A novel assay for studying the involvement of blood cells in whole blood thrombin generation

Abstract

Background Fluorogenic thrombin generation (TG) assays are commonly used to determine global coagulation phenotype in plasma. Whole blood (WB)‐TG assays reach one step closer to physiology by involving the intrinsic blood cells, but erythrocytes cause variable quenching of the fluorescence signals, hampering its routine application. Objective To develop a new assay for continuous WB‐TG measurement. Methods In the new WB‐TG assay, the erythrocyte‐caused distortion of signal was solved by continuously mixing the sample during the measurement. The assay was validated by evaluating the reproducibility and comparing with the paper‐based WB‐TG assay. Reconstituted human blood and WB from 119 healthy donors was tested to explore the influences of hematocrit and platelet count on TG. Results This novel WB‐TG assay showed good reproducibility while being less affected by contact activation compared with the previous paper‐based assay. Reconstitution experiments showed that the lag time of TG was shortened by the addition of platelets but not erythrocytes. Increasing hematocrit strongly augmented the peak thrombin, even in the presence of high platelet counts. The lag time and peak of WB‐TG of 119 healthy donors were positively related to erythrocyte count after adjusting for age, sex, and oral contraceptive use with multiple linear regression analyses. The reference range and interindividual variation of WB‐TG were determined in the healthy cohort. Conclusions A novel WB‐TG assay was developed, which is a straightforward tool to measure the involvement of platelets and erythrocytes in TG and may assist the research of blood cell‐associated coagulation disorders.

Interplay between platelets and coagulation

“…The disadvantage of viscoelastic tests is that they are too a-specific to be a serious alternative for plasma coagulation tests or platelet function tests. Our group recently developed a whole blood thrombin generation test that shows good correlations with plasma thrombin generation tests, with platelet numbers and with the use of different platelet inhibitors [212]. Although the WB-TG seems to be the first serious method to study involvement of blood cells in thrombosis, the technique is in its infancy.…”

“…Global tests such as TEG and ROTEM have too many shortcomings to be a serious candidate for the measurement of the interplay between platelets and coagulation [211]. Recently, a novel whole blood thrombin generation test (WB-TG) was developed that theoretically should give a precise insight in the interplay between platelets and coagulation, but the test needs to be clinically validated before real studies on the interplay between platelets and coagulation can be initiated [212].…”

Added Value of Blood Cells in Thrombin Generation Testing

“…Studies on the influence of platelet counts on PRP-TG using reconstituted PRPs have shown that platelet counts correlate with TG acceleration if the platelet numbers are below 100 Â 10 9 /L: increasing platelet counts reduce the lag time of TG and increase the peak thrombin level and ETP. 32,74,87 If the platelet numbers are above 100 Â 10 9 /L, then platelet numbers still correlate with the peak height, but the ETP becomes independent of the numbers. Currently, to control the pre-analytical varia-tion of platelet count introduced during PRP preparation, platelet-dependent TG is often tested with platelet count adjusted to 150 Â10 9 /L, 51 but this may mask the impact of individual platelet count differences, especially in patients with thrombocytopenia.…”

“…For example, the use of CTI was shown to improve the reproducibility of plasma-TG when the assay was triggered with low concentration of TF. 48,51 WB-TG is typically triggered with low amounts of TF (0.5 or 1 pM) 87,99 so that coagulation is dependent on physiological PL provided by blood cells and on feedback loops, but whether the use of CTI would also impact the reproducibility of the WB-TGA requires further assessment. The assay trigger should also be optimized for different settings; for instance, a trigger that contains low or even no TF may be necessary to reflect the procoagulant effect of TF/phosphatidylserine on cancer cells or MPs.…”

Delicate interactions between plasma factors and blood cells affect thrombin generation

“…Normal platelet count in humans is between 150 to 450 *10 9 /L and altered numbers have been seen in thrombocytopenia and thrombocythemia. Studies on the influence of platelet counts on PRP-TG using reconstituted PRPs have shown that platelet counts correlate with TG acceleration if the platelet numbers are below 100 *10 9 /L: increasing platelet counts reduce the lag time of TG and increase the peak thrombin level and ETP [32,74,87] . If the platelet numbers are above 100 *10 9 /L, then platelet numbers still correlate with the peak height but the ETP becomes independent of the numbers.…”