Prediction of the Hematocrit of Dried Blood Spots via Potassium Measurement on a Routine Clinical Chemistry Analyzer

Abstract

The potential of dried blood spot (DBS) sampling as an alternative for classical venous sampling is increasingly recognized, with multiple applications in, e.g., therapeutic drug monitoring and toxicology. Although DBS sampling has many advantages, it is associated with several issues, the hematocrit (Hct) issue being the most widely discussed challenge, given its possible strong impact on DBS-based quantitation. Hitherto, no approaches allow Hct prediction from nonvolumetrically applied DBS. Following a simple and rapid extraction protocol, K(+) levels from 3 mm DBS punches were measured via indirect potentiometry, using the Roche Cobas 8000 routine chemistry analyzer. The extracts' K(+) concentrations were used to calculate the approximate Hct of the blood used to generate DBS. A linear calibration line was established, with a Hct range of 0.19 to 0.63 (lower limit of quantification, LLOQ, to upper limit of quantification, ULOQ). The procedure was fully validated; the bias and imprecision of quality controls (QCs) at three Hct levels and at the LLOQ and ULOQ was less than 5 and 12%, respectively. In addition, the influence of storage (pre- and postextraction), volume spotted, and punch homogeneity was evaluated. Application on DBS from patient samples (n = 111), followed by Bland and Altman, Passing and Bablok, and Deming regression analysis, demonstrated a good correlation between the "predicted Hct" and the "actual Hct". After correcting for the observed bias, limits of agreement of ±0.049 were established. Incurred sample reanalysis demonstrated assay reproducibility. In conclusion, potassium levels in extracts from 3 mm DBS punches can be used to get a good prediction of the Hct, one of the most important "unknowns" in DBS analysis.

Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots

“…Lower DBS concentrations were found at low Hct levels, while higher concentrations were measured at high Hct levels, with differences ranging from -29.3 to 25.4 %. A similar pattern of decreasing uncorrected DBS concentrations with decreasing Hct was observed when differences (%) were plotted against calculated Hct values (Figure 2b), illustrating the validity of K + -based Hct prediction [7].…”

“…Fig. 1 Hematocrit (Hct) distribution of blood samples collected from healthy volunteers and hospital patients in which caffeine (a) and paraxanthine (b) concentrations were measured *Calculated Hct: Hct of the blood used to prepare the DBS, as calculated using the K+ concentration measured in the DBS [7] 12 …”

“…Further details of the analytical methods are described elsewhere [9]. K + concentrations in DBS extracts were measured by indirect potentiometry, using the ionselective electrode of a Roche Cobas 8000 routine chemistry analyzer (Roche Diagnostics, Mannheim, Germany), as previously described [7]. K + was extracted from 3-mm DBS punches by two-fold addition of 50 µL of a 2.5 mM KCl solution in ultrapure water and shaking for 5 minutes.…”

“…To this end, a reliable marker is required that allows to predict the Hct of the blood used to prepare a given DBS. We previously demonstrated that potassium (K + ), an endogenous compound that correlates with the amount of red blood cells, allows to predict the Hct over a wide range with acceptable accuracy and precision [7]. This method was thoroughly validated and application on real patient samples showed a good correlation between the estimated Hct (based on the K + content) and the real Hct.…”

Why Dried Blood Spots Are an Ideal Tool for CYP1A2 Phenotyping

“…To evaluate the effect of varying hematocrit (Hct) on the concentration of caffeine and paraxanthine, we prepared blood samples with Hct values of 0.20, 0.25, 0.30, 0.43, 0.54 and 0.60 as described before [27]. These were used to prepare low and high quality control samples (QCs) versus peripheral punches (n=6) using an independent samples t-test (α=0.05).…”