Biomedical Engineering Reference
In-Depth Information
3.3.2
Paper/Card Dependent Distribution of Analytes on Dried Blood Spots
There are various factors that can influence the distribution of analytes in a dried
blood spot. Water-soluble chemicals uniformly coated on DBS cards would redis-
tribute when the blood was spotted. The redistribution of chemicals may depend on
their properties, viscosity of blood, the volume spotted, and the technique used for
spotting. Another factor is the viscosity of the blood. Viscosity is normally depen-
dent on the blood composition (hematocrit, protein, lipid levels), and it can affect
the physical spread of the blood spot in that the same volume of a less viscous blood
will form a larger diameter spot than that of a more viscous blood sample. Viscosity,
combined with the chemical redistribution on the sample cards, will increase the
complexity of the analyte distribution.
In order to study the radial distribution of the six analytes on dried blood spots,
smaller DBS punches (1 mm. i.d.) were extracted and quantified against blood stan-
dards. Six DBS 1 mm punches were taken from the centers of six separate spots and
combined into one well of a 96-well plate. The next DBS punch was taken adjacent to
the center one and each subsequent punch was taken adjacent to the previous one radi-
ally moving outward towards the edge as shown in Fig.
4
. Additional punch was taken
from just outside of the visual edge of the blood spot as a control sample to verify that
analytes are not moving outside of the visual spot. All results from the control samples
confirmed the absence of the tested compounds outside of the visual border of spots.
Three types of paper/card and six compounds were used to evaluate the paper impact on
the analyte distribution, but only FTA Elute and VWR 237 cards are presented here.
Figure
5
shows the radial distribution of analytes on FTA Elute cards and VWR
237 paper using two spotting volumes (15 and 50 mL). The general impression from
the obtained results is that FTA Elute cards show a more significant change in concen-
tration across the spot especially when crossing the line between the darker and lighter
(halo) regions of the spot. The VWR 237 paper shows minimal change in distribution
of the analytes across the spots. One observation relates to the distribution of the ana-
lytes within the dark center of the spots on the FTA Elute cards where the higher log
D
compound F shows a gradual change upward and then downward in concentration
from center to the edge of the dark circle as shown in Fig.
5b
(for both 15 and 50 m L
spotting volumes), while the low log
D
compound A shows no obvious upward or
downward change as shown in Fig.
5a
. This was also seen for the rest of the com-
pounds studied when looking at their log
D
values. It is possible that this phenomenon
may be due to the redistribution of the water-soluble chemical components of the
paper, as previously described, as well as to the specific properties of the analytes.
3.3.3
Paper/Card Dependent Matrix Effect
Accurate quantitation of analytes requires consistent measurements independent
from matrix lot variation. Matrix lot to lot variation becomes critical for successfully
implementing DBS. In addition to the analyte distribution across dried blood spots,
matrix effect was also evaluated. In addition to the blood pool used for standards and
