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Measuring carbonic anhydrase IX as a hypoxia biomarker: differences in concentrations in serum and plasma using a commercial enzyme-linked immunosorbent assay due to influences of metal ions.

Wind TC, Messenger MP, Thompson D, Selby PJ, Banks RE - Ann. Clin. Biochem. (2011)

Bottom Line: This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay.These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid.This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

ABSTRACT

Background: There is increasing interest in measuring the soluble forms of carbonic anhydrase IX (CA IX) in blood as a marker of hypoxia for prognostic purposes or for predictive use in therapeutic trials in various cancers. Following our initial observations of marked differences in the measured concentrations of CA IX in EDTA plasma versus serum, we sought to investigate these further in order to determine their effects on results in published studies and to ensure accurate measurement in future studies.

Methods: Serum and EDTA plasma samples from healthy controls and patients with renal cancer were used in the validation of two commercially available enzyme-linked immunosorbent assays (ELISAs) for CA IX with examination of recovery, parallelism and specificity and comparison of paired plasma and serum.

Results: Successful validation of one of the ELISAs was not achieved with particular problems with parallelism and marked differences in measured CA IX concentrations between EDTA plasma and serum. This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay. The other commercially available ELISA examined was successfully validated and showed no difference in CA IX between EDTA plasma and serum.

Conclusions: These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid. This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

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Related in: MedlinePlus

Graphs showing the relationship between concentrations of CA IX in EDTA plasma or serum (n = 15 pairs) assessed using either the (a) Siemens or (b) R&D assays. The dotted line shows the line of equivalence with a slope of 1. (c) Effects of adding 20 mmol/L CaCl2 to EDTA plasma or of adding 1.8 mg/mL EDTA to serum on the CA IX concentrations as measured using the Siemens assay. (d) The reversibility of the effect is shown by sequential addition of CaCl2 and EDTA to EDTA plasma or recombinant CA IX. CA, carbonic anhydrase; rCA, recombinant CA
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ACB-10-240F3: Graphs showing the relationship between concentrations of CA IX in EDTA plasma or serum (n = 15 pairs) assessed using either the (a) Siemens or (b) R&D assays. The dotted line shows the line of equivalence with a slope of 1. (c) Effects of adding 20 mmol/L CaCl2 to EDTA plasma or of adding 1.8 mg/mL EDTA to serum on the CA IX concentrations as measured using the Siemens assay. (d) The reversibility of the effect is shown by sequential addition of CaCl2 and EDTA to EDTA plasma or recombinant CA IX. CA, carbonic anhydrase; rCA, recombinant CA

Mentions: Comparison of EDTA plasma and serum concentrations of CA IX in 15 matched pair samples showed significantly higher concentrations in EDTA plasma samples compared with serum (P < 0.001) when measured using the Siemens assay and although a significant correlation was seen (P < 0.001; r2 = 0.961), the slope of the line was only 0.538 with several samples lying distantly (Figure 3a). Using the Siemens assay, concentrations in EDTA plasma ranged from 34.5 to 1476.4 pg/mL (mean 305.8 pg/mL), while concentrations in serum were <2.5 to 770.6 pg/mL (mean 127.9 pg/mL). In contrast, no significant difference between the two sample types was found using the R&D assay and a significant correlation (P < 0.001; r2 = 0.998) and a slope of 0.905 were seen (Figure 3b). Using the R&D assay, concentrations in EDTA plasma were 17.7 to 482.9 pg/mL (mean 91.3 pg/mL) and concentrations in serum were 18.2 to 436.6 pg/mL (mean 87.4 pg/mL).


Measuring carbonic anhydrase IX as a hypoxia biomarker: differences in concentrations in serum and plasma using a commercial enzyme-linked immunosorbent assay due to influences of metal ions.

Wind TC, Messenger MP, Thompson D, Selby PJ, Banks RE - Ann. Clin. Biochem. (2011)

Graphs showing the relationship between concentrations of CA IX in EDTA plasma or serum (n = 15 pairs) assessed using either the (a) Siemens or (b) R&D assays. The dotted line shows the line of equivalence with a slope of 1. (c) Effects of adding 20 mmol/L CaCl2 to EDTA plasma or of adding 1.8 mg/mL EDTA to serum on the CA IX concentrations as measured using the Siemens assay. (d) The reversibility of the effect is shown by sequential addition of CaCl2 and EDTA to EDTA plasma or recombinant CA IX. CA, carbonic anhydrase; rCA, recombinant CA
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104817&req=5

ACB-10-240F3: Graphs showing the relationship between concentrations of CA IX in EDTA plasma or serum (n = 15 pairs) assessed using either the (a) Siemens or (b) R&D assays. The dotted line shows the line of equivalence with a slope of 1. (c) Effects of adding 20 mmol/L CaCl2 to EDTA plasma or of adding 1.8 mg/mL EDTA to serum on the CA IX concentrations as measured using the Siemens assay. (d) The reversibility of the effect is shown by sequential addition of CaCl2 and EDTA to EDTA plasma or recombinant CA IX. CA, carbonic anhydrase; rCA, recombinant CA
Mentions: Comparison of EDTA plasma and serum concentrations of CA IX in 15 matched pair samples showed significantly higher concentrations in EDTA plasma samples compared with serum (P < 0.001) when measured using the Siemens assay and although a significant correlation was seen (P < 0.001; r2 = 0.961), the slope of the line was only 0.538 with several samples lying distantly (Figure 3a). Using the Siemens assay, concentrations in EDTA plasma ranged from 34.5 to 1476.4 pg/mL (mean 305.8 pg/mL), while concentrations in serum were <2.5 to 770.6 pg/mL (mean 127.9 pg/mL). In contrast, no significant difference between the two sample types was found using the R&D assay and a significant correlation (P < 0.001; r2 = 0.998) and a slope of 0.905 were seen (Figure 3b). Using the R&D assay, concentrations in EDTA plasma were 17.7 to 482.9 pg/mL (mean 91.3 pg/mL) and concentrations in serum were 18.2 to 436.6 pg/mL (mean 87.4 pg/mL).

Bottom Line: This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay.These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid.This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

View Article: PubMed Central - PubMed

Affiliation: Clinical and Biomedical Proteomics Group, Cancer Research UK Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK.

ABSTRACT

Background: There is increasing interest in measuring the soluble forms of carbonic anhydrase IX (CA IX) in blood as a marker of hypoxia for prognostic purposes or for predictive use in therapeutic trials in various cancers. Following our initial observations of marked differences in the measured concentrations of CA IX in EDTA plasma versus serum, we sought to investigate these further in order to determine their effects on results in published studies and to ensure accurate measurement in future studies.

Methods: Serum and EDTA plasma samples from healthy controls and patients with renal cancer were used in the validation of two commercially available enzyme-linked immunosorbent assays (ELISAs) for CA IX with examination of recovery, parallelism and specificity and comparison of paired plasma and serum.

Results: Successful validation of one of the ELISAs was not achieved with particular problems with parallelism and marked differences in measured CA IX concentrations between EDTA plasma and serum. This appeared to be due to a metal ion-dependent epitope on CA IX recognized by the detection antibody in this assay. The other commercially available ELISA examined was successfully validated and showed no difference in CA IX between EDTA plasma and serum.

Conclusions: These results have important consequences for published studies using this assay where the conclusions drawn from the measurements made may be invalid. This study highlights the need for stringent validation of commercially available assays, including examination of various sample types, before use in research studies.

Show MeSH
Related in: MedlinePlus