Platelet aggregation studies play an important role in the assessment of hereditary and acquired platelet function defects. The first aggregation test introduced into laboratory practice used platelet-rich plasma (PRP) where aggregation was detected by an optical method. The assessment of platelet function using whole blood (WB) aggregation by an impedance method followed up nearly 20 years later. The WB impedance aggregation assay appears to be superior to the optical method because it 1) evaluates platelets in a physiologic milieu in the presence of red and white blood cells, which are known to modulate platelet function; 2) is faster; 3) has higher sensitivity; and 4) does not require centrifugation, thus avoiding injury to platelets and loss of giant thrombocytes. These two assays were compared. Clearly, the WB impedance aggregation methodology has many advantages over the optical PRP assay for the assessment of the hyperactive platelet syndrome and the effects of anti-platelet drugs.

Platelet aggregation inhibition by clopidogrel may be suboptimal in 4-30% of patients. Traditionally, optical aggregometry is used to assess clopidogrel's anti-platelet effects by inhibition of ADP-induced aggregation in platelet rich plasma. Red blood cells are an important source of ADP and, thus, are known to modulate platelet function. Because the whole blood aggregation by impedance method assesses platelet function in a physiological milieu, we compared clopidogrel response by this method with the optical method in platelet rich plasma (PRP) and the Platelet Function Analyzer (PFA-100). Platelet function studies were performed in 17 healthy subjects at baseline and after 10 days of clopidogrel intake (75 mg/day). Optical and impedance aggregometry were performed after addition of ADP (10 and 20 microM) and collagen (1 and 2 microg/mL). For PFA-100 analysis, whole blood closure time was measured in collagen-coated cartridges with ADP and epinephrine. All subjects except one showed a decrease in ADP-induced aggregation using both aggregation methods. However, ADP-induced platelet aggregation was significantly inhibited when assessed in whole blood as compared to the optical method (71+/- 34% vs. 34.2+/- 23%, p = 0.0002); this suggests that whole blood aggregometry is more sensitive in the detection of clopidogrel effect in the presence of red cells, which are known to modulate platelet function. The PFA-100 ADP closure time was slightly prolonged above the reference interval in only 5/17 (29%) subjects, suggesting that this instrument is not able to detect clopidogrel effect. We conclude that whole blood aggregation appears to be more sensitive in detecting clopidogrel effect compared with the platelet rich plasma method; the PFA-100 was unable to detect clopidogrel effect in the majority of the subjects.

To cite this article: Depasse F., Gerotziafas G. T., Busson J., Van Dreden P., Samama M.M. Assessment of three chromogenic and one clotting assays for the measurement of synthetic pentasaccharide fondaparinux (Arixtra Synthetic pentasaccharide fondaparinux (Arixtra 1) is the ®rst indirect (antithrombin-dependent) synthetic speci®c factor (F)Xa inhibitor approved in several countries for the prevention of deep vein thrombosis (DVT) in major orthopedic surgery. In addition, ongoing clinical trials aim to prove its ef®cacy for the treatment of DVT and in coronary heart disease (for review see [1,2]). Fondaparinux plasma concentrations obtained in patients treated at prophylactic and therapeutic doses range from 0.1 to 0.5 mg mL À1 and from 0.6 to 1.5 mg mL À1, respectively; in healthy volunteers, the subcutaneous injection of the established prophylactic dose (2.5 mg) is associated with plasma levels at 0.34 AE 0.04 mg mL À1 [3±5]. Although no coagulation monitoring is advocated, accurate assays dedicated to anti-Xa activity measurement should be available. This could be useful in practical use at least in some particular groups of patients, e.g. with renal impairment, during pregnancy, body weight < 50 kg as recommended for LMWHs or in the elderly. Nevertheless, no special assay has been developed for this purpose and the commercial available assays are designed for either unfractionated or low molecular weight heparins. The aim of this work was to evaluate the possibility of using commercially available assays. Three different chromogenic and one clotting assays for the measurement of fondaparinux anti-Xa activity in plasma were evaluated.Normal pool plasma (Normapool 1 , Hyphen BioMed, Neuville sur Oise, France) was spiked with increasing amounts of fondaparinux (Sano®, Paris, France) in order to obtain plasma concentrations ranging from 0 to 10 mg mL À1 . The antithrombin activity level was measured in this range of concentrations using the Berichrom 1 Heparin was used with addition of exogenous bovine antithrombin as provided with the assay and without addition of exogenous antithrombin. In addition to the results expressed in LMWH anti-Xa IU mL À1 , results were registered as raw data, i.e. optical density variation per minute (DOD min À1 ) and clotting time for the chromogenic and the clotting assays, respectively. FXa inhibition for the chromogenic assays was calculated by reporting the DDO min À1 of the considered sample to the DDO min À1 of the normal pool plasma free of fondaparinux considered as baseline. A statistical analysis was performed using an analysis of variance (ANOVA, F-test).All the antithrombin activity levels measured remained in the normal range and were not signi®cantly affected by the addition of fondaparinux.Results expressed in LMWH anti-Xa IU/mL differed signi®cantly (P < 0.01) from an assay to another for the same fondaparinux plasma concentration, e.g. (mean AE SD, n 10) 0.93 AE 0.03 IU mL A linear relationship was obtained for fondaparinux plasma concentrations ranging from 0 up ...

No abstract