Evaluation of Amount of Blood in Dry Blood Spots: Ring-Disk Electrode Conductometry

A fixed area punch in dried blood spot (DBS) analysis is assumed to contain a fixed amount of blood, but the amount actually depends on a number of factors. The presently preferred approach is to normalize the measurement with respect to the sodium level, measured by atomic spectrometry. Instead of sodium levels, we propose electrical conductivity of the extract as an equivalent nondestructive measure. A dip-type small diameter ring-disk electrode (RDE) is ideal for very small volumes. However, the conductance (G) measured by an RDE depends on the depth (D) of the liquid below the probe. There is no established way of computing the specific conductance (a) of the solution from G. Using a COMSOL Multiphysics model, we were able to obtain excellent agreement between the measured and the model predicted conductance as a function of D. Using simulations over a large range of dimensions, we provide a spreadsheet-based calculator where the RDE dimensions are the input parameters and the procedure determines the 99% of the infinite depth conductance (G(99)) and the depth D-99 at which this is reached. For typical small diameter probes (outer electrode diameter similar to <2 mm), D-99 is small enough for dip-type measurements in extract volumes of similar to 100 mu L. We demonstrate the use of such probes with DBS extracts. In a small group of 12 volunteers (age 20-66), the specific conductance of 100 mu L aqueous extracts of 2 mu L of spotted blood showed a variance of 17.9%. For a given subject, methanol extracts of DBS spots nominally containing 8 and 4 mu L of blood differed by a factor of 1.8-1.9 in the chromatographically determined values of sulfate and chloride (a minor and major constituent, respectively). The values normalized with respect to the conductance of the extracts differed by similar to 1%. For serum associated analytes, normalization of the analyte value by the extract conductance can thus greatly reduce errors from variations in the spotted blood volume/unit area.