Estimation of hematocrit in filter paper dried bloodspots by potassium measurement: advantage of use of perimeter ring samples over circular center sub-punch samples

Quantitative assays using dried filter paper bloodspots (DBS) may be adversely affected by hematocrit (HCT) as an unknown variable. Studies have demonstrated the utility of the measurement of potassium (K) from DBS punches to estimate HCT. Because there is significant accrual of RBCs at the DBS perimeter, we investigated whether K measurement from ring-shaped specimens inclusive of the perimeter might provide an advantage over conventional interior circular sub-punch samples to estimate HCT. Primary samples were Li-heparin whole blood, with HCT as measured on concurrently-drawn K-EDTA specimens. DBS were formed by bolus addition of 40 μL whole blood to filter paper cards. Total bloodspot area was determined by image analysis. Removal of center sub-punch (P) samples of fixed area produced remainder ring (R) samples inclusive of the perimeter. Samples were extracted in K-EDTA (2.5 mmol/L) and measured for diluent-corrected K per area (α, μmol K/cm2). Forty-three patient samples were utilized. α was normally distributed: α(P)=1.23±0.26 μmol K/cm2; α(R)=1.86±0.41 μmol K/cm2; α(R)/α(P)=1.51±0.15. α was correlated with HCT: α(P)=0.030 HCT(%)+0.015 μmol K/cm2 (r2=0.795); α(R)=0.052 HCT(%)+0.010 μmol K/cm2 (r2 = 0.912), but with higher resolution and lesser error for α(R). K per area (α) was significantly higher in R samples vs. P samples, with higher resolution for α(R) vs. HCT. Use of ring samples inclusive of the perimeter to estimate HCT for DBS via K measurement can provide an advantage over use of center sub-punch samples.