Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system.

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.

Whole blood analysis of reticulated platelets: improvements of detection and assay stability.

The flow cytometric analysis of reticulated platelets based on the fluorescent derivatization of their RNA content is increasingly used for the diagnostic classification of patients with thrombocytopenia as well as the monitoring of thrombopoiesis recovering under therapy. Many different modifications of the analytical protocol have been published following the first description in 1990 but consensus on the method has not yet been established. We have now reevaluated the assay's methodology in order to optimize sensitivity and specificity and reduce the time length of incubation and washing procedures. In the modified experimental approach native whole blood is incubated for 15 min with an increased amount of thiazole orange (1 microg/ml) in the presence of phycoerythrin labeled antibodies directed against the constitutively surface expressed antigen GPlb. Data acquisition on the flow cytometer can be started immediately after stopping and stabilization of the reaction by paraformaldehyde fixation. Thiazole orange fluorescence was not significantly changed in thrombin-activated, degranulated platelets compared to resting platelets indicating no significant non-specific staining of platelet granules under the selected test conditions. In addition, experiments employing RNAse digestion demonstrated specificity of thiazole orange staining for platelet RNA.

Spermatic granuloma of vas deferens after vasectomy in rhesus monkeys and men: light and electron microscopic study.

Spermatic granuloma of the vas deferens is a common complication of vasectomy which has received scant morphologic study. This study investigated the light and electron microscopic structure of such granulomas detected in rhesus monkeys (Macaca mulatta) and man after various modes of vasectomy and postoperative periods. Unilateral experimental vasectomy in monkeys was performed by either silk ligation or clasp occlusion; in 4 of 13 ligated animals and 5 of 5 clasp vasectomized animals granulomas developed at the site of fasectomy. In man, portions of the vas deferens were excised adjacent to the site of vasectomy preparatory to vasovasostomy. Of 5 patients studied, unilateral spermatic granulomas developed in 3. Such granulomas in both monkey and man were characterized by (1) masses of sperm surrounded by epithelioid cells and connective tissue, and (2) multiple epithelial-lined channels which often contained sperm and spermiophages. In both species, fine structural characteristics of the epithelium lining such channels closely resembled those of the principal cells of the normal vas. Spermiophagic cells included macrophages, epithelioid cells, and, in the monkey only, neutrophils. Lymphocytic invasion was a common feature of the human granulomas but was found only occasionally in the monkey granulomas. As a greater number of granulomas are studied in humans and monkeys, it is hoped that the processes underlying granuloma formation and the role of such granulomas in the development of complications after vasectomy will be clarified.

Vasectomy in rhesus monkeys. III. Light microscopic studies of testicular morphology.

Rhesus monkeys were randomly assigned to undergo various surgical procedures. The animals were followed from one to sixty-six weeks postvasectomy, at which time they were sacrificed and their tissues prepared for light and electron microscopy. Vasectomy in the rhesus monkey, as in certain other species, appears to be a procedure not attended with widespread testicular atrophy or histologic evidence of impaired spermatogenic potential utilizing the procedures and postoperative periods studied. Why certain animals exhibited focal degenerative changes is unclear; perhaps a certain population, yet to be defined, is more sensitive to such procedures, resulting in testicular alterations. It is important that such a population and such changes be defined to predict more accurately the possibility of successful vasovasostomy and reestablishment of fertility.