There has been a great expansion in literature on bloodspots in the past few years, driven primarily by interest of pharma to investigate utility of such specimens in animal studies and clinical trials (1). Interpatient variation in hematocrit, however, has been identified as a preanalytical factor that often can influence quantitative bloodspot analyses (2). There can be many reasons for this, but one, for instance, is the influence of hematocrit to affect the volume of blood per area of a filter paper bloodspot. A given punch size may not carry the same volume of blood across patients, because higher hematocrit = smaller bloodspot area per volume of blood applied (3).
Capiau et al. (4) recently demonstrated success in estimation of sample hematocrit using K+ extracted from bloodspot punches. We recently performed a follow-on to their work, presented as an abstract at AACC (5). Our follow-on was based on a simple hypothesis: given that red cells accrete at the perimeter of filter paper bloodspots, measurement of K+ from the perimeter should provide higher resolution of the relationship of K+ to hematocrit than can measurement from center punch samples.
Perimeter accretion of red cells in filter paper bloodspots is an interesting phenomenon. It is consistent with a form of size exclusion chromatography (6). Simply put, the liquid fraction of whole blood (plasma) can occupy a greater fractional volume of the interior of the filter paper substrate than can red cells; the net effect is that red cells become more and more concentrated at the leading edge of a bloodspot as it spreads during formation. This discontinuity in red cell concentration at the perimeter is often readily visible in dried bloodspots. The effect is certainly measurable; for instance, the concentration of lead (Pb), a red cell-associated analyte, is at least 1.5x greater at the perimeter of a bloodspot than at the center (7).
On this basis, we investigated the relationship of K+/area between perimeter and center bloodspot samples. Perimeter samples were annular specimens – donut-shaped specimens remaining after removal of the majority of the bloodspot interior via punch. The comparison between perimeter and center punch specimens was similar to results for Pb: there was a 1.5x greater concentration (K+/area) in the perimeter specimens than in the center punch. For hematocrit determination, this meant simply that there was higher resolution of the correlation of K+/area to hematocrit for the perimeter sample data, and lesser residual error, than existed for the center punch data.
The upshot of the study is simply this: for estimation of hematocrit via K+ measurement, a perimeter sample rather than a center punch sample is likely to be superior for this purpose. Thus, there is a potential use for a “usedâ€, remainder bloodspot. It is important to note, however, that use of a new, simple device for volumetric application of blood to filter paper to enable whole-spot analysis may in future obviate any need to estimate sample hematocrit from bloodspots (8).
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