serum method for the analysis of β-Transferrin (β-Tf) in
1284
ABSTRACTS / Clinical Biochemistry 41 (2008) 1263–1287
cerebrospinal fluid (CSF). β-Tf (asialo-Tf found only in CSF, ocular fluids, and perilymph) is a marker of CSF leakage. Method: CSF pools (clear, haemolysed and xanthochromic) were prepared. Total protein and total transferrin (Tf) concentrations were determined using the Roche Modular and the Dade Behring Protein Analyzer, respectively. Various isoforms of sialylated transferrin were analyzed using the CEofix™ CDT kit (Analis) by capillary electrophoresis (CE). Studies followed NCCLS EP5-A and EP12-P guidelines. Results: Total protein and total Tf concentrations in the clear pool were 3.83 and 0.23 g/L respectively. Quantification was referenced to the 4-sialo-Tf peak, typically the highest, which migrated consistently at 6.08 min. The within-run and total imprecision varied dependent on the sialo-Tf isoform. Specificity of the migration time for β-Tf was verified by Neuraminidase and anti-transferrin antibody treatment. The detection limit for β-Tf was 0.46 mg/L. Usual CSF concentrations of β-Tf are 2–40 mg/L. Neither hemoglobin nor bilirubin co-migrated with β-Tf. Conclusions: The CDT kit can be used in CE for detecting CSF β-Tf. The method resolves β-Tf from other Tf isoforms. The procedure is fast, specific and sensitive. Sample type
Isoform
QC (serum)
0-Tf 0.01 2-Tf 0.04 β-Tf (33.7 mg/L) 0.25 β-Tf (2.59 mg/L) 0.11
CSF
Within-run imprecision Area ratio CV % to 4-Tf 10.2 3.3 2.7 21.7
Total imprecision MT CV % Area ratio CV % MT CV % to 4-Tf 5.58 5.84 5.51 5.53
0.09 0.07 0.05 0.07
0.01 0.04 0.24 0.13
41.5 2.7 8.3 26.9
5.59 5.85 5.52 5.53
0.41 0.07 0.15 0.09
doi:10.1016/j.clinbiochem.2008.08.056
161 Quantitation of calcitriol in plasma samples using triple quadrupole tandem mass spectrometry coupled to orthogonal liquid chromatography Casetta B.1, Impey G.2, Sapp L.3 1 Applied Biosystems, Monza (MI), Italy 2 Sciex, Toronto, Canada 3 Applied Biosystems, Framingham, MA, USA Objectives: Calcitriol (1,25-dihydroxycholecalciferol) is the active form of vitamin D3 endogenous to humans. Highly sensitive assays are required to study this analyte in humans as the concentration levels are significantly low, below 10 pg/mL, which are several orders of magnitude lower than the monohydroxylated species. Here we describe an analytical strategy that incorporates a new two-dimensional chromatography analysis coupled to tandem mass spectrometry that minimizes the sample preparation and significantly improves the chromatographic resolution important for reaching low level detection. Methods: Sample preparation is limited to a protein precipitation step. Supernatant is directly injected in a chromatographic system centered on a perfusion chromatographic column for the first dimension. The plug carrying the
analyte is trapped and introduced in the second chromatographic dimension coupled to electrospray ionization tandem mass spectrometry for the quantitative measurement. Results are generated through calibration with standard solutions of calcitriol with the use of d6-calcitriol as the internal standard. Results: With the proposed configuration, total analysis time per sample results in 16 min with a sample consumption of 30 μL and with a LOQ of 5 pg/mL. Linearity has been tested from the LOD (estimated as 2 pg/mL) up to over 2 ng/mL. The range of the total assay precision is 5–10%. (n = 6), and the average accuracy, tested through comparison with RIA, is around 11% for measurements above 10 pg/mL. Conclusion: The proposed methodology enables reliable quantitation of calcitriol in plasma samples after performing just a simple protein precipitation. doi:10.1016/j.clinbiochem.2008.08.057
162 Evaluation of the Piccolo Xpress chemistry analyzer for point of care testing of plasma liver function markers, electrolytes and metabolites Schnabl K.L., Tarek M., Cursio C., Yip P.M. University Health Network, Toronto, Ontario, Canada M5G 2C4 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L6 Objective: Analytical performance of the Piccolo Xpress, a tabletop chemistry analyzer for near patient testing, was evaluated. Plasma assays are contained in single-use reaction discs as liver function (ALT, albumin, ALP, amylase, AST, GGT, total bilirubin and protein), electrolyte and metabolite (creatinine, glucose, urea) panels. Methods: The evaluation was performed according to modified CLSI guidelines. Precision was assessed using 2 levels of Bio-Rad Lyphocheck controls measured over 5 days with 2 runs per day in duplicate. Method comparison studies were performed on the Piccolo Xpress with patient samples (n = 40) over a broad range of analyte concentrations and were compared to the Abbott Architect c8000. Results: Total imprecision met laboratory goals for all measured analytes except ALT, ALP, AST and creatinine (Table 1). Table 1 Piccolo Xpress total imprecision Analyte
QC level
Mean ± SD
Measured % CV
Goal % CV
ALT (U/L)
Low High Low High Low High Low High
27 ± 3.3 81 ± 3.2 98 ± 6.3 485 ± 18 46 ± 3.0 189 ± 3.8 46 ± 7.4 495 ± 10
13 4.4 6.5 3.7 7.6 2.1 16 2.1
5.0
ALP (U/L) AST (U/L) Creatinine (μmol/L)
5.0 5.0 6.7
Deming linear regression showed good correlation between Piccolo Xpress and Architect c8000 for all analytes (R2; 0.88–
results, the difference between which represents error for not adjusting for albumin concentrations. Results:
1283
contamination, not all medications containing EtOH also contain PG and vice versa. doi:10.1016/j.clinbiochem.2008.08.054
Clinical impact of single-value protein compensation of Jaffe creatinine assay results on eGFR values Patient group
Patient records
Age/sex
Total
1 19–70 y, F 6201 2 19–70 y, M 5689 Total: 11890
Error in eGFR in patients with creatinine ≥ 88.4 μmol/L (1 mg/dL) Creatinine Total ≥88.4 range % μmol/L 1018 2242 3260
25th Median 75th Percentage Percentage percentile % percentile N 5% N 10% % %
−7.7 to 18.2 0.8 −8.7 to 16.7 − 0.2
2.1 1.4
4 19.0 3.1 14.2 Average: 16.6
4.3 3.5 3.9
Conclusion: For creatinine concentrations ≥ 88.4 μmol/L, 3.9% of the eGFR values have N 10% error and 16.6% have N 5% error due exclusively to not compensating for actual albumin concentrations. This undermines achieving reliable clinical interpretation of eGFR. doi:10.1016/j.clinbiochem.2008.08.053
158 Ethanol and propylene glycol contamination of blood specimens drawn from indwelling lines following intravenous medication administration Perkins S. L. 1,2, Forsythe J. E.1, Bookalam S. N.1 1 The Ottawa Hospital, Canada 2 University of Ottawa, Ottawa, Canada Case history: A 34-year-old male with known seizure disorder was brought to Emergency after two seizures. Blood samples analyzed by GC revealed 86 mmol/L propylene glycol (PG), 27.3 mmol/L ethanol (EtOH), and no other volatiles. Analysis of a second sample (same draw) and a fresh specimen detected no EtOH, PG or other volatiles. Investigation: We reviewed the intravenous medications and found PG and EtOH to be preservatives and solvents in two formulations: lorazepam (benzyl alcohol, polyethylene glycol, PG), phenytoin (40% PG, 10% EtOH). The patient had one indwelling line for administration of IV medications. It is our hospital's practice to use this saline lock to procure blood samples, however it must be thoroughly flushed and one “discard” tube drawn prior to sample procurement. We hypothesize that in the busy ER the saline lock may not be adequately flushed. We calculated that the phenytoin solution contained 5200 mmol/L PG and 2173 mmol/L EtOH. Small contamination of the blood sample drawn through the saline lock could have led to PG and EtOH detection in the first specimen. Conclusions: We believe the first specimen was contaminated with PG and EtOH during procurement through the indwelling line. The presence of small concentrations of PG is of little clinical consequence, however EtOH contamination could result in erroneous medical management and medical/ legal implications for impaired driving cases. Although the presence of PG in a sample may be a surrogate marker of sample
159 Are vitamin B12 results too low? Khajuria A.1, Higgins T.N.1, Prosser C.I.2, Janzen P.1 1 DynaLIFEDX, Edmonton, Alberta, Canada 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada Objectives: To explore the validity of low vitamin B12 results assayed by the Siemens' method after referring physicians expressed concern that results reported were too low and were not reflected in hematological changes. Method: Samples were separated into three groups based on Siemens' total vitamin B12 and the MCV:. 1) vitamin B12 (N150 pmol/L) and MCV (80–100 fL), 2) vitamin B12 (b 150 pmol/L) and MCV (80–100 fL), 3) vitamin B12 (b 150 pmol/L) and MCV (N 100 fL). All specimens were also analyzed for total vitamin B12 by Abbott Architect and Beckman Coulter DxI methods. In addition holo-cobalamin (Abbott), homocysteine (Siemens), intrinsic factor antibody and gastrin assays were performed on all samples. Results: Total B12 concentrations by the Siemens' method were higher than Abbott and Beckman Coulter methods (Bias − 18% and − 5% respectively). Patients with low holocobalamin (b 35 pmol/L) had increased homocysteine (N13.5 μmol/L). Total vitamin B12 compared reasonably well with holo-cobalamin and homocysteine independent of MCV. There was little association between low total vitamin B12 and either gastrin or anti-intrinsic factor results. Conclusions: The low B12 results seen on the Siemens method were confirmed by other methods. In patients with low total B12 and normal MCV, holo-cobalamin results were decreased and homocysteine results increased suggesting that low total B12 results are reasonable from a physiological perspective. Because MCV was elevated only at levels of vitamin B12 less then 70 pmol/L, decreased B12 results may precede hematological changes. doi:10.1016/j.clinbiochem.2008.08.055
160 Evaluation of a commercially available carbohydrate deficient transferrin kit to detect β2-transferrin in cerebrospinal fluid using capillary electrophoresis Rodríguez-Capote K.1, Turner J.2, Macri J.1 1 Department of Laboratory Medicine, HRLMP and McMaster University, Canada 2 Special Chemistry, HRLMP, Canada Objective: To evaluate a carbohydrate deficient transferrin (CDT) serum method for the analysis of β-Transferrin (β-Tf) in
1284
ABSTRACTS / Clinical Biochemistry 41 (2008) 1263–1287
cerebrospinal fluid (CSF). β-Tf (asialo-Tf found only in CSF, ocular fluids, and perilymph) is a marker of CSF leakage. Method: CSF pools (clear, haemolysed and xanthochromic) were prepared. Total protein and total transferrin (Tf) concentrations were determined using the Roche Modular and the Dade Behring Protein Analyzer, respectively. Various isoforms of sialylated transferrin were analyzed using the CEofix™ CDT kit (Analis) by capillary electrophoresis (CE). Studies followed NCCLS EP5-A and EP12-P guidelines. Results: Total protein and total Tf concentrations in the clear pool were 3.83 and 0.23 g/L respectively. Quantification was referenced to the 4-sialo-Tf peak, typically the highest, which migrated consistently at 6.08 min. The within-run and total imprecision varied dependent on the sialo-Tf isoform. Specificity of the migration time for β-Tf was verified by Neuraminidase and anti-transferrin antibody treatment. The detection limit for β-Tf was 0.46 mg/L. Usual CSF concentrations of β-Tf are 2–40 mg/L. Neither hemoglobin nor bilirubin co-migrated with β-Tf. Conclusions: The CDT kit can be used in CE for detecting CSF β-Tf. The method resolves β-Tf from other Tf isoforms. The procedure is fast, specific and sensitive. Sample type
Isoform
QC (serum)
0-Tf 0.01 2-Tf 0.04 β-Tf (33.7 mg/L) 0.25 β-Tf (2.59 mg/L) 0.11
CSF
Within-run imprecision Area ratio CV % to 4-Tf 10.2 3.3 2.7 21.7
Total imprecision MT CV % Area ratio CV % MT CV % to 4-Tf 5.58 5.84 5.51 5.53
0.09 0.07 0.05 0.07
0.01 0.04 0.24 0.13
41.5 2.7 8.3 26.9
5.59 5.85 5.52 5.53
0.41 0.07 0.15 0.09
doi:10.1016/j.clinbiochem.2008.08.056
161 Quantitation of calcitriol in plasma samples using triple quadrupole tandem mass spectrometry coupled to orthogonal liquid chromatography Casetta B.1, Impey G.2, Sapp L.3 1 Applied Biosystems, Monza (MI), Italy 2 Sciex, Toronto, Canada 3 Applied Biosystems, Framingham, MA, USA Objectives: Calcitriol (1,25-dihydroxycholecalciferol) is the active form of vitamin D3 endogenous to humans. Highly sensitive assays are required to study this analyte in humans as the concentration levels are significantly low, below 10 pg/mL, which are several orders of magnitude lower than the monohydroxylated species. Here we describe an analytical strategy that incorporates a new two-dimensional chromatography analysis coupled to tandem mass spectrometry that minimizes the sample preparation and significantly improves the chromatographic resolution important for reaching low level detection. Methods: Sample preparation is limited to a protein precipitation step. Supernatant is directly injected in a chromatographic system centered on a perfusion chromatographic column for the first dimension. The plug carrying the
analyte is trapped and introduced in the second chromatographic dimension coupled to electrospray ionization tandem mass spectrometry for the quantitative measurement. Results are generated through calibration with standard solutions of calcitriol with the use of d6-calcitriol as the internal standard. Results: With the proposed configuration, total analysis time per sample results in 16 min with a sample consumption of 30 μL and with a LOQ of 5 pg/mL. Linearity has been tested from the LOD (estimated as 2 pg/mL) up to over 2 ng/mL. The range of the total assay precision is 5–10%. (n = 6), and the average accuracy, tested through comparison with RIA, is around 11% for measurements above 10 pg/mL. Conclusion: The proposed methodology enables reliable quantitation of calcitriol in plasma samples after performing just a simple protein precipitation. doi:10.1016/j.clinbiochem.2008.08.057
162 Evaluation of the Piccolo Xpress chemistry analyzer for point of care testing of plasma liver function markers, electrolytes and metabolites Schnabl K.L., Tarek M., Cursio C., Yip P.M. University Health Network, Toronto, Ontario, Canada M5G 2C4 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L6 Objective: Analytical performance of the Piccolo Xpress, a tabletop chemistry analyzer for near patient testing, was evaluated. Plasma assays are contained in single-use reaction discs as liver function (ALT, albumin, ALP, amylase, AST, GGT, total bilirubin and protein), electrolyte and metabolite (creatinine, glucose, urea) panels. Methods: The evaluation was performed according to modified CLSI guidelines. Precision was assessed using 2 levels of Bio-Rad Lyphocheck controls measured over 5 days with 2 runs per day in duplicate. Method comparison studies were performed on the Piccolo Xpress with patient samples (n = 40) over a broad range of analyte concentrations and were compared to the Abbott Architect c8000. Results: Total imprecision met laboratory goals for all measured analytes except ALT, ALP, AST and creatinine (Table 1). Table 1 Piccolo Xpress total imprecision Analyte
QC level
Mean ± SD
Measured % CV
Goal % CV
ALT (U/L)
Low High Low High Low High Low High
27 ± 3.3 81 ± 3.2 98 ± 6.3 485 ± 18 46 ± 3.0 189 ± 3.8 46 ± 7.4 495 ± 10
13 4.4 6.5 3.7 7.6 2.1 16 2.1
5.0
ALP (U/L) AST (U/L) Creatinine (μmol/L)
5.0 5.0 6.7
Deming linear regression showed good correlation between Piccolo Xpress and Architect c8000 for all analytes (R2; 0.88–