The effect of sample storage on the performance and reproducibility of the galactomannan EIA test

Abstract

Galactomannan enzyme immune assay (GM EIA) is a nonculture test for detecting invasive aspergillosis (IA) forming a key part of diagnosis and management. Recent reports have questioned the reproducibility of indices after sample storage. To investigate this, 198 serum samples (72 from cases and 126 from controls) and 61 plasma samples (24 from cases and 37 from controls), initially tested between 2010 and 2013, were retested to determine any change in index. Data were also collected on circulatory protein levels for false-positive serum samples. Serum indices significantly declined on retesting (median: initial, 0.50, retest, 0.23; P < 0.0001). This was shown to be diagnosis dependent as the decline was apparent on retesting of control samples (median: initial 0.50, retest 0.12; P< 0.0001), but was not evident with case samples (median: initial, 0.80, retest, 0.80; P = 0.724). Plasma samples showed little change on reanalysis after long-term storage at 4°C. Retesting after freezing showed a decrease in index values for controls (median: initial 0.40, retest 0.26; P = 0.0505), but no significant change in cases. Circulatory proteins showed a correlation between serum albumin concentration and difference in index value on retesting. Overall, this study suggests that a lack of reproducibility in GM EIA positivity is only significant when disease is absent. Retesting after freezing helps to differentiate false-positive GM EIA results and, with consecutive positivity, could help to improve accuracy in predicting disease status. The freezing of samples prior to testing could potentially reduce false-positivity rates and the need to retest.

Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

“…As these patients had no other evidence of invasive fungal disease, these samples represent falsepositive results. The change in qualitative results for these four samples was not likely a result of DS2 automation but a result of the variability in measured ODIs seen for false-positive samples, which was reported in a similar study (11). A lack of repeatability for samples with ODI values close to the positivity threshold of the assay was also reported by Marr et al, as 10.2% of positive serum samples included in their study, all with ODIs of 0.5 to 0.7, tested negative on repeat testing (8).…”

“…The storage conditions of specimens appear to have an impact on reproducibility, with a significant decline in the sample ODI reported after storage at Ϫ80°C (11). An IA diagnosis also appears to be important, with nonreproducibility observed more frequently for retesting of false-positive samples from patients without IA (11,12). A significant correlation between the serum albumin concentration and the difference in ODI value on retesting was also recently reported.…”

“…A significant correlation between the serum albumin concentration and the difference in ODI value on retesting was also recently reported. A larger reduction in indices was observed on retesting sera with increasing albumin concentrations (11).…”

“…In particular, samples with an optical density index (ODI) at or around the positivity threshold (Х0.5) of the assay were regularly found to be negative on repeat testing (8,11). The storage conditions of specimens appear to have an impact on reproducibility, with a significant decline in the sample ODI reported after storage at Ϫ80°C (11).…”

“…In particular, samples with an optical density index (ODI) at or around the positivity threshold (Х0.5) of the assay were regularly found to be negative on repeat testing (8,11). The storage conditions of specimens appear to have an impact on reproducibility, with a significant decline in the sample ODI reported after storage at Ϫ80°C (11). An IA diagnosis also appears to be important, with nonreproducibility observed more frequently for retesting of false-positive samples from patients without IA (11,12).…”