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Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid
CLB-09261; No. of pages: 4; 4C: Clinical Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid Clarisse Benard 1, Jasmine Chauzeix 1, Antoine Blancher, Bénédicte Puissant-Lubrano ⁎ Laboratoire d'Immunologie, CHU de Toulouse, France
a r t i c l e
i n f o
Article history: Received 20 January 2016 Received in revised form 24 March 2016 Accepted 26 March 2016 Available online xxxx Keywords: Turbidimetry Analytical performances IgG Albumin Cerebrospinal fluid
a b s t r a c t Objectives: The intrathecal production of IgG is part of the diagnosis criteria for Multiple Sclerosis. Its assessment requires both quantitative (quantification of IgG and albumin in serum and cerebrospinal fluid (CSF)) and qualitative assays (isoelectric focusing). We have evaluated the analytical performances of the SPAPLUS® immunoturbidimeter (The Binding Site®) for the quantification of IgG and albumin in serum and CSF. Design and methods: Within-day and between-day precision, linearity and carry-over were assessed. Results obtained with SPAPLUS® were compared to those obtained with the nephelometer IMMAGE® 800, including albumin quotient and CSF IgG index. Isoelectric focusing was performed and considered as the gold standard for assessment of intrathecal production of IgG. Results: The within-day and the between-day precisions were obtained at two concentration levels and were below the recommendations of the manufacturer and of the French Society of Clinical Biology. Our evaluation confirmed the linearity of the assays and the absence of contamination. An agreement above 94% was observed between the results obtained with SPAPLUS® and those obtained with IMMAGE® 800. The use of the new reference material DA470k did not significantly modify IgG and albumin values. The confrontation of CSF IgG index and isoelectric focusing results led to a sensitivity of 79% and a specificity of 97% of CSF IgG index quantified on SPAPLUS® for the presence of oligoclonal bands at IEF. The sensitivity of intrathecal IgG calculated with the Reiber's hyperbolic formula was 47.4% and specificity was 97% for the presence of oligoclonal bands at IEF. Automatic rerun managed by the device for concentrations outside the measuring range was satisfactory. Conclusion: The SPAPLUS® immunoturbidimeter displays good analytical performances for the parameters evaluated in this work. © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction The intrathecal production of IgG is a useful parameter in case of Multiple Sclerosis (MS) suspicion to confirm the inflammatory nature of the disease and/or to exclude alternative diagnosis [1]. The intrathecal production of IgG is part of the diagnosis criteria for primary progressive Multiple Sclerosis which is characterized by a chronic progression from diagnosis without identified relapse [1,2]. It is therefore particularly indicated after one year of disease progression in association with the evidence of dissemination in space [1]. The evaluation of IgG production in cerebrospinal fluid (CSF) requires both quantitative and qualitative assays. The quantitative tests consist of quantifying IgG and albumin in serum and CSF. This allows the calculation of the albumin quotient (albumin CSF/albumin serum) which reflects the state of the blood–brain barrier, and that of CSF IgG index ([IgG CSF/albumin CSF]/[IgG serum/albumin serum]) which reflects ⁎ Corresponding author at: Laboratoire d'Immunologie, CHU Rangueil, 1, avenue Jean Poulhès, TSA 50032, 31059 Toulouse cedex 9, France. E-mail address: [email protected] (B. Puissant-Lubrano). 1 These authors have contributed equally to the work.
the abnormal intrathecal production of IgG. However, the sensitivity of the CSF IgG index is insufficient to affirm the intrathecal production of IgG [3,4], therefore the isoelectric focusing (IEF) of IgG in serum and CSF is required. Recent improvement of immunoturbidimetry led to consider this technique as a validated alternative to immunonephelometry, which is the reference method for the determination of specific proteins [5]. We have evaluated the analytical performances of SPAPLUS® immunoturbidimeter (The Binding Site) for the quantification of IgG and albumin in serum and CSF. This evaluation was performed according to CLSI (Clinical and Laboratory Standards Institute) protocols with minor modifications (between-day precision was assessed on 15 operating days and linearity was not assessed in duplicate).
2. Methods and results 2.1. Reagents For each parameter, reagent kits include a specific antiserum, a reaction buffer, six calibrators and two levels of internal quality control.
http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008 0009-9120/© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
2
C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
Calibrators consist of mixtures of human sera. IgG and albumin assays are calibrated with the International DA470k reference material.
calculated as follows: (B1 − B3) ∗ 100 / (average H − B3). Alternating samples with high concentrations with low concentration samples did not reveal any contamination (Supplemental Table 2).
2.2. Evaluation of precision Albumin (Vialebex®, LFB, Courtaboeuf, France) and therapeutic immunoglobulins (TEGELINE®, LFB) were diluted at various concentrations in PBS (Gibco Life Technologies, Saint Aubin, France) to obtain samples in sufficient quantity, with concentrations corresponding to a normal serum, a normal CSF and a pathological CSF (i.e. with high albumin quotient and high CSF IgG index). These three samples were assayed 30 times the same day in the same series. The within-run precision in serum gave satisfactory results (Table 1) with CV less or equal to 3% which met the acceptability criteria of the manufacturer and of the French Society of Clinical Biology (SFBC) [6]. The CV obtained in CSF were b4% for IgG and albumin, leading to CV b 5.8% for albumin quotient and CSF IgG index. For each parameter, the between-day precision was evaluated on two levels of internal quality controls provided in the kits. They were quantified twice a day during 14 to 15 consecutive working days. CV were below 5% for all the parameters studied, including calculated indexes (Table 1) and are in accordance with the SFBC's and manufacturer's recommendations. 2.3. Evaluation of linearity Linearity was assessed in CSF obtained from two patients having IgG or albumin concentration close to the upper limit of the measuring range of SPAPLUS®. Because no sera were available with such characteristics, we used a mixture of albumin and therapeutic immunoglobulins for the assessment of linearity in serum. Six to seven diluted points covering the measuring range given by the supplier were quantified on SPAPLUS®. The linear regressions between measured concentrations and theoretical values had slopes near 1 and intercept close to zero, except for albumin in CSF. Excellent correlation coefficients (N0.995) were obtained (Supplemental Table 1). 2.4. Evaluation of carry-over The carry-over was evaluated by measuring three times a sample with a high level for a parameter (H1, H2, H3) and then a sample with a low level for this same parameter (B1, B2, B3). Carry-over was
2.5. Agreement with results from IMMAGE® 800 nephelometer Fifty-three coupled samples of sera and CSF were obtained from 53 patients suspected of Multiple Sclerosis, of neurodegenerative disease, or of other inflammatory diseases or infections of the central nervous system. These samples were residues of routine analysis performed at our laboratory. IgG and albumin were quantified the same day on SPAPLUS® and on nephelometer IMMAGE® 800 (the device routinely used in our laboratory, Beckman Coulter, Villepinte, France). The comparison was performed on ten operating days. On IMMAGE, the IgG and albumin assays are calibrated with the international reference material CRM470. The slopes of linear regressions were between 0.92 and 0.97 and the correlation coefficients were above 0.87 for the four analytes measured (Fig. 1A). With Bland and Altman analysis, IgG and albumin biases were below 0.3 g/L in serum and b2 mg/L in CSF (Fig. 1B). The agreement between the two assays was greater than 94% for the four measured parameters and N90% for the two calculated indexes. It is interesting to note that the use of the new reference material DA470k did not significantly modify IgG and albumin values. Agreement was also evaluated for classification as normal or elevated albumin quotient (N6.5) and CSF IgG index (N0.65) [7] (Supplemental Table 3). Agreements between SPAPLUS® and IMMAGE® 800 for albumin quotient and CSF IgG index were 92% and 89%, respectively (see details below). 2.6. Agreement with isoelectric focusing results Isoelectric focusing followed by IgG immunoblotting [8] was used as the reference technique for the assessment of intrathecal IgG production [3]. Six patients had normal CSF IgG index with results from SPAPLUS® while having elevated CSF IgG index with results from IMMAGE® 800. Among those, two patients had no oligoclonal bands (OCB) as determined by IEF (they had uveitis and unreported diagnosis, respectively), and four had OCB at IEF. All four had CSF IgG index obtained from SPAPLUS® very close to the threshold (values from 0.61 to 0.63 for a threshold at 0.65). Two of them suffered from Multiple Sclerosis.
Table 1 Evaluation of within-run and between-day precision. For each parameter, the table shows the number of determinations, the mean of obtained values, the standard deviation and the CV, as well as the acceptability criteria from the French Society of Clinical Biology (SFBC) [6] and from the manufacturer (TBS = The Binding Site). IQC: internal quality control. If not stated, the evaluation was performed on a patient sample. Precision
Samples
Parameter
N dosages
Mean
Standard deviation
CV (%)
Within-run
Serum
Serum (IQC)
IgG (g/L) Albumin (g/L) IgG (mg/L) Albumin (mg/L) Albumin quotient CSF IgG index IgG (mg/L) Albumin (mg/L) Albumin quotient CSF IgG index IgG (g/L)
Serum (IQC)
Albumin (g/L)
CSF (IQC)
IgG (mg/L)
CSF (IQC)
Albumin (mg/L)
30 30 30 30 30 30 30 30 30 30 28 28 29 29 30 30 29 29
6.5 32.45 16.39 190.18 5.87 0.43 68.88 380.41 11.73 0.91 7.1 13.6 30.07 58.74 29.8 85.8 325.8 752.5
0.14 0.99 0.64 6.37 0.27 0.02 1.85 8.15 0.46 0.04 0.28 0.40 1.25 2.12 1.2 2.9 9.35 27.32
2.08 3.05 3.89 3.35 4.68 5.77 2.69 2.14 3.95 4.93 3.86 2.96 4.16 3.61 4.04 3.38 2.87 3.63
Acceptability criteria SFBC
Normal CSF
Pathological CSF
Between-day
4.5 3.8
TBS 5 6 5 5
5 5
5 5 5 4
5 5 8 8 8 8 10 10
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
3
Fig. 1. Agreement between SPAPLUS® and IMMAGE® 800. (A) For each parameter, the figure shows the correlation line (solid line) between the concentrations obtained with the reference laboratory technique (Nephelometer IMMAGE® 800) and the SPAPLUS®, as well as the line of identity (dotted line) between the two assays. (B) The figure shows the graphs of the differences (Bland and Altman test): each point represents the difference between the concentrations obtained by the two assays according to the average of determinations obtained by the two assays. The black line shows the ideal difference of 0 between the two assays. The blue line shows the average of the differences obtained and corresponds to the bias between the two assays. Blue dotted lines correspond to the lower and upper limits of the confidence interval at 95% of the differences between both assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Because only five patients among the 53 studied had a Multiple Sclerosis, it was not possible to calculate the sensitivity and the specificity of quantitative intrathecal IgG production for MS. We therefore calculated the sensitivity and the specificity of both CSF IgG index and intrathecal IgG calculated with Reiber's formula for the presence of OCB at IEF.
Indeed, the hyperbolic formula of Reiber can also be used to assess the intrathecal production of IgG and has shown superior performance than CSF IgG index (less false positive results) [7]. Among the 42 patients with normal intrathecal IgG with the Reiber's formula, ten had OCB (= false negative), the 32 remaining patients are true negative
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
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C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
2.8. Management of automatic rerun Automatic reruns at a dilution different from the initial working dilution were triggered for CSF samples with concentrations of albumin (n = 9) or IgG (n = 4) outside the measuring range. Final results (from 94.6 to 167 mg/L for albumin and from 169 to 348 mg/L for IgG) were close to those obtained with IMMAGE® 800 (see the results of the agreement between the two assays).
3. Discussion In conclusion, the immunoturbidimeter SPAPLUS® provided consistent results for precision, linearity and contamination for IgG and albumin assays in serum and CSF. The automatic rerun was well managed by the device. The agreement of the SPAPLUS® results with those of the nephelometer IMMAGE® 800 was good, including the calculated albumin quotient and CSF IgG index. The CSF IgG index calculated from SPAPLUS® had good sensitivity and specificity for determining the presence of OCB at IEF. The SPAPLUS® is therefore a valuable tool to measure albumin and IgG in sera and CSF. This device allows the quantification of 49 biochemical and immunological parameters.
Fig. 2. IgG CSF/serum quotient diagrams with hyperbolic graphs according to Reiber. Areas are defined as follows: 1 normal; 2 blood-CSF barrier dysfunction; 3 intrathecal IgG synthesis plus blood-CSF barrier dysfunction; 4 intrathecal IgG synthesis; 5 methodological error. Triangles show patients with no oligoclonal bands at IEF while open squares show patients with presence of oligoclonal bands at IEF.
Acknowledgments The reagents were supplied by The Binding Site.
Appendix A. Supplementary data (Fig. 2). Among the 11 patients with elevated intrathecal IgG with the Reiber's formula, 10 had OCB at IEF (true positive), the remaining patient is a false positive. These results led to a sensitivity of 47.4% and a specificity of 97% of intrathecal IgG calculated from SPAPLUS® with the Reiber's formula for the presence of OCB at IEF. These results are slightly lower than previously reported as for sensitivity, but were similar as for specificity [9]. Among the 37 patients with normal CSF IgG index on SPAPLUS®, four had OCB (the same four detailed above with elevated CSF IgG index on IMMAGE = false negative), the 33 remaining patients are true negative. Among the 16 patients with elevated CSF IgG index on SPAPLUS®, 15 had OCB at IEF (93%, true positive), the remaining patient is a false positive. This frequency of true positive is slightly better than frequencies previously reported [10]. These results led to a sensitivity of 79% and a specificity of 97% of CSF IgG index calculated from SPAPLUS® for the presence of OCB at IEF. These results are better than those obtained by other studies [11,12]. This could be explained in part because we use a threshold of 0.65 [7] while Lunding and Mares used a threshold of 0.72 and 0.70, respectively. These results are consistent with the fact that CSF IgG index is less sensitive than IEF to assess intrathecal production of IgG [12]. 2.7. Practical characteristics of the assays The CSF volume needed for the analysis on SPAPLUS® is 215 μl. This includes 35 μl + 30 μl for IgG and albumin quantification, respectively, and 150 μl of dead-volume (when using tubes, 120 μl if using cups). The initial measuring ranges are 4–135 mg/L for IgG (no dilution on the device) and 170–2700 mg/L for albumin (dilution 1/10). For IgG, rerun can be performed at 1:10 by the device (30 μl sample required) leading to a measuring range of 40–1350 mg/L. For albumin, rerun can be performed at 1:1 by the device (30 μl sample required) leading to a measuring range of 17–270 mg/L. On IMMAGE® 800, CSF volume needed is 300 μl. The initial measuring ranges are 9.3–167 mg/L and 37–278 mg/L for IgG and albumin, respectively while extended measuring ranges after rerun are 9.3–36,000 mg/L and 6.2–60,000 mg/L.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clinbiochem.2016.03.008.
References [1] C.H. Polman, S.C. Reingold, B. Banwell, M. Clanet, J.A. Cohen, M. Filippi, et al., Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria, Ann. Neurol. 69 (2011) 292–302. [2] W.I. McDonald, A. Compston, G. Edan, D. Goodkin, H.P. Hartung, F.D. Lublin, et al., Recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis, Ann. Neurol. 50 (2001) 121–127. [3] M. Andersson, J. Alvarez-Cermeno, G. Bernardi, I. Cogato, P. Fredman, J. Frederiksen, et al., Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report, J. Neurol. Neurosurg. Psychiatry 57 (1994) 897–902. [4] A. Bourahoui, J. De Seze, R. Guttierez, B. Onraed, B. Hennache, D. Ferriby, et al., CSF isoelectrofocusing in a large cohort of MS and other neurological diseases, Eur. J. Neurol. 11 (2004) 525–529. [5] T.B. Ledue, M.F. Collins, R.F. Ritchie, Development of immunoturbidimetric assays for fourteen human serum proteins on the Hitachi 912, Clin. Chem. Lab. Med. 40 (2002) 520–528. [6] A. Vassault, D. Grafmeyer, J. de Graeve, R. Cohen, A. Beaudonnet, J. Bienvenu, Quality specifications and allowable standards for validation of methods used in clinical biochemistry, Ann. Biol. Clin. 57 (1999) 685–695. [7] S. Ohman, J. Ernerudh, P. Forsberg, A. Henriksson, H. von Schenck, M. Vrethem, Comparison of seven formulae and isoelectrofocusing for determination of intrathecally produced IgG in neurological diseases, Ann. Clin. Biochem. 29 (Pt 4) (1992) 405–410. [8] L.M. Villar, J. Masjuan, M.C. Sadaba, P. Gonzalez-Porque, J. Plaza, A. Bootello, et al., Early differential diagnosis of multiple sclerosis using a new oligoclonal band test, Arch. Neurol. 62 (2005) 574–577. [9] M. Auer, H. Hegen, A. Zeileis, F. Deisenhammer, Quantitation of intrathecal immunoglobulin synthesis — a new empirical formula, Eur. J. Neurol. 25 (2016 Jan) (Epub ahead of print). [10] J. Mares, R. Herzig, K. Urbanek, V. Sladkova, J. Sklenarova, V. Bekarek, et al., Correlation of the IgG index and oligoclonal bands in the cerebrospinal fluid of patients with multiple sclerosis, Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 152 (2008) 247–249. [11] I. Mayringer, B. Timeltaler, F. Deisenhammer, Correlation between the IgG index, oligoclonal bands in CSF, and the diagnosis of demyelinating diseases, Eur. J. Neurol. 12 (2005) 527–530. [12] J. Lunding, R. Midgard, C.A. Vedeler, Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index, Acta Neurol. Scand. 102 (2000) 322–325.
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Short Communication
Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid Clarisse Benard 1, Jasmine Chauzeix 1, Antoine Blancher, Bénédicte Puissant-Lubrano ⁎ Laboratoire d'Immunologie, CHU de Toulouse, France
a r t i c l e
i n f o
Article history: Received 20 January 2016 Received in revised form 24 March 2016 Accepted 26 March 2016 Available online xxxx Keywords: Turbidimetry Analytical performances IgG Albumin Cerebrospinal fluid
a b s t r a c t Objectives: The intrathecal production of IgG is part of the diagnosis criteria for Multiple Sclerosis. Its assessment requires both quantitative (quantification of IgG and albumin in serum and cerebrospinal fluid (CSF)) and qualitative assays (isoelectric focusing). We have evaluated the analytical performances of the SPAPLUS® immunoturbidimeter (The Binding Site®) for the quantification of IgG and albumin in serum and CSF. Design and methods: Within-day and between-day precision, linearity and carry-over were assessed. Results obtained with SPAPLUS® were compared to those obtained with the nephelometer IMMAGE® 800, including albumin quotient and CSF IgG index. Isoelectric focusing was performed and considered as the gold standard for assessment of intrathecal production of IgG. Results: The within-day and the between-day precisions were obtained at two concentration levels and were below the recommendations of the manufacturer and of the French Society of Clinical Biology. Our evaluation confirmed the linearity of the assays and the absence of contamination. An agreement above 94% was observed between the results obtained with SPAPLUS® and those obtained with IMMAGE® 800. The use of the new reference material DA470k did not significantly modify IgG and albumin values. The confrontation of CSF IgG index and isoelectric focusing results led to a sensitivity of 79% and a specificity of 97% of CSF IgG index quantified on SPAPLUS® for the presence of oligoclonal bands at IEF. The sensitivity of intrathecal IgG calculated with the Reiber's hyperbolic formula was 47.4% and specificity was 97% for the presence of oligoclonal bands at IEF. Automatic rerun managed by the device for concentrations outside the measuring range was satisfactory. Conclusion: The SPAPLUS® immunoturbidimeter displays good analytical performances for the parameters evaluated in this work. © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
1. Introduction The intrathecal production of IgG is a useful parameter in case of Multiple Sclerosis (MS) suspicion to confirm the inflammatory nature of the disease and/or to exclude alternative diagnosis [1]. The intrathecal production of IgG is part of the diagnosis criteria for primary progressive Multiple Sclerosis which is characterized by a chronic progression from diagnosis without identified relapse [1,2]. It is therefore particularly indicated after one year of disease progression in association with the evidence of dissemination in space [1]. The evaluation of IgG production in cerebrospinal fluid (CSF) requires both quantitative and qualitative assays. The quantitative tests consist of quantifying IgG and albumin in serum and CSF. This allows the calculation of the albumin quotient (albumin CSF/albumin serum) which reflects the state of the blood–brain barrier, and that of CSF IgG index ([IgG CSF/albumin CSF]/[IgG serum/albumin serum]) which reflects ⁎ Corresponding author at: Laboratoire d'Immunologie, CHU Rangueil, 1, avenue Jean Poulhès, TSA 50032, 31059 Toulouse cedex 9, France. E-mail address: [email protected] (B. Puissant-Lubrano). 1 These authors have contributed equally to the work.
the abnormal intrathecal production of IgG. However, the sensitivity of the CSF IgG index is insufficient to affirm the intrathecal production of IgG [3,4], therefore the isoelectric focusing (IEF) of IgG in serum and CSF is required. Recent improvement of immunoturbidimetry led to consider this technique as a validated alternative to immunonephelometry, which is the reference method for the determination of specific proteins [5]. We have evaluated the analytical performances of SPAPLUS® immunoturbidimeter (The Binding Site) for the quantification of IgG and albumin in serum and CSF. This evaluation was performed according to CLSI (Clinical and Laboratory Standards Institute) protocols with minor modifications (between-day precision was assessed on 15 operating days and linearity was not assessed in duplicate).
2. Methods and results 2.1. Reagents For each parameter, reagent kits include a specific antiserum, a reaction buffer, six calibrators and two levels of internal quality control.
http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008 0009-9120/© 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
2
C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
Calibrators consist of mixtures of human sera. IgG and albumin assays are calibrated with the International DA470k reference material.
calculated as follows: (B1 − B3) ∗ 100 / (average H − B3). Alternating samples with high concentrations with low concentration samples did not reveal any contamination (Supplemental Table 2).
2.2. Evaluation of precision Albumin (Vialebex®, LFB, Courtaboeuf, France) and therapeutic immunoglobulins (TEGELINE®, LFB) were diluted at various concentrations in PBS (Gibco Life Technologies, Saint Aubin, France) to obtain samples in sufficient quantity, with concentrations corresponding to a normal serum, a normal CSF and a pathological CSF (i.e. with high albumin quotient and high CSF IgG index). These three samples were assayed 30 times the same day in the same series. The within-run precision in serum gave satisfactory results (Table 1) with CV less or equal to 3% which met the acceptability criteria of the manufacturer and of the French Society of Clinical Biology (SFBC) [6]. The CV obtained in CSF were b4% for IgG and albumin, leading to CV b 5.8% for albumin quotient and CSF IgG index. For each parameter, the between-day precision was evaluated on two levels of internal quality controls provided in the kits. They were quantified twice a day during 14 to 15 consecutive working days. CV were below 5% for all the parameters studied, including calculated indexes (Table 1) and are in accordance with the SFBC's and manufacturer's recommendations. 2.3. Evaluation of linearity Linearity was assessed in CSF obtained from two patients having IgG or albumin concentration close to the upper limit of the measuring range of SPAPLUS®. Because no sera were available with such characteristics, we used a mixture of albumin and therapeutic immunoglobulins for the assessment of linearity in serum. Six to seven diluted points covering the measuring range given by the supplier were quantified on SPAPLUS®. The linear regressions between measured concentrations and theoretical values had slopes near 1 and intercept close to zero, except for albumin in CSF. Excellent correlation coefficients (N0.995) were obtained (Supplemental Table 1). 2.4. Evaluation of carry-over The carry-over was evaluated by measuring three times a sample with a high level for a parameter (H1, H2, H3) and then a sample with a low level for this same parameter (B1, B2, B3). Carry-over was
2.5. Agreement with results from IMMAGE® 800 nephelometer Fifty-three coupled samples of sera and CSF were obtained from 53 patients suspected of Multiple Sclerosis, of neurodegenerative disease, or of other inflammatory diseases or infections of the central nervous system. These samples were residues of routine analysis performed at our laboratory. IgG and albumin were quantified the same day on SPAPLUS® and on nephelometer IMMAGE® 800 (the device routinely used in our laboratory, Beckman Coulter, Villepinte, France). The comparison was performed on ten operating days. On IMMAGE, the IgG and albumin assays are calibrated with the international reference material CRM470. The slopes of linear regressions were between 0.92 and 0.97 and the correlation coefficients were above 0.87 for the four analytes measured (Fig. 1A). With Bland and Altman analysis, IgG and albumin biases were below 0.3 g/L in serum and b2 mg/L in CSF (Fig. 1B). The agreement between the two assays was greater than 94% for the four measured parameters and N90% for the two calculated indexes. It is interesting to note that the use of the new reference material DA470k did not significantly modify IgG and albumin values. Agreement was also evaluated for classification as normal or elevated albumin quotient (N6.5) and CSF IgG index (N0.65) [7] (Supplemental Table 3). Agreements between SPAPLUS® and IMMAGE® 800 for albumin quotient and CSF IgG index were 92% and 89%, respectively (see details below). 2.6. Agreement with isoelectric focusing results Isoelectric focusing followed by IgG immunoblotting [8] was used as the reference technique for the assessment of intrathecal IgG production [3]. Six patients had normal CSF IgG index with results from SPAPLUS® while having elevated CSF IgG index with results from IMMAGE® 800. Among those, two patients had no oligoclonal bands (OCB) as determined by IEF (they had uveitis and unreported diagnosis, respectively), and four had OCB at IEF. All four had CSF IgG index obtained from SPAPLUS® very close to the threshold (values from 0.61 to 0.63 for a threshold at 0.65). Two of them suffered from Multiple Sclerosis.
Table 1 Evaluation of within-run and between-day precision. For each parameter, the table shows the number of determinations, the mean of obtained values, the standard deviation and the CV, as well as the acceptability criteria from the French Society of Clinical Biology (SFBC) [6] and from the manufacturer (TBS = The Binding Site). IQC: internal quality control. If not stated, the evaluation was performed on a patient sample. Precision
Samples
Parameter
N dosages
Mean
Standard deviation
CV (%)
Within-run
Serum
Serum (IQC)
IgG (g/L) Albumin (g/L) IgG (mg/L) Albumin (mg/L) Albumin quotient CSF IgG index IgG (mg/L) Albumin (mg/L) Albumin quotient CSF IgG index IgG (g/L)
Serum (IQC)
Albumin (g/L)
CSF (IQC)
IgG (mg/L)
CSF (IQC)
Albumin (mg/L)
30 30 30 30 30 30 30 30 30 30 28 28 29 29 30 30 29 29
6.5 32.45 16.39 190.18 5.87 0.43 68.88 380.41 11.73 0.91 7.1 13.6 30.07 58.74 29.8 85.8 325.8 752.5
0.14 0.99 0.64 6.37 0.27 0.02 1.85 8.15 0.46 0.04 0.28 0.40 1.25 2.12 1.2 2.9 9.35 27.32
2.08 3.05 3.89 3.35 4.68 5.77 2.69 2.14 3.95 4.93 3.86 2.96 4.16 3.61 4.04 3.38 2.87 3.63
Acceptability criteria SFBC
Normal CSF
Pathological CSF
Between-day
4.5 3.8
TBS 5 6 5 5
5 5
5 5 5 4
5 5 8 8 8 8 10 10
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
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Fig. 1. Agreement between SPAPLUS® and IMMAGE® 800. (A) For each parameter, the figure shows the correlation line (solid line) between the concentrations obtained with the reference laboratory technique (Nephelometer IMMAGE® 800) and the SPAPLUS®, as well as the line of identity (dotted line) between the two assays. (B) The figure shows the graphs of the differences (Bland and Altman test): each point represents the difference between the concentrations obtained by the two assays according to the average of determinations obtained by the two assays. The black line shows the ideal difference of 0 between the two assays. The blue line shows the average of the differences obtained and corresponds to the bias between the two assays. Blue dotted lines correspond to the lower and upper limits of the confidence interval at 95% of the differences between both assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Because only five patients among the 53 studied had a Multiple Sclerosis, it was not possible to calculate the sensitivity and the specificity of quantitative intrathecal IgG production for MS. We therefore calculated the sensitivity and the specificity of both CSF IgG index and intrathecal IgG calculated with Reiber's formula for the presence of OCB at IEF.
Indeed, the hyperbolic formula of Reiber can also be used to assess the intrathecal production of IgG and has shown superior performance than CSF IgG index (less false positive results) [7]. Among the 42 patients with normal intrathecal IgG with the Reiber's formula, ten had OCB (= false negative), the 32 remaining patients are true negative
Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008
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C. Benard et al. / Clinical Biochemistry xxx (2016) xxx–xxx
2.8. Management of automatic rerun Automatic reruns at a dilution different from the initial working dilution were triggered for CSF samples with concentrations of albumin (n = 9) or IgG (n = 4) outside the measuring range. Final results (from 94.6 to 167 mg/L for albumin and from 169 to 348 mg/L for IgG) were close to those obtained with IMMAGE® 800 (see the results of the agreement between the two assays).
3. Discussion In conclusion, the immunoturbidimeter SPAPLUS® provided consistent results for precision, linearity and contamination for IgG and albumin assays in serum and CSF. The automatic rerun was well managed by the device. The agreement of the SPAPLUS® results with those of the nephelometer IMMAGE® 800 was good, including the calculated albumin quotient and CSF IgG index. The CSF IgG index calculated from SPAPLUS® had good sensitivity and specificity for determining the presence of OCB at IEF. The SPAPLUS® is therefore a valuable tool to measure albumin and IgG in sera and CSF. This device allows the quantification of 49 biochemical and immunological parameters.
Fig. 2. IgG CSF/serum quotient diagrams with hyperbolic graphs according to Reiber. Areas are defined as follows: 1 normal; 2 blood-CSF barrier dysfunction; 3 intrathecal IgG synthesis plus blood-CSF barrier dysfunction; 4 intrathecal IgG synthesis; 5 methodological error. Triangles show patients with no oligoclonal bands at IEF while open squares show patients with presence of oligoclonal bands at IEF.
Acknowledgments The reagents were supplied by The Binding Site.
Appendix A. Supplementary data (Fig. 2). Among the 11 patients with elevated intrathecal IgG with the Reiber's formula, 10 had OCB at IEF (true positive), the remaining patient is a false positive. These results led to a sensitivity of 47.4% and a specificity of 97% of intrathecal IgG calculated from SPAPLUS® with the Reiber's formula for the presence of OCB at IEF. These results are slightly lower than previously reported as for sensitivity, but were similar as for specificity [9]. Among the 37 patients with normal CSF IgG index on SPAPLUS®, four had OCB (the same four detailed above with elevated CSF IgG index on IMMAGE = false negative), the 33 remaining patients are true negative. Among the 16 patients with elevated CSF IgG index on SPAPLUS®, 15 had OCB at IEF (93%, true positive), the remaining patient is a false positive. This frequency of true positive is slightly better than frequencies previously reported [10]. These results led to a sensitivity of 79% and a specificity of 97% of CSF IgG index calculated from SPAPLUS® for the presence of OCB at IEF. These results are better than those obtained by other studies [11,12]. This could be explained in part because we use a threshold of 0.65 [7] while Lunding and Mares used a threshold of 0.72 and 0.70, respectively. These results are consistent with the fact that CSF IgG index is less sensitive than IEF to assess intrathecal production of IgG [12]. 2.7. Practical characteristics of the assays The CSF volume needed for the analysis on SPAPLUS® is 215 μl. This includes 35 μl + 30 μl for IgG and albumin quantification, respectively, and 150 μl of dead-volume (when using tubes, 120 μl if using cups). The initial measuring ranges are 4–135 mg/L for IgG (no dilution on the device) and 170–2700 mg/L for albumin (dilution 1/10). For IgG, rerun can be performed at 1:10 by the device (30 μl sample required) leading to a measuring range of 40–1350 mg/L. For albumin, rerun can be performed at 1:1 by the device (30 μl sample required) leading to a measuring range of 17–270 mg/L. On IMMAGE® 800, CSF volume needed is 300 μl. The initial measuring ranges are 9.3–167 mg/L and 37–278 mg/L for IgG and albumin, respectively while extended measuring ranges after rerun are 9.3–36,000 mg/L and 6.2–60,000 mg/L.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clinbiochem.2016.03.008.
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Please cite this article as: C. Benard, et al., Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid, Clin Biochem (2016), http://dx.doi.org/10.1016/j.clinbiochem.2016.03.008