Laboratory diagnosis of renal failure: urine conductivity and tubular function.

AIM: Conductivity is a measure of a material's ability to conduct an electric current and it works thanks to movable charges. Conductivity in urine is directly proportional to ionic contents. The aim of this study was to evaluate the significance of urine conductivity by using the Sismex UF-100 analyser in correlations with other surrogate parameters of osmolality and renal diuresis, relative density, electrolytes and creatinine concentration. METHODS: For this study 140 urine samples were submitted for diagnostic urinalysis to the Clinical Pathology laboratory. Samples were collected from 70 healthy subjects, 42 diabetics with poor metabolic control and significant glicosuria, 28 patients with monoclonal gammopathy of uncertain significance, with significant proteinuria. All the samples were assessed for conductivity (UF-100 Sysmex), relative density (refract meter Zeiss), sodium, potassium, chlorine, creatinine, urea, glucose, protein (Olympus AU-2700). RESULTS: Urine conductivity appears to be related to ionic concentration but not to glucose and/or protein presence. CONCLUSIONS: This study results suggest that conductivity determination should be useful in diabetic patients to study the tubular function minimising interferences due to osmotic action of glucose.

Laboratory assessment of hypercoagulable state. A study in a group of patients with venous thromboembolism born in Chioggia.

AIM: Authors performed a Laboratory assessment for thrombophilia risk factors in a group of patients with previous deep venous thrombosis. METHODS: 123 consecutive patients were considered. The following parameters were investigated by using commercially available methods: PT, aPTT, TT, Fibrinogen, D-Dimer, Anti thrombin 3 (AT), Protein C (PC), Protein S (PS), activated C protein (APC) resistance, Lupus anticoagulant (LA), FV Leiden (G1691a mutation), Prothrombin G20210A mutation, MTHFR mutation (G677T mutation), anti Prothrombin auto-antibodies (PR) IgG and IgM, anti Beta 2 glycoprotein 1 (B2GP1) auto-antibodies IgG and IgM, anti Cardiolipin (CL) auto-antibodies IgG and IgM, homocysteine. RESULTS: In the 123 patients considered we observed: two AT deficiency, one PC deficiencies, one PS deficiency, 60 FV Leiden mutation (six homozygous), 1 Prothrombin gene mutations (heterozygous), 71 MTHFR mutations (15 homozygous). Study of anti phospholipid auto antibodies showed 10 patients positive for LA, 9 for anti CL antibodies (6IgG and 3IgM), 10 for anti B2GP1 antibody (5 IgG and 5IgM), 3 for anti PR antibody (IgG). Thirty nine patients showed hyper homocysteinemia. CONCLUSION: In our study only 19 patients were free of demonstrable thrombophilia risk factor. In 51 subjects a single risk factor was identified and in 53 multiple (from 2 to 5) risk factors were identified. In our opinion a Laboratory assessment of thrombophilia risk factors after a previous episode of deep venous thrombosis is a diagnostic tool of great importance. As a matter of fact, in our experience, by using a standard analytical panel, it was possible to highlight one or more risk factors in about 85% of the patients considered.

Accuracy of semiquantitative immunoenzymatic methods in quantitation of anti-topoisomerase I (Scl-70) antibodies.

Reports of a possible correlation between anti-Scl-70 antibody concentration and clinical manifestations in systemic sclerosis patients have recently appeared in the scientific literature. The goal of our study was to evaluate, by means of a multicenter study, the analytical reliability of immunoassay systems in the quantitative measurement of Scl-70 antibodies. Three blind samples (H, M, L) at different anti-Scl-70 antibody concentrations, and a low concentration antibody serum (LPC) used as a common calibrator, were sent three times in a 6-month time span to 39 Italian clinical laboratories. Each laboratory was asked to calculate dosages following the enzyme-linked immunosorbent assay (ELISA) method they used and report the optical density values of each sample (ODs), of the cutoff serum provided by the manufacturer of the kit used (ODco) and of LPC (ODLPC). The overall analytical imprecision (between methods and between laboratories) of the three different determinations of the values respectively expressed in ODs, ODs/ODco and ODs/ODLPCratio was 47.1, 52.8 and 34.0% for sample H, 56.2, 47.4% and 34% for sample M and 84.6, 86.0 and 86.6% for sample L. The average intra-method analytical imprecision was, respectively, 20.7, 29.8 and 18.6% for sample H, 24.6, 26.5 and 19.3% for sample M, and 30.6, 28.1 and 20.2% for sample L. The commercial ELISA methods currently used to determine the presence of anti-Scl-70 autoantibodies show considerable differences in the quantitative determination. The best results for reproducibility analyses have been obtained when the values were expressed as a ratio between the ODs of the sample and of the common calibrator (ODs/ODLPC). Forward-looking clinical studies that can clarify the usefulness of quantitative determination of anti-Scl-70 antibodies in the monitoring of diffuse scleroderma patients can be performed only when standard serum with a known antibody concentration and calibration curves for quantitative ELISA measurements are made available.

Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

Field evaluation of a second-generation cytometer UF-100 in diagnosis of acute urinary tract infections in adult patients.

AIMS: The authors evaluated the analytical performance of the Sysmex UF-100 cytometer vs. the diagnosis of urinary tract infections (UTI). METHODS: We considered 2010 subjects, aged between 18 and 78, 870 males and 1140 females. The majority (90.2%) of the samples were voided urine specimens collected by using the midstream technique. Each sample was subjected to microbiological evaluation (culture + residual antibacterial activity), dipstick tests, UF-100 examination and microscopic observation. In order to obtain a final diagnosis of UTI these laboratory results were taken into consideration together with clinical data and patients' characteristics. The analytical performance of the laboratory tests was obtained by adopting this diagnosis as standard practice. RESULTS: Out of the total 2010 subjects considered a clinical diagnosis of UTI was obtained in 529 cases (26.32%). The UF-100-based screening had sensitivity, 0.94; specificity, 0.93; positive predictive value, 0.83; negative predictive value, 0.98; and correctly classified incidence, 0.93. CONCLUSIONS: In our experience the results of the UF-100-based screening show a very good correlation with the diagnosis of acute UTI in adults patients.

Angiotensin-converting enzyme content of human spermatozoa and its release during capacitation.

In this study, we demonstrated, by using known detergents, the presence of angiotensin-converting enzyme (ACE) within human spermatozoa. We determined that maximal angiotensin-converting activity is expressed by sperm incubated in capacitating conditions, whereas this activity is negligible in saline-incubated spermatozoa. We further demonstrated that not acrosomes but cytoplasmic residues contain ACE. Because follicular fluid provides the necessary conditions for a maximal angiotensin-converting activity and for capacitations' metabolic activation, we hypothesize that ACE may play its physiologic role within the female reproductive tract.

Possible significance of transferrin levels in seminal plasma of fertile and infertile men.

Human seminal plasma contains large amounts of transferrin, which is a protein secreted mostly by Sertoli cells. It has been suggested that the concentration of transferrin may serve as a possible clinical marker of Sertoli cell function. Therefore the concentration of this protein in human seminal plasma from fertile and infertile men has been evaluated in order to find a relationship between transferrin concentrations and human semen parameters and plasma FSH levels. Findings show that seminal transferrin in subjects with oligozoospermia or azoospermia is significantly lower than in controls, and that it is strongly related to sperm count. Results also indicate that transferrin secretion can be impaired when plasma FSH levels are still normal, suggesting that seminal transferrin is an early and specific marker of Sertoli cell function. These results, however, do not clarify whether impairment of transferrin secretion by Sertoli cells is due to an organic dysfunction or to an organic secretory alteration.

Tissue distribution of methotrexate in rats. A comparison between intravenous injection as bolus or drip infusion.

Concentrations of methotrexate (MTX, 30 mg/kg) in rat sera and tissues were compared after the drug was administered by i.v. route as bolus injection or by drip infusion. Plasma and tissue specimens were collected after 1 h and 4 h and assayed for MTX by RIA. After 1 h the continuous infusion gave higher MTX levels in plasma, liver, bowel, lung, kidney, testicle and muscle; bolus injection gave higher levels in brain. No differences in levels were found in fat. After 4 h the differences between the two methods of administration were still appreciable only in liver, testicle and muscle. The differences were confirmed by determination of AUC for sera and tissue levels (1-4 h). These data suggest that continuous infusion may be more useful to obtain the highest levels of MTX in tissues with low or medium blood flow (as is probably the case in cancer tissue). Bolus injection, on the other hand, promotes the flow of the drug across the blood-brain barrier.

[Modifications induced on thrombin and plasmin activity by voluntary interruption of pregnancy]. / Modificazioni indotte dall'interruzione volontaria di gravidanza sull'attivazione trombinica e plasminica.

Pregnancy is characterized by plasmatic variations of coagulative factors' concentration and by different haemostatic-fibrinolytic balance. At present it is possible, with EIA methods, to measure fibrinogen (FgDP) and fibrin (FbDP) degradation products with precision and accuracy, as direct indexes of fibrinolysis and the thrombin-antithrombin III complex (TAT) as indirect index of thrombophilia. We have considered the course of those indexes in 61 pregnant women within the tenth week of gestation, before and after voluntary pregnancy interruption (VPI) resulted without complications. The results don't show any peculiar variation of the examined parameters between the pregnant women before VPI and a control group. Comparing the basal data with those obtained three hours after VPI, all indexes are increased, particularly FbDP. After 24 hours the concentration of FgDP, FbDP and TDP decreased in comparison with the three hours control drawing, nevertheless staying higher than the values obtained in the basal drawing. The evolution of FDP and of TAT, in our study, points out that, in the first weeks of pregnancy, the haemostatic-fibrinolytic balance does not differ significantly from the physiological balance. Three hours after VPI fibrinolytic mechanisms prevail as regards the fibrinogenolytic ones. TAT increases after 3 hours and returns to the rules after 24 hours, proposing itself as an indirect index of thrombinic activation and as a direct index of antithrombinic activity.

[Foetal-maternal alloimmunizations in the South-East area of the Venice province]. / Alloimmunizzazioni feto-materne nel Sud-Est del Veneto.

BACKGROUND: This study report the results obtained in a retrospective analysis of the foetal-maternal alloimmunizations observed from 1993 to 1999 in the South-East area of the Venice province. METHODS: The data concerning 17,000 pregnancy observed in this area from 1993-1999 have been collected. For each pregnancy data concerning maternal ABO, Rh, Kk and IAT as well as foetal ABO, Rh, Kk and DAT were available. Further data (mainly antibodies concentration and specificity) were available if a foetal-maternal alloimmunization was detected and if transfusional support was given after the birth. RESULTS: The authors observed 465 alloimmunizations (prevalence 2.7%): 381 (82%) of these were due to an ABO foetal-maternal incompatibility, 23 due to D incompatibility and the other 61 due to other blood groups antigens. Only 6 cases needed transfusional support: 5 exchange transfusion (a patient needed 2 exchanges) and a delayed transfusion. CONCLUSIONS: Foetal-maternal alloimmunizations are today a rare but not exceptional event (about 3% of pregnancy), the great majority of these alloimmunizations are due to an ABO incompatibility. Despite the prevention of alloimmunization in D negative women by using Rh immune globulin, anti-D alloimmunization is still observed. A great number of other blood groups antigens are involved in foetal-maternal alloimmunization mainly within the Rh system (CcEe, etc.). In the authors' experience the great majority of foetal-maternal alloimmunizations were clinically silent, only 6 cases (1.3% of patients with a positive DAT) needed transfusional therapy.

[Laboratory diagnosis of autoimmune thyroid disease]. / Diagnostica di laboratorio delle tireopatie autoimmuni.

Assay of the anti-thyroperoxidase (TPO), anti-thyroglobulin (Tg), and anti-TSH receptor antibodies constitutes the basis of the laboratory diagnostic work-up for autoimmune thyroid diseases. However, although these antibodies have been routinely assayed in serum for more than 40 years with increasingly reliable methods, it still exists a wide analytical variability that influences their correct diagnostic use. We analysed the biochemical, physiopathological and clinical aspects of the thyroid antigen-anti body systems, and propose the following guidelines for using autoantibody tests and analytical assay methods: a) assay of anti-TPO with third generation ultrasensitive method should constitute the main test for the diagnosis of autoimmune thyroid diseases. Assay of anti-M antibodies should be considered obsolete because even purified microsome preparations contain traces of contaminants (Tg and other antigens) that make the test aspecific; b) assay of anti-Tg antibodies may be limited to patients with suspect AITD and negative for anti-TPO antibodies, and patients undergoing thyroglobulin assay, because anti-Tg autoantibodies may interfere in the assay of the molecule in the immunometric test; c) assay of the anti-TSH receptor antibodies is used to diagnose Basedow's disease, and atrophic chronic thyroiditis; d) it is appropriate to use normal reference values adjusted for age and gender.

[Detection of anti-ENA autoantibodies in patients with systemic connective tissue diseases. Analytical variability and diagnostic sensitivity of 4 methods]. / La determinazione degli autoanticorpi anti-ENA in pazienti con connettivite sistemica. Variabilità analitica e sensibilità diagnostica di quattro metodi.

This study was designed to assess the analytical sensitivity and rate of agreement between commercial methods and reagents, among the most used in Italy for the detection of autoantibodies to extractable nuclear antigens (ENA). Sixty-eight serum samples from patients with clinically diagnosed systemic rheumatic diseases were aliquoted and distributed to 4 hospital laboratories; three ELISA (Elias, Shield, Inova) and 1 immunoblot method (Euroimmun) were used. Overall agreement between the test reagents, for each anti-ENA specificity, was 69.1% for Ro/SSA, 83.3% for La/SSB, 70.6% for RNP, 73.5% for Sm, 91.1% for Jo1, and 82.3% for Scl70. Lack of specificity (i.e., false positive reactions) was the most important cause of low concordance. When the data were analysed according to the clinical diagnosis, total agreement and specificity improved. However, a significant difference in terms of sensitivity was observed in the SLE group (30 sera) for RNP (positivity ranged from 20% to 43%) and for Sm (from 7% to 37%), and in the Sjögren's syndrome group (13 sera) for anti-La/SSB (from 8% to 38%). Comparable data were obtained for anti-Ro/SSA (from 70% to 77%) both in the SLE and the Sjögren's syndrome group. Sensitivity of all 4 reagents was good in detecting anti-Scl70 autoantibodies in the 8 patients with diffuse systemic sclerosis, as well as anti-Jo1 autoantibody in the 5 polymyositis patients, with a 100% and a 95% agreement, respectively. These data suggest the need of a better standardization of commercial reagents and analytical procedures, and the opportunity that every laboratory should perform anti-ENA determination by at least two different methods, since none of the methods tested was completely reliable in detecting all anti-ENA autoantibody specificities.

Evaluation of the GeneXpert assay in the detection of Factor V Leiden and prothrombin 20210 in stored, previously classified samples.

BACKGROUND: In this study, we evaluated the GeneXpert HemosIL Factor II and Factor V assay, an innovative assay for the detection of Factor V Leiden (FVL) and prothrombin G20210A mutation (GPRO). PATIENTS AND METHODS: We evaluated 132 patients that were previously classified (with a concordant result) using two commercial real-time PCR assays supplied, by Applied Biosystems and Roche Molecular Biochemicals. The cohort comprised 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO subjects. All of the samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay. RESULTS: All of the samples were correctly identified using the GeneXpert HemosIL Factor II and Factor V assay; therefore, in this patient series, the specificity and sensitivity of the test under evaluation was 1.00. DISCUSSION: We have shown that the GeneXpert HemosIL Factor II and Factor V assay, a rapid fully automated assay, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms with specificity and sensitivity that are comparable to other current real-time PCR-based methods. The theoretical advantages of such an assay include improved standardisation across varying healthcare environments, more thorough sample manipulation and reduced human error.

Prevalence of anti-hepatitis C virus antibodies among teenagers in the Venetian area: a seroepidemiological study.

OBJECTIVES: To establish the prevalence of circulating anti-hepatitis C virus antibodies and investigate hepatitis B virus serology, in adolescents in Chioggia (Venitia), Italy. METHODS: We conducted a population based survey of 1,015 fourteen-year-old school children, 529 boys and 486 girls. All were testing for hepatitis C antibodies with second generation enzyme-linked immunosorbent assay (ELISA). Four immunoblotting tests were used to confirm positive ELISA tests. Hepatitis B (HB) markers, HBs, anti-HBs and anti-HBc, were tested with commercial ELISA kits. RESULTS AND COMMENTS: Fourteen children were positive for anti-HCV antibodies, after confirmation with four immunoblotting tests. No significant difference was seen between the prevalence rate in males (0.41%) and females (0.38%) or between urban (0.43%) or rural (0.31%) dwellers. A significant variation of rate (12.5% to 0.2%) was seen in subjects with or without previous HBV infection. A positive relationship between age and prevalence of antibodies to HCV was noted, suggesting a late acquisition of infection in our district. Nine subjects had had a history of apparent parenteral exposure and only 1 was anti-HCV positive. Among HCV positive subjects only 25% had a history positive for parenteral exposure.

Hepatitis E virus infection in north-east Italy: serological study in the open population and groups at risk.

Developed western countries are considered to be relatively free from endemic foci of hepatitis E virus (HEV) infections. The aim of this study was to assess the seroepidemiology of HEV in north-east Italy. Of the 2361 individuals studied 1889 were representative of the general population and 472 were from groups at high risk for viral infections: 279 drug users and 193 patients on chronic haemodialysis. All sera were tested for hepatitis C virus antibody (HCVAb), human immunodeficiency virus antibody (HIVAb) and for hepatitis B virus (HBV) serology. Two solid-phase enzyme-linked immunosorbent assays (ELISA) were used to study the seroepidemiology of HEV IgG, the first (using recombinant antigens) for confirmation of initially reactive samples. The prevalence of circulating hepatitis E virus antibody (HEVAb) was 2.6% in the open population, 5.4% among drug users and 9.3% among patients on chronic haemodialysis. In the open population a positive relationship between age and prevalence of HEVAb was observed. A relationship between presence of HEVAb and serological evidence of previous HBV or HCV infections was also observed in this study. It was concluded that HEV infections are present in north-east Italy and are more frequent among subjects at risk for blood-borne viral infections. The positive correlation, observed in the open population, between age and prevalence of HEVAb suggests the presence of a cohort effect.

Antibodies to non-structural hepatitis C virus antigens: a link with liver disease activity?

OBJECTIVES: The aim of our study was to evaluate the aetiopathogenetic role of hepatitis C virus (HCV) in the genesis of chronic liver disease and hepatocellular carcinoma and the relationship between presence of circulating antibodies against non-structural HCV antigens and disease activity. METHODS: Using a new enzyme-immunoassay capable of detecting, either separately or together, circulating antibodies to structural and non-structural hepatitis C virus antigens, we tested 208 chronic hepatitis B surface antigen (HBsAg) carriers and 144 patients suffering from chronic liver disease. RESULTS: Among the chronic HBsAg carriers with normal transaminase activity, there were 4% who proved to be anti-HVC positive, compared with 14.5% among those with high serum transaminase. Among subjects with chronic liver disease, 20% of patients with chronic persistent hepatitis, 54% with chronic active hepatitis, 35% with liver cirrhosis and 47% with hepatocellular carcinoma proved to be anti-HCV positive. With regard to the aetiology of liver disease, 13% and 25% of chronic HBV infected patients, with or without anti-delta virus superinfection respectively, had anti-hepatitis C virus antibody. This prevalence was 22% among alcoholic liver disease and 89% among patients with cryptogenic liver diseases. The percentage of subjects with circulating antibodies against non-structural HCV proteins was higher among HBsAg chronic carriers with raised alanine aminotransferase and among patients with chronic active hepatitis and hepatocellular carcinoma. CONCLUSIONS: Many subjects with chronic liver disease have circulating anti-HCV antibodies. Anti-HCV positive subjects, especially those with antibodies to non-structural antigens, are more frequently found among patients with aggressive liver diseases. Among HBsAg chronic carriers the prevalence of anti-HCV was lower in "healthy carriers" and HBeAg positive rather than in HBeAg negative subjects with high alanine aminotransferase. We therefore suggest that there may be a relation between HCV replication, activeness of the liver disease and presence of antibodies to non-structural viral antigens.

Erythrocyte Na+-Li+ countertransport and blood viscosity in arterial hypertension.

The aim of this study was to evaluate together the main hemorheologic parameters and one of the transmembrane ion transport systems in erythrocytes of subjects with normal and elevated blood pressure. Three sex-, age-, and weight-matched groups consisting of 15 normotensive subjects (NT) with no parental hypertension, 15 patients with essential hypertension (EH) at stage 1-II WHO, and eight patients with secondary hypertension (Sec.H), respectively, were studied. Red blood cell Na+-Li+ countertransport (CTT), blood viscosity (eta B) at shear rates of 230 X S-1, 115 X S-1, and 46 S-1 and plasma viscosity (eta P) at shear rate of 46 X S-1 were measured. Plasma proteins and fibrinogen were also evaluated. CTT was higher in EH than in NT (P less than 0.01), while no significant difference was found between NT and Sec. H patients. eta B at 115 X S-1 and 46 X S-1 was higher in EH, but not in Sec. H, than in NT patients (P less than 0.05). No difference in eta P, plasma proteins and fibrinogen levels was observed between EH and NT. Elevated eta B and/or CTT may indicate a structural alteration in the erythrocyte membrane of some essential hypertensive patients. This is consistent with the hypothesis that a widespread membrane disorder is involved in the pathogenesis of primary hypertension.

In subjects with antibody to hepatitis C virus a high serum level of interleukin-2 soluble receptor suggests activity of liver disease.

The hepatitis C virus (HCV) is an RNA virus without apparent cytopathic effects, and hepatocellular damage in chronic infection is generally believed to be immune-mediated by cytotoxic T lymphocytes. Activated T cells release the soluble form of the interleukin-2 (IL-2) receptor (sIL-2R) and its concentration is correlated with the degree of lymphocyte activation. We measured sIL-2R in 69 subjects: 24 healthy repeat blood donors (group I), 17 HCV carriers without liver damage (group II) and 28 patients with HCV-related chronic active hepatitis (group III). There was no significant difference between sIL-2R levels in patients of group I (36.5 +/- 14.6 U ml-1) and group II (46.8 +/- 17.4 U ml-1), and the levels for both of these groups were significantly lower than those observed in the patients with active HCV, group III (176.9 +/- 59.5 U ml-1). Hence, among HCV-infected subjects (HCV RNA positive) with persistently normal alanine aminotransferase (ALT) levels, the plasma levels of sIL-2R are normal, but, in patients (HCV RNA positive) with HCV-related chronic active hepatitis there are increased plasma levels of sIL-2R. We conclude that in HCV infection high levels of sIL-2R are related to activity of the disease rather than to virus replication. In patients with HCV-related chronic liver disease, the sIL-2R concentration may be a useful marker of disease activity.

Assessment of in vitro platelet activation by Advia 120 platelet parameters.

The Advia 120 Hematology System provides new platelet parameters that have been proposed as useful markers of platelet activation. The aim of this study was to investigate platelet parameter variations after adenosine diphosphate (ADP) activation of platelet-rich plasma (PRP) in vitro, with particular interest in the mean platelet component (MPC), which was compared with two well-known degranulation antigens. Changes in platelet parameters that were induced by the activation of PRP with different concentrations of ADP were examined first. The time course of parameter values up to 60 minutes after maximal ADP activation and the relationships between the MPC and P-selectin and granulophysin expression as determined by flow cytometry were then investigated. After 10 minutes of ADP stimulation, the MPC presented a dose-dependent increase. At the maximal ADP concentration, the initial increase of the MPC was followed by a progressive decrease, leading the MPC to become significantly lower with respect to the baseline after 60 minutes of incubation. Significant variations in other parameters are also described. Finally, a negative correlation was found between the MPC change with respect to time 0 and both P-selectin and granulophysin expression. The present study suggests that platelet parameter variation, particular in the MPC, may be used to assess platelet activation in PRPs stimulated by ADP.