Hemostatic imbalance induced by tamoxifen in estrogen receptor-positive breast cancer patients: An observational study.

BACKGROUND: Estrogen receptor (ER)-positive (ER+) breast cancer accounts for approximately 75% of all breast cancers. Tamoxifen, a selective estrogen receptor modulator, is the standard adjuvant treatment. Although better tolerated than aromatase inhibitors, tamoxifen increases the risk of venous thromboembolism (VTE) 1.4-fold. AIM: To assess the hemostatic imbalance induced by tamoxifen in adjuvant treatment of ER+ breast cancer. METHOD: Twenty-five patients in remission from ER+ breast cancer under tamoxifen were included. One hundred and thirty one age- and BMI-matched healthy controls were included to establish reference ranges of thrombin generation assay (TGA) parameters. TGA was performed in the absence and presence of exogenous activated protein C (APC) to calculate the normalized APC sensitivity ratio (nAPCsr), a marker of APC resistance. RESULTS: All TG parameters except the endogenous thrombin potential (ETP) (-APC) were significantly impacted by tamoxifen (p < 0.001). In absence of APC, regardless of TGA parameters, at least 50% of results were outside the reference ranges except for ETP, which was above the upper reference limit in only two individuals. The most impacted parameter was the Peak Height with 52% (-APC) and 80% (+APC) of results above the upper reference range limit, respectively. The nAPCsr was significantly higher in tamoxifen users (mean ± standard deviation = 3.18 ± 0.91) compared to the control group (2.19 ± 0.92, p < 0.0001). CONCLUSION: This observational study showed that patients in remission from ER+ breast cancer taking tamoxifen had altered thrombin generation, as well as an acquired APC resistance. Moreover, this is the first study using the validated ETP-based APC resistance assay in tamoxifen-treated patients.

Feasibility of a mean platelet volume standard: an international council for standardization in hematology (ICSH) inter-laboratory study.

We have evaluated a commercial-fixed porcine platelet preparation (with and without added fixed human red blood cells (RBC)) for the potential standardization of mean platelet volume (MPV) measurements. The standards (Biotechne) were distributed internationally to 19 laboratories including all major hematology instrument manufacturers and academic/pathology laboratories. Overall, the standards demonstrated excellent stability up to 1 month within both MPV values and platelet counts when stored at 4°C. The presence of RBC significantly increased the platelet count and MPV values compared to platelets alone. However, as expected, there were differences in MPV values between different instruments and manufacturers. MPV values were also significantly higher in the whole blood standard compared to the platelet standard in the majority of instruments except with some instruments, where MPV values were significantly higher in the platelet only preparation. To further investigate this phenomenon, two different Platelet MPV preparations (with low and high MPV) in combination with 3 different RBC MCV preparations (with low, normal or high MCVs) were tested to try and further elucidate how RBC populations may impact upon platelet analysis (count, MPV, and PDW) using a single impedance analyzer. Both MPV and MCV values showed good stability over the course of the study for up to 50 days. As expected, the RBC preparation with the lowest MCV had the greatest impact on the MPV. However, this was not observed with an increase in MCV of the RBC or by a larger MPV of the platelet population. To further understand how different gating strategies may also influence results, we investigated the effect of either fixed or floating gate strategies upon MPV raw data from patient samples in a single impedance analyzer. Overall, it was clear that floating and fixed gate strategies also significantly impact upon MPV values. In conclusion, we have demonstrated the potential of an MPV standard with good stability characteristics for calibrating and comparing full blood counters that use different analysis principles, gating and MPV calculations. This may facilitate future instrument calibration and harmonization of results between different technologies.

Analytical Sensitivity of Six SARS-CoV-2 Rapid Antigen Tests for Omicron versus Delta Variant.

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to <4.9 log10 copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.

Analytical performance of the endogenous thrombin potential-based activated protein C resistance assay on the automated ST Genesia system.

Background: The evaluation of activated protein C (APC) resistance based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives in women. In 2019, this assay was validated on the calibrated automated thrombogram (CAT) device. However, in view of its screening potential, its automation is essential. Objectives: To transfer the ETP-based APC resistance assay on the ST Genesia system using reagent STG-ThromboScreen with exogenous APC added. Method: Dose-response curves were performed to define APC concentration leading to 90% ETP inhibition on healthy donors. Intra- and interrun reproducibility was assessed. The normal range was defined on the basis of 56 samples from healthy individuals. The sensitivity was assessed on 40 samples from women using combined oral contraceptives (COCs). A method comparison with the validated ETP-based APC resistance on the CAT system was performed. Results were expressed in normalized APC sensitivity ratio (nAPCsr). Results: The APC concentration leading to 90% ETP inhibition was 652 mU/mL. Intra- and interrun reproducibility showed standard deviation <4%. The nAPCsr normal range stood between 0.00 and 2.20. Analyses of 40 samples from women using COCs confirmed the good sensitivity of the assay. Compared to the CAT system, nAPCsr values were slightly higher on the automated system. Conclusion: This study is the first reporting the analytical performances of the ETP-based APC resistance assay on an automated platform. Results support the concept that this test, when incorporated into clinical routine, could become a promising regulatory and clinical tool to document on the thrombogenicity of female hormonal therapies.

Study of in vitro thrombin generation after neutralization of heparin.

INTRODUCTION: Thrombin generation (TG) documents hypercoagulability. TG in platelet-poor plasma is exquisitely sensitive to heparins, which thus must be neutralized before testing. Heparinase and hexadimethrine bromide (polybrene) have been used for that purpose, but their effects per se on TG have been poorly studied so far. METHODS: (i) TG was studied in commercial normal pooled plasma (NPP; CryoCheck® , Cryopep) in absence or presence of neutralizing agents. (ii) NPP was spiked with increasing concentrations of unfractionated heparin (UFH; up to 1.0 IU/mL) or low-molecular-weight heparin (LMWH; enoxaparin up to 1.2 IU/mL) and TG studied after incubation of heparinase (Hepzyme® ; 15 minutes) or polybrene (0.025 mg/mL; 10 minutes). RESULTS: (i) With ThromboScreen reagent to initiate TG, addition of heparinase was associated with increased peak, whereas polybrene caused lengthening of lag time and time to peak, compared with nonsupplemented NPP. (ii) With polybrene, TG was completely restored over the whole range of UFH and LMWH studied. By contrast, heparinase failed to fully restore TG in presence of UFH concentrations ≥0.8 IU/mL or LMWH concentrations ≥1.0 IU/mL. Those effects were matched with detectable tiny residual amounts of non-neutralized heparin (as assessed with an anti-Xa assay) and were less pronounced with a higher picomolar concentration of tissue factor (DrugScreen reagent). CONCLUSION: Polybrene fully restored TG of heparinized plasma at the expense of an alteration of TG, pointing to the need to use adapted reference ranges. Heparinase failed to do so in presence of high concentrations of both heparins.

Interlaboratory variability of activated protein C resistance using the ETP-based APC resistance assay.

BACKGROUND AND OBJECTIVE: Although the endogenous thrombin potential (ETP)-based activated protein C (APC) resistance is recommended for the development of steroid contraceptive agents, one of the main limitations of this technique was its lack of standardization, which hampered study-to-study comparison. A validated methodology that meets all the regulatory requirements in terms of analytical performances has been developed recently. To ensure a wide implementation of this test, the assessment of the interlaboratory variability was needed. METHOD: The assay was implemented in three testing laboratories. First, dose-response curves were performed to locally define APC concentration leading to 90% of ETP inhibition on healthy donors. Intra- and inter-run repeatability were assessed on a reference plasma and three quality controls. To investigate the variability in results among the different testing units, 60 donor samples were analyzed at each site. RESULTS: The APC concentration leading to 90% of ETP inhibition was defined at 1.21 µg/ml and 1.14 µg/ml in the two receiving units. Intra- and inter-run repeatability showed standard deviation below 3%. Analyses of the 60 donor samples showed no statistically significant difference. The sensitivity of the test in the different laboratories was maintained and subgroup analyses still reported significant differences depending on hormonal status of donors. CONCLUSION: This study is the first reporting the interlaboratory variability of the ETP-based APC resistance assay. Data revealed excellent intra- and interlaboratory reproducibility. These results support the concept that this blood coagulation test provides an appropriate sensitivity irrespective of the laboratory in which analyses are performed.

Assessment of acquired activated protein C resistance with the FibWave and comparison with the ETP-based APC resistance.

INTRODUCTION: Activated protein C (APC) resistance is a major risk factor of venous thrombosis which may be acquired by hormonal therapy or other causes. The FibWave, a sensitive global clot-based assay design to analyze the coagulation kinetics in plasma, may be a good candidate to assess this prothrombotic state. This study aims to assess the suitability of the FibWave to differentiate the coagulation kinetics of women on oral contraceptives. MATERIALS AND METHODS: Fifty-four healthy volunteers were divided into 5 groups: men [n = 13], women not using hormonal contraception [n = 12], women using second [n = 12] or third generation [n = 12] combined oral contraceptives, and women using progestin only contraceptive [n = 5]. Patients with coagulation abnormalities were also assessed [n = 8]. The APC resistance was assessed on the FibWave using exogenous APC or Protac, and on the Calibrated Automated Thrombogram using the ETP-based APC resistance assay. RESULTS: Either in presence or in absence of APC or Protac, the FibWave was able to detect a hypercoagulable state in plasma samples. All combined oral contraceptives showed a lower FW-Max1 , FW-Max2, and FW-Min2 percentage of inhibition and a lower FW-Ttpeak ratio than the other groups. The sensitivity of the FibWave was similar to the one of the ETP-based APC resistance assay. CONCLUSION: The FibWave is able to differentiate APC resistance levels observed in women on combined oral contraceptive. The FW-Max1 , FW-Max2, and to a lesser degree FW-Min2 were identified as the most sensitive parameters with a similar performance to the ETP-based APC resistance assay.

Characteristics of L-PRP preparations for treating Achilles tendon rupture within the PATH-2 study.

Platelet-rich plasma (PRP) is an autologous preparation that has been claimed to improve healing and mechanobiological properties of tendons both in vitro and in vivo. In this sub-study from the PATH-2 (PRP in Achilles Tendon Healing-2) trial, we report the cellular and growth factor content and quality of the Leukocyte-rich PRP (L-PRP) (N = 103) prepared using a standardized commercial preparation method across 19 different UK centers. Baseline whole blood cell counts (red cells, leukocyte and platelets) demonstrated that the two groups were well-matched. L-PRP analysis gave a mean platelet count of 852.6 x 109/L (SD 438.96), a mean leukocyte cell count of 15.13 x 109/L (SD 10.28) and a mean red blood cell count of 0.91 x 1012/L (SD 1.49). The activation status of the L-PRP gave either low or high expression levels of the degranulation marker CD62p before and after ex-vivo platelet activation respectively. TGF-ß, VEGF, PDGF, IGF and FGFb mean concentrations were 131.92 ng/ml, 0.98 ng/ml, 55.34 ng/ml, 78.2 ng/ml and 111.0 pg/ml respectively with expected correlations with both platelet and leukocyte counts. While PATH-2 results demonstrated that there was no evidence L-PRP is effective for improving clinical outcomes at 24 weeks after Achilles tendon rupture, our findings support that the majority of L-PRP properties were within the method specification and performance.