Results show that the NGF was present in aqueous humor in a concentration below 1 pg/ml. These levels are below the sensitivity limit of the kit used, thus they were considered undetectable. However, since the standard curve as well as external positive control values were linear below the 7.8 pg/ml sensitivity levels, it is possible that the detected levels represent actual NGF values. The results from aqueous samples are considered reliable because the positive controls give predictable values. Quantity of sample size available from patients posed a problem; however, even when 100 μL of aqueous was available the NGF levels were undetectable. The same experiment was performed two more times with additional samples and the results were comparable. This is consistent with low quantities of proteins present in aqueous [5].
Several methods have been described in the literature to enhance detectability of a protein [6]. Even in the presence of low levels of starting proteins, sample dilutions, possibly by diluting other interfering proteins, can some times improve detectability. However, even with dilution in pilot studies NGF levels in aqueous remained below sensitivity levels. Methods have been described to extract NGF in samples by acidification. This method requires acid treatment and readjustment of the pH [7]. Pretreatment or pH shock extraction was not feasible because of the small sample size. Other possibility is to concentrate the sample with freeze-dry method. This method could only be used if we pooled the samples from different patients. This will however preclude our aim of detecting NGF levels in individual subjects.
The samples with higher values were considered outliers (with respect to patient groups of control and POAG) because (1) these values were rare and highly inconsistent with others in the group (2) they were found with equal frequency in control and glaucomatous eyes. Histories of outliers were reviewed to identify any apparent cause but none was identified.
Our results highlight the difficulties in assessing NGF levels in aqueous. An alternate strategy could be to pool the samples from different patients (but from same group) in order to increase the sample volume and therefore the detectability. However, results also show that outliers may skew the results obtained by pooling the samples. The outliers may be a result of variables at the time of collection of sample (e.g. per operative trauma, bleeding, iris handling etc). There are reports in the literature where NGF levels have been assessed in animals [3, 8] and in humans [1].
Our result highlights the limitations in collection of 100 μl of human aqueous sample for Elisa assay, to determine the nerve growth factor levels. This result differs from limited reports where NGF levels have been reported in aqueous samples [1, 3, 8]. However, in these reports methodological details are not clear if 100 microlitres of aqueous was obtained by pooling the samples. Considering these limitations, other technologies such as multiplex assays might be more useful.