Simultaneous assessment of blood coagulation and hematocrit levels in dielectric blood coagulometry

Abstract

Background: In a whole blood coagulation test, the concentration of any in vitro diagnostic agent in plasma is dependent on the hematocrit level but its impact on the test result is unknown.

Objective: The aim of this work was to clarify the effects of reagent concentration, particularly Ca2+, and to find a method for hematocrit estimation compatible with the coagulation test.

Methods: Whole blood coagulation tests by dielectric blood coagulometry (DBCM) and rotational thromboelastometry were performed with various concentrations of Ca2+ or on samples with different hematocrit levels. DBCM data from a previous clinical study of patients who underwent total knee arthroplasty were re-analyzed.

Results: Clear Ca2+ concentration and hematocrit level dependences of the characteristic times of blood coagulation were observed. Rouleau formation made hematocrit estimation difficult in DBCM, but use of permittivity at around 3 MHz made it possible. The re-analyzed clinical data showed a good correlation between permittivity at 3 MHz and hematocrit level (R2=0.83).

Conclusions: Changes in the hematocrit level may affect whole blood coagulation tests. DBCM has the potential to overcome this effect with some automated correction using results from simultaneous evaluations of the hematocrit level and blood coagulability.

Keywords: Dielectric spectroscopy; interfacial polarization; rotational thromboelastometry; rouleau formation; whole blood coagulation test.

Fig. 1.

A typical dielectric spectrum (dielectric dispersion curve) of whole blood from a healthy volunteer before the blood coagulation process proceeds (a), and the change in normalized dielectric spectra during the progression of blood coagulation (b). The normalization was done with the dielectric dispersion curve at the first time point.

Fig. 2.

Dielectric permittivity changes at 10 MHz with blood coagulation for the same blood sample with different final concentrations of reagent (Ca²⁺) at 6.3 mM (black circles), 12.5 mM (blue squares), and 21.9 mM (red triangles). The permittivity data are normalized by the minimum and maximum values of the permittivity, and ${\mathit{t}}_{\mathit{x}}$ was determined as the intersection of two extrapolated lines (dashed lines).

Fig. 3.

Reagent (Ca²⁺) concentration dependences of ${\mathit{t}}_{\mathit{x}}$ obtained by DBCM (asterisks and circles) or clotting time (CT) by ROTEM^® (squares). The colors of the symbols indicate three individual subjects, where blue asterisks and blue circles show the data for the same subject but on different dates of experiments.

Fig. 4.

Rouleau formation and dielectric response observed in a previous study [3]. Panel (a) shows the dielectric dispersion curves just after pipet mixing and 5 min after. Panels (b), (c), and (d) show changes of the normalized permittivity at 10, 3, and 1 MHz, respectively. The normalization was done with permittivity at the first time point. The measurements of these changes were started just after mixing was stopped and started again after resuspension, three times in a row. The arrows indicate the points at which mixing was stopped. (Rearranged from [3] under the open access license granted by American Chemical Society.)

Fig. 5.

Conductivity of blood samples (${\mathit{\sigma }}_{\mathit{s}}$) at 50 kHz observed 1 minute from the start of DBCM measurement against the hematocrit level for patients who underwent total knee arthroplasty [7] (full square). The solid line shows the result of linear regression where the square of the coefficient of correlation (${\mathit{R}}^2$) is 0.76. The dashed curve shows the relationship of conductivity and cell volume fraction in Hanai’s theory (Eq. (2)) assuming constant conductivity of the supernatant as ${\mathit{\sigma }}_{\mathit{b}}=1.6$ S/m. A plot for a healthy subject is also presented (full diamond) with error bar (SD = 0.065 S/m, CV = 9.8%, $\mathit{n}=7$) to show the repeatability.

Fig. 6.

Dielectric permittivity of blood samples at 3 MHz observed 1 minute from the start of DBCM measurement against hematocrit level for patients who underwent total knee arthroplasty [7] (full square). The solid line shows the result of linear regression where the square of the coefficient of correlation (${\mathit{R}}^2$) is 0.83. A plot for a healthy subject is also presented (full diamond) with error bar (SD = 64.0, CV = 6.8%, $\mathit{n}=7$) to show the repeatability. Note that there can be systematic errors in permittivity values mainly due to uncertainty of the correction for stray capacitance. In the worst case, for example, the presented permittivity may be underestimated by 5%. Nevertheless, this effect was not critical to the correlation, because always the same systematic errors appeared even in this case.

Fig. 7.

The characteristic coagulation time, such as ${\mathit{t}}_{\mathit{x}}$ in DBCM (stars) and CT for ROTEM^® (squares), are plotted against the hematocrit level, where platelet counts for the hematocrit-manipulated samples were almost constant between 12.6 and 13.8 × 10⁴/µl. To show the repeatability, a plot for a healthy subject in DBCM is also presented (full diamond) with error bar (SD = 2.7 min, CV = 11%, $\mathit{n}=7$).