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 “used”, 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).
- Spooner N. A glowing future for dried blood spot sampling. Bioanalysis 2010;2:1343-4.
- De Kesel PM, Sadones N, Capiau S, Lambert WE, Stove CP. Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions. Bioanalysis 2013;5:2023-41.
- McCloskey LJ, Yoo JH, Stickle DF. Interpatient distributions of bloodspot area per fixed volume of application: comparison between filter paper and non-cellulose dried matrix spotting cards. Clin Chim Acta 2014;437:187-90.
- Capiau S, Stove VV, Lambert WE, Stove CP. Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer. Anal Chem 2013;85:404-10.
- Rufail ML, McCloskey LJ, Stickle DF. Potassium vs. hematocrit in filter paper bloodspots: effect of large differences in potassium per unit area between center circular punch samples vs. outer annular remainder samples. AACC, Atlanta, July 2015, #A-131.
- Stickle DF, Rawlinson NJ, Landmark JD. Increased perimeter red cell concentration in filter paper bloodspot samples is consistent with constant-load size exclusion chromatography occurring during application. Clin Chim Acta 2009;401:42-5.
- El-Hajjar DF, Swanson KH, Landmark JD, Stickle DF. Validation of use of annular once-punched filter paper bloodspot samples for repeat lead testing. Clin Chim Acta 2007;377:179-84.
- Leuthold LA, Heudi O, Déglon J, Raccuglia M, Augsburger M, Picard F, Kretz O, Thomas A. New microfluidic-based sampling procedure for overcoming the hematocrit problem associated with dried blood spot analysis. Anal Chem 2015;87:2068-71.