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 (σ)
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 (G99) and the depth D99 at which this is reached. For typical small
diameter probes (outer electrode diameter ∼ <2 mm), D99 is small enough for dip-type measurements
in extract volumes of ∼100 μL. 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 μL aqueous
extracts of 2 μL of spotted blood showed a variance of 17.9%.
For a given subject, methanol extracts of DBS spots nominally containing
8 and 4 μL 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 ∼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.