A device for dried blood microsampling in quantitative bioanalysis Methodology
Gravimetric determination of blood volume absorbed Prior
to performing the blood absorption experi-
ments, the balance and weighing areas were cleaned and dried to ensure there was no loose liquid on the balance plate, as evaporation would lead to poor bal- ance stability. Moreover, to improve precision during weighing, vials were placed in the same place on the balance. Finally, to increase humidity and reduce the rate of evaporation, a wet tissue was placed in a beaker and placed within the balance enclosure, but not on the balance plate. Before weighing, each balance was set to zero and a
2-ml aliquot of the 20, 45 or 65% (including 60 and 69% for two laboratories) HCT blood was placed in a scintillation vial, which was capped and placed in the center of the balance plate. To determine the weight of an accurately pipet-
ted volume (control protocol) at each HCT level, the initial weight of a capped vial containing 2 ml of blood was recorded. The vial was then uncapped and 10 μl of blood was removed using a pipette. The vial was then recapped, reweighed and the weight was recorded. This procedure was conducted six times per HCT at each laboratory. Once completed, the average density of each HCT at each laboratory was calculated as follows:
Blood density(ng/ml) 10
=
Mean weight of blood in 10 laliquot (g) 1000 µ
# To determine the weight of the blood absorbed by
the VAMS tip at each HCT level (VAMS protocol), the initial weight of a capped vial containing 2 ml of blood was recorded. The vial was then uncapped and a fresh VAMS was carefully ‘dipped’ into the blood such that only the tip was exposed to the blood. The tip was held in the blood until it appeared to be full (i.e., no white portions were observed) and then for an extra approximately 2 s before being removed. Care was taken when filling the tips not to immerse the tip past the shoulder, as this could result in excess blood being retained and hence introduce a positive bias to any measurements. Furthermore, upon removing the tip after filling, care was taken to ensure the sampler or tip had not contacted the walls of the vial, as this could have caused errors in the result. The VAMS was then discarded. As in the control experiment, each vial was recapped, reweighed and the weight was recorded. This procedure was conducted six times per HCT at each laboratory. The blood volume absorbed on the VAMS tip for each HCT at each laboratory was calculated as follows:
future science group
Tip shoulder
Handle Absorbant tip
Figure 1. Volumetric absorbtive microsampler before (left) and after (right) filling with blood.
Volume of bloodinVAMSTip (µl) Mean weight of bloodin10l aliquot(g)
=
Mean weight of blood absorbed by VAMS Tip(g) µ
Laboratories 1, 4, 5 and 6 performed the operation to
determine the mean weight of blood in a 10-μl aliquot for the above experiments as described earlier, while laboratories 2 and 3 performed the operation by weigh- ing a vial containing 2 ml of blood and then dispensed 10-μl aliquots of blood into it from a separate vial con- taining blood at the same HCT as that being weighed, and reweighing the target vial after each addition.
Results & discussion Volume of blood absorbed at different HCTs The volume of human and rat blood with different HCTs absorbed by the VAMS was investigated in six different laboratories by determining changes in weight after dipping the device into control blood (Figure 2). The average volume of human or rat blood collected across the approximate HCT range of 20–65% was 10.6 ± 0.4 μl (error determined as [2 × standard devia- tion]/square root of the count). This is in good agree- ment with the 10.5 ± 0.1 μl determined using radio- active 14
by measurement of CO2
C caffeine spiked into human blood followed after oxidation of the dried
tip [26]. Furthermore, the slope of the blood volume against HCT plot is close to zero, demonstrating that the blood volume collected by the tip showed little variance with different blood HCT values. The variability in the blood volume collected across
laboratories and HCTs was minimal (CV: 8.7%) and well within the 15% that is routinely accepted for the validation of quantitative bioanalyical methods. However, there was notable variance in the minimum and maximum volumes absorbed (between 9.1 and 13.1 μl). While the reasons for this variance are not
www.future-science.com 655
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128 |
Page 129 |
Page 130 |
Page 131 |
Page 132 |
Page 133 |
Page 134 |
Page 135 |
Page 136 |
Page 137 |
Page 138 |
Page 139 |
Page 140 |
Page 141 |
Page 142 |
Page 143 |
Page 144 |
Page 145 |
Page 146 |
Page 147 |
Page 148 |
Page 149 |
Page 150 |
Page 151 |
Page 152 |
Page 153 |
Page 154