Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P F 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r 0.69, P F 0.001, n 20) and the percentage (r 0.72, P F 0.001, n 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood. Cytometry (Comm.

Background: Monitoring of platelet activation by the ADVIA® 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro. Methods: Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 °C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates. Results: In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 °C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 °C. Conclusions: When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 °C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.

Platelet activation results in changes in a number of cell surface molecules including an increase in P‐Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose‐dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post‐venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti‐coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = −0.69, P < 0.001, n = 20) and the percentage (r = −0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood. Cytometry (Comm. Clin. Cytometry) 38:250–255, 1999.  © 1999 Wiley‐Liss, Inc.

Summary Introduction The 4Kscore Test is a prebiopsy blood test that incorporates four prostate protein biomarkers along with patient clinical information to determine a man's risk for high‐grade, aggressive (Gleason ≥7) prostate cancer. However, some men likely to benefit from the test may be seen in primary care settings where the digital rectal examination (DRE) information is not always obtained. In this study, we assessed the clinical validity of the 4Kscore Test when the DRE information was not included in the algorithm. Methods The Prospective 4Kscore Validation Study enrolled 1012 men scheduled for prostate biopsy across 26 urology practices in the United States. The 4Kscore was calculated for each patient with and without DRE information. The primary outcome was Gleason ≥7 prostate cancer on prostate biopsy. The contribution of DRE to the predictive accuracy of the test was evaluated by area under the receiver operating curve (AUC‐ROC), risk calibration and clinical consequences. Results High‐grade, aggressive prostate cancer was found in 231 (23%) of the 1012 patients. Both versions of the 4Kscore Test, with and without DRE, showed excellent discrimination (AUC=0.821 with DRE and AUC=0.818 without DRE input) and excellent calibration. No clinically significant difference was found between the two versions of the 4Kscore. Conclusions The 4Kscore Test algorithm, whether DRE findings are available or not, performs well in predicting a man's risk of high‐grade, aggressive prostate cancer. Patients who are suspected of having aggressive prostate cancer can safely have their risk better defined by 4Kscore even if a DRE has not been performed recently.

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In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

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