Quantitative detection of platelet aggregates in whole blood without fixation

Platelet counting detects lesser degrees of platelet aggregation than conventional aggregometry. In order to prevent progressive platelet aggregation or disaggregation after sampling it is customary to fix blood samples. However fixation may introduce other artefacts. We first compared stability of platelet counts in EDTA-, citrate- and r-hirudin-anticoagulated blood from healthy volunteers. Second, the stability of platelet counts in unfixed EDTA- and hirudin-anticoagulated blood was compared with glutaraldehyde-fixed blood in the same anticoagulants. Third, the effect of in vivo heparin administration on platelet counts in EDTA- and hirudin-anticoagulated blood was studied. Platelet counts within 2 h of collection were significantly higher in EDTA- than in hirudin- or citrate-anticoagulated blood (P = 0.002 vs. hirudin and P = 0.001 vs. citrate). Twenty-four hour counts in hirudin and EDTA were unchanged (P = 0.3 and P = 0.2, respectively, vs. earlier counts). Counts in citrate increased significantly (P = 0.007; n = 10). Platelet counts in fixed blood did not differ significantly from those in unfixed blood. Heparin administered for cardiopulmonary bypass reduced platelet counts in hirudin-anticoagulated blood from (mean +/- 1 standard deviation) 180 +/- 45 to 162 +/- 30 x 10(9) l-1 (P = 0.01; n = 14), without significantly lowering counts with EDTA-anticoagulation, consistent with increased platelet aggregation. Hirudin and EDTA provided stable platelet counts, suggesting that fixation is unnecessary.     

Time dependent reduction in platelet aggregation using the multiplate analyser and hirudin blood due to platelet clumping

The Multiplate is a popular instrument that measures platelet function using whole blood. Potentially considered a point of care instrument, it is also used by hemostasis laboratories. The instrument is usually utilized to assess antiplatelet medication or as a screen of platelet function. According to the manufacturer, testing should be performed within 0.5–3 hours of blood collection, and preferably using manufacturer provided hirudin tubes. We report time-associated reduction in platelet aggregation using the Multiplate and hirudin blood collection tubes, for all the major employed agonists. Blood for Multiplate analysis was collected into manufacturer supplied hirudin tubes, and 21 consecutive samples assessed using manufacturer supplied agonists (ADP, arachidonic acid, TRAP, collagen and ristocetin), at several time-points post-sample collection within the recommended test time period. Blood was also collected into EDTA as a reference method for platelet counts, with samples collected into sodium citrate and hirudin used for comparative counts. All platelet agonists showed a diminution of response with time. Depending on the agonist, the reduction caused 5–20% and 22–47% of responses initially in the normal reference range to fall below the reference range at 120min and 180min, respectively. Considering any agonist, 35% and 67% of initially “normal” responses became ‘abnormal’ at 120 min and 180 min, respectively. Platelet counts showed generally minimal changes in EDTA blood, but were markedly reduced over time in both citrate and hirudin blood, with up to 40% and 60% reduction, respectively, at 240 min. The presence of platelet clumping (micro-aggregate formation) was also observed in a time dependent manner, especially for hirudin. In conclusion, considering any platelet agonist, around two-thirds of samples can, within the recommended 0.5–3 hour testing window post-blood collection, yield a reduction in platelet aggregation that may lead to a change in interpretation (i.e., normal to reduced). Thus, the stability of Multiplate testing can more realistically be considered as being between 30–120 min of blood collection for samples collected into hirudin.     

Giant platelet disorder in the Cavalier King Charles Spaniel

OBJECTIVE: The aim of this study was to describe the clinical, functional, and morphologic characteristics of platelets in Cavalier King Charles Spaniel dogs (Cavaliers). MATERIALS AND METHODS: Blood from 69 clinically normal Cavaliers was collected and anticoagulated with ethylenediamine-tetraacetic acid (EDTA) and citrate. Automated and manual platelet counts were obtained. Percent platelet aggregation in response to ADP (2, 4, 8, 16, and 32 microM) was determined. Electron microscopy was performed to examine platelet internal morphology and dense granule distribution. A cardiologist recorded the quality of murmurs. RESULTS: Thrombocytopenia (<100,000/microL) was present in 51.43% (36/69) of Cavaliers. Macrothrombocytes (>3 microm) were present in 33.33% (22/69). Mean manual platelet count was 118,770/microL. Manual (EDTA blood) and automated (EDTA and citrated blood) methods of platelet counting were correlated. Prevalence of cardiac murmurs was 38% (26/69). There was no association between affected dogs and murmur, signalment, or coat color. Mean percent platelet aggregation was significantly higher in controls than in Cavaliers (79% vs 38%, p=0.001). Response to ADP was unaffected by thrombocytopenia, macrothrombocytes, murmur, or any combination thereof. Platelet electron microscopy showed normal and giant sized platelets with normal internal morphology. CONCLUSIONS: A benign inherited giant platelet disorder affects approximately 50% of Cavalier King Charles Spaniels. It is characterized by thrombocytopenia, macrothrombocytes, or decreased platelet aggregation in response to ADP. Platelet ultrastructure is normal. Citrated or EDTA blood provides accurate platelet counts. Further studies are indicated to determine platelet glycoprotein structure and any association with mitral endocardiosis. Cavaliers may be useful models of inherited giant platelet disorders.     

Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume

Platelet counts on whole blood samples collected into tripotassium salt of EDTA, trisodium citrate (Na3 citr), citrate phosphate dextrose adenine formula 1 (CPDA-1) and acid citrate dextrose formula A (ACD-A), all showed a statistically significant drop (P less than 0.01) after 1 h standing at room temperature (RT) as compared with the immediate (within 30 min) counts. After 1 h the enumeration became stable in the EDTA samples but the drop continued up to 4-6 h in those samples taken into citrate. The decreases in citrate were significant (18-30%, P less than 0.001). The addition of EDTA (1.5 mg/ml) to the citrated samples after the sixth hour count created a significant rise (6-22%, P less than 0.01) in the counts between the sixth and the seventh hour. Our observations show that platelet counts in citrated blood samples are lower than those in EDTA and highlight the necessity to present citrated samples mixed wtih dried EDTA when characterization or quality control of blood and blood components is required. Analysis of the mean platelet volume (MPV) showed significantly lower values (6-13%, P less than 0.05) in the citrated samples as compared to the same samples in EDTA, and a significant increase (4-6%, P less than 0.01) on the addition of EDTA to the citrated samples after the sixth hour analysis.     

Platelet concentrates transfusion in cardiac surgery in relation to preoperative point-of-care assessment of platelet adhesion and aggregation

Platelet dysfunction is an important cause of bleeding early after cardiac surgery. Whole-blood multiple electrode aggregometry (MEA), investigating the adhesion and aggregation of activated platelets onto metal electrodes, has shown correlations with platelet concentrates transfusion in this setting. Platelet activity in vivo is dependent on shear stress, an aspect that cannot be investigated with MEA, but with the cone and plate(let) analyzer (CPA) Impact-R that measures the interaction of platelets and von Willebrand factor (vWF) in whole blood under shear. We hypothesized that preoperative CPA may show better correlation with platelet concentrates transfusion post-cardiac surgery than MEA, since it is dependent on both platelet activity and platelet interaction with vWF multimers. Blood was obtained preoperatively from 30 patients undergoing aorto-coronary bypass (ACB) and 20 patients with aortic valve (AV) surgery. MEA was performed in hirudin-anticoagulated blood. The Impact-R analyses were performed in blood anticoagulated with hirudin, heparin or the standard anticoagulant citrate. For the light microscopy images obtained, the parameter surface coverage (SC) was calculated. Preoperative Impact-R results were abnormally decreased in AV patients and significantly lower than in ACB patients. For the Impact-R analysis performed in citrated blood, no correlation with platelet concentrates transfusion was observed. In contrast, MEA was comparable between the groups and correlated significantly with intraoperative platelet concentrates transfusion in both groups (rho between ?0.47 and ?0.62, p < 0.05). Multiple electrode aggregometry appeared more useful and easier to apply than CPA for preoperatively identifying patients with platelet concentrates transfusion in cardiac surgery.     

Different behaviour of soluble CD40L concentrations can be reflected by variations of preanalytical conditions.

INTRODUCTION: Biomarkers reflecting an inflammatory or immunological response are increasingly offered to improve the risk stratification of patients. For example, current evidence suggests that soluble CD40 ligand (sCD40L) is elevated in patients with acute coronary syndrome. But only a few data are available to evaluate the influence of preanalytic conditions on sCD40L values. METHODS: Blood samples of seven healthy blood donors were collected in tubes without additives and in EDTA- or citrate-filled tubes at various storage conditions. Platelet count was modified by serum dilution, and sCD40L was measured in platelet-rich-plasma and in whole blood. sCD40L levels were determined by an commercially available ELISA-Kit. RESULTS: Immediately after blood sample assessment, sCD40L levels in serum samples were elevated (1258+/-820 pg/ml) compared to EDTA (64+/-32 pg/ml) and citrate (60+/-8.5 pg/ml) values. Additionally, sCD40L levels were dependent on storage duration. After platelet activation, sCD40L levels were significantly increased to 8278+/-2453 pg/ml and were significantly correlated to platelet count (r=0.96). CONCLUSIONS: Soluble CD40L levels were clearly influenced by preanalytical conditions and were dependent on storage duration, sample technique and platelet count. These influences should be considered by the determination and evaluation of sCD40L concentrations.     

Flow cytometric analysis of platelet activation under calcium ion-chelating conditions

Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA.     

Flow cytometric analysis of platelet activation under calcium ion‐chelating conditions

Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre‐ and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre‐ and postagitation. Heparin‐treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA‐dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA.     

Thrombin regulation of platelet interaction with damaged vessel wall and isolated collagen type I at arterial flow conditions in a porcine model: effects of hirudins, heparin, and calcium chelation

The role of thrombin inhibition in platelet vessel wall interaction and thrombus growth was studied under controlled flow conditions. Natural hirudin and recombinant hirudin (r-hirudin), which are specific thrombin inhibitors, were compared with heparinized blood (1.8 +/- 0.2 U/mL) and Ca(2+)-chelated blood in their potential to inhibit platelet interaction and thrombus growth on two biologic vascular surfaces and one immobilized vessel wall component. The substrates were perfused by flowing blood at shear rates typical of patent and stenosed arteries (212 to 1,690/s) for 5 minutes. Platelet deposition was measured by In-111-labeled platelets. We found that both natural and r-hirudin have similar effects on platelet-substrate interaction. As compared with heparin, platelet deposition to mildly damaged vessel wall and digested collagen type I was not reduced by hirudin or citrate. However, hirudin and citrate significantly reduced platelet deposition to severely damaged vessel wall (platelets x 10(6)/cm2: 93 +/- 10 in heparinized blood v 50 +/- 7 in blood treated with 100 U/mL r-hirudin). Therefore, thrombus growth on areas of severe wall damage is in part dependent on local thrombin production at the site of vascular damage. We also found that hirudin added to heparinized blood reduced platelet deposition to severely injured wall but not to subendothelium or collagen-coated slides. Hirudin added to citrated blood did not affect platelet deposition. Our study indicates that local thrombin generation at the site of severe injury will induce platelet activation and deposition even in the presence of average therapeutic heparin levels that inhibit blood coagulation.     

Evidence that high-density lipoprotein cholesterol is an independent predictor of acute platelet-dependent thrombus formation

Plasma total and low-density lipoprotein (LDL) cholesterol are established risk factors for atherosclerotic vascular disease and may also contribute to a prothrombotic risk via enhanced platelet reactivity. This study examines whether high-density lipoprotein (HDL) cholesterol, which is inversely correlated with coronary artery disease, is associated with a reduced thrombogenic potential. Platelet thrombus formation was evaluated by exposing porcine aortic media placed in Badimon perfusion chambers to flowing nonanticoagulated venous blood for 5 minutes at a shear rate of 1,000 s(-1). Forty-five subjects, 23 normal (LDL 104 +/- 31, HDL 50 +/- 15 mg/dl) and 22 hypercholesterolemic (LDL 181 +/- 45, HDL 41 +/- 10 mg/dl) patients without coronary artery disease were studied. Platelet aggregation and CD62 antigen expression, and assay for circulating prothrombotic factors were also performed. In univariate analysis platelet thrombus formation correlated with weight (r = 0.33, p = 0.03), diastolic blood pressure (r = 0.39, p = 0.01), HDL cholesterol (r = -0.45, p = 0.003), total/HDL cholesterol (r = 0.43, p = 0.004) and LDL/HDL (r = 0.38, p = 0.01) ratios, and platelet CD62 expression (r = 0.41, p = 0.02). In multiple regression analysis only HDL cholesterol showed significant correlation with platelet thrombus formation (p = 0.03). Platelet aggregation and circulating prothrombotic factors did not correlate with platelet thrombus formation. A comparison between normal and hypercholesterolemic subjects revealed enhanced thrombus area (0.026 +/- 0.20 vs 0.045 +/- 0.039 mm2/mm; p = 0.04), resting CD62 expression (6 +/- 7% vs 15 +/- 10% positive platelets, p = 0.02), and platelet aggregation (16.7 +/- 5.2 vs 21.7 +/- 6.7 ohms, p = 0.04) in hypercholesterolemic subjects. Our results demonstrate that HDL cholesterol is a significant independent predictor of ex vivo platelet thrombus formation.     

The platelet count in EDTA-anticoagulated blood from patients with thrombocytopenia may be underestimated when measured in routine laboratories

Spuriously low platelet counts (PCs) can be observed in normal blood samples anticoagulated with ethylenediamine tetra-acetic acid (EDTA)and, much less frequently, with citrate-tris-pyridossalphosphate (CPT),due to time-dependent in vitro platelet agglutination. Accuracy in PC determination is essential as PC is one of the parameters that usually guides treatment for thrombocytopenic patients. PCs of 93 thrombocy to penic patients were measured in EDTA- or CPT-anticoagulated blood samples immediately after sampling (t0) and 90 min (t90) after storage at room temperature. The presence of platelet agglutinates in blood samples was determined by examining blood smears using optical microscopy.PCs decreased at t90 with both anticoagulants. Platelet agglutinates were present at t90 in 27% of EDTA-samples vs. 2% of CPT-samples with decreased PCs (P < 0.001). Based on PCs in EDTA-samples, 15 patients (16%) shifted from a lower bleeding risk at t0 to a higher bleeding risk category at t90 (P 5 0.019), compared to 5 (5%) patients, based on PCs in CPT-samples. Therefore, time-dependent in vitro platelet agglutination in EDTA-blood samples may cause underestimation of PCs in thrombocytopenic patients, possibly leading to improper management.     

Selective decrease in platelet dense granule adenine nucleotides during recovery from acute experimental thrombocytopenia and ensuing thrombocytosis in baboons

Serial measurements of platelet volume, platelet content of adenine nucleotides, beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and ex vivo platelet aggregation were made in baboons under basal, steady-state conditions of normal platelet production, and during recovery from acute thrombocytopenia induced by the exposure of flowing blood to spherical glass microbeads. The mean basal platelet count of 509 +/- 107 X 10(9)/l (+/- 1 SD; n = 4) fell acutely to 36.8 +/- 12.6 X 10(9)/l after the insertion of glass bead columns and blood filters, placed distally, for 60 min into surgically implanted arteriovenous-shunts in heparinized baboons. After the irreversible removal of up to 90% of the baseline circulating platelet population, recovery from thrombocytopenia was characterized by a constant rate of increase in circulating platelet counts (115 +/- 11 X 10(9)/l/d) and a rebound thrombocytosis to 1.5 times the basal platelet count after 7 d. Steadystate thrombocytopoiesis was achieved by 3-4 weeks after the onset of thrombocytopenia. Platelet dense granule ADP and ATP decreased significantly from 3.89 +/- 0.20 and 2.33 +/- 0.25 mumol/10(11) platelets respectively at baseline to 2.17 +/- 0.37 and 1.68 +/- 0.37 mumol/10(11) platelets respectively after 7 d (P less than 0.001 in both cases) and normalization was achieved only after 4 weeks. By contrast, the mean platelet volume and platelet content of beta-TG and PF4 did not change significantly throughout the course of study (P greater than 0.1 in both cases). Platelet function, assessed by platelet aggregation ex vivo, demonstrated that platelet function was not impaired despite the significant decrease in dense granule ADP. We conclude that a selective, temporal reduction in platelet dense granule adenine nucleotides reflects changes in the thrombocytopoietic control mechanism secondary to induction of acute thrombocytopenia.     

Clopidogrel anti-platelet effect: an evaluation by optical aggregometry, impedance aggregometry, and the platelet function analyzer (PFA-100)

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.     

Increased peripheral platelet destruction and caspase-3-independent programmed cell death of bone marrow megakaryocytes in myelodysplastic patients

To investigate underlying mechanisms of thrombocytopenia in myelodysplastic syndrome (MDS), radiolabeled platelet studies were performed in 30 MDS patients with platelet counts less than 100 x 10(9)/L. Furthermore, plasma thrombopoietin and glycocalicin index (a parameter of platelet or megakaryocyte destruction) were determined. Mean platelet life (MPL), corrected for the degree of thrombocytopenia, was reduced in 15 of 30 patients (4.3 +/- 0.9 days [mean +/- SD] vs 6.0 +/- 1.3, P = .0003). Platelet production rate (PPR) was reduced in 25 of 30 patients (68 +/- 34 x 10(9)/d vs 220 +/- 65, P < .0001). Thrombopoietin levels were not significantly correlated with the PPR. However, the glycocalicin index was significantly higher compared with controls (15 +/- 16 vs 0.7 +/- 0.2, P = .001) and significantly correlated with the PPR (P = .02, r = -0.5), but not with the MPL (P = 1.8). Ultrastructural studies demonstrated necrosis-like programmed cell death (PCD) in mature and immature megakaryocytes (n = 9). Immunohistochemistry of the bone marrow biopsies demonstrated no positive staining of MDS megakaryocytes for activated caspase-3 (n = 24) or cathepsin D (n = 21), while activated caspase-8 was demonstrated in a subgroup of patients (5/21) in less than 10% of megakaryocytes. These results indicate that the main cause of thrombocytopenia in MDS is caspase-3-independent necrosis-like PCD resulting in a decreased PPR in conjunction with an increased glycocalicin index.     

Platelet contractile force (PCF) and clot elastic modulus (CEM) are elevated in diabetic patients with chest pain.

AIMS: Platelet function and clot structure may be altered in diabetes. We have noted increased platelet contractile force (PCF) and clot elastic modulus (CEM) in patients presenting to the emergency department with chest pain. Twenty-six of the chest pain patients were diabetic. Here, we compare the PCF, CEM and platelet aggregation in diabetic chest pain patients, non-diabetic patients with chest pain and asymptomatic controls. PATIENTS AND METHODS: PCF, CEM and collagen whole blood aggregations were measured in 100 chest pain patients and 25 asymptomatic controls. RESULTS: Platelet concentrations for diabetic patients, non-diabetic patients and controls were identical. PCF was significantly (P < 0.05) elevated in diabetic chest pain patients (9.42 +/- 0.59 kdynes) vs. controls (7.40 +/- 0.32 kdynes). CEM in diabetic patients (29.96 +/- 2.19 kdynes/cm2) was significantly elevated relative to that in non-diabetic chest pain patients (25.22 +/- 0.84 kdynes/cm2) and normal controls (23.18 +/- 0.74 kdynes/cm2). Collagen-induced whole blood aggregation was decreased (P < 0.05) in diabetic chest pain patients vs. controls. PCF values (10.23 +/- 0.76 kdynes) in diabetic patients with haemoglobin A1c > 7% were higher than in any other group. CONCLUSION: PCF and CEM are elevated in diabetic chest pain patients. The significance of these laboratory findings awaits additional clinical studies.     

Impact of body mass index on platelet aggregation after administration of a high loading dose of 600 mg of clopidogrel before percutaneous coronary intervention

This study assessed the effect of body mass index (BMI) on platelet aggregation after administration of a high loading dose of clopidogrel 600 mg. Blood samples of 402 patients before percutaneous coronary intervention were collected >or=2 hours after administration of clopidogrel 600 mg. Platelet aggregation was measured in response to adenosine diphosphate (ADP; 5 and 20 microM). Patients were categorized as normal weight (BMI <25 kg/m(2)) or overweight (BMI >or=25 kg/m(2)). ADP-induced platelet aggregation was significantly higher in overweight patients than in normal-weight patients (46.0 +/- 21.8% vs 38.2 +/- 19.3% for ADP 5 microM, p = 0.0007; 55.1 +/- 22.7% vs 45.2 +/- 21.7% for ADP 20 microM, p <0.0001). Multivariate analyses demonstrated high BMI as the only independent predictor for increased ADP-induced platelet aggregation (p 相似文献   

肝硬化患者血小板计数与血小板生成素及脾脏指数间的关系

目的 研究肝硬化患者外周血血小板计数、血清血小板生成素(TPO)水平及脾脏指数间的关系,以明确肝硬化患者血小板减少的原因。方法 血清TPO水平采用酶联吸附法检测,脾脏指数由同一医生在相同的条件下用彩色多普勒超声仪测量。结果 健康对照组与肝硬化组外周血小板计数分别为(169.63±26.60)×10~(12)/L、(109.20±53.39)×10~(12)/L,t=3.630,P<0.05;血清TPO水平分别为(412.63±132.80)pg/ml、(436.42±258.97)pg/ml,t=0.272,P>0.05。随着肝脏损伤的加重,血清TPO水平依次下降,Child-Pugh A级、B级、C级分别为(526.13±317.44)pg/ml、(445.22±214.90)pg/ml、(311.45±182.66)pg/ml,A级与C级之间差异有显著性,而其它指标(血小板计数、脾脏指数、门静脉宽度)A级与B级、C级之间差异有显著性。血小板正常组(35例)与血小板减少组(36例)血清TPO水平分别为(529.43±282.64)pg/ml、(351.27±228.25)pg/ml,t=-2.926,P<0.01;而两组间脾脏指数分别为(19.65±12.00)cm~2、(36.35±12.68)cm~2,t=1.891,P>0.05。结论 血清TPO水平降低可能是肝硬化患者血小板减少的原因之一,脾脏在血小板减少中的作用有待进一步研究。     

Gorog Thrombosis Test: analysis of factors influencing occlusive thrombus formation.

We used the Gorog Thrombosis Test to analyze the factors influencing the occlusion time, which represents platelet activation and subsequent occlusive thrombus formation, in 132 healthy Japanese volunteers (116 men, 16 women; mean age, 45.0 +/- 12.0 years). The Gorog Thrombosis Test was designed to evaluate platelet aggregation and thrombolytic activity under a high shear stress condition (175 dynes/cm) in a native blood sample in vitro. The mean +/- SD occlusion time was 154.8 +/- 64.7 s (men, 153.4 +/- 64.2 s and women, 165.4 +/- 56.5 s). The occlusion time was inversely correlated with von Willebrand factor ristocetin cofactor activity (VWF:Rco) (r = -0.242, P = 0.0055) and von Willebrand factor antigen (r = -0.230, P = 0.0080). The mean occlusion time in the group with VWF:Rco of at least 170% (137 s) was significantly shorter than that in the group with VWF:Rco less than 170% (156 s, P < 0.05). Platelet counts, other coagulation markers and smoking showed no significant correlations with occlusion time. Red blood cells (r = -0.177, P = 0.0365), hemoglobin (r = -0.191, P = 0.0245) and hematocrit (r = -0.182, P = 0.0329) also showed inverse correlations with the occlusion time. This report is the first to clearly demonstrate the role of von Willebrand factor in the formation of occlusive thrombi in the Gorog Thrombosis Test.     

'Spontaneous' platelet aggregation in whole blood in diabetic patients with and without microvascular disease.

Consistent abnormalities of agonist-induced platelet aggregation, in either whole blood or platelet rich plasma, have not been demonstrated in diabetic patients without microvascular disease. In the present study platelet aggregation in the absence of exogenous agonists ('spontaneous' aggregation) was compared between 22 non-diabetic subjects and 23 Type 1 diabetic patients with (n = 12) and without (n = 11) microvascular disease. 'Spontaneous' aggregation was determined by measuring the percentage fall in single platelet number in aliquots of whole blood shaken for 60 min. Diabetic patients without microvascular disease had fewer single platelets remaining (greater aggregation) than non-diabetic subjects at all time-points (69.7 +/- 6.6 vs 82.3 +/- 7.3% at 60 min p less than 0.001), but more platelets remaining than in diabetic patients with microvascular disease at all time-points (69.7 +/- 6.6 vs 61.0 +/- 7.8% at 60 min p less than 0.02). No significant correlations were observed between platelet aggregation and plasma glucose, blood cell counts, or glycated haemoglobin levels. The study suggests that platelet abnormalities antedate the appearance of microvascular disease in diabetic patients.     

Paraformaldehyde fixation induces a systematic activation of platelets

In whole blood flow cytometric platelet assays sample fixation using paraformaldehyde (PFA) is considered very advantageous to prevent spontaneous activation of platelets in vitro. However, fixation is an important variable in activation assays and its influence on platelets is poorly understood. Using a direct immunofluorescence labelling technique and whole blood flow cytometry, the effect of PFA fixation was investigated for 4 different epitopes on platelet surface each of which mirrors a different aspect of platelet activation, namely P-selectin (CD62P), GP IIbIIa complex (CD41), the fibrinogen binding site of the activated GP IIaIIIb complex (PAC-1) and GP Ib-V-IX complex (CD42b). Platelets fixed with PFA (0.5%) before antibody labelling showed significant (P<0.01) increases in mean fluorescence intensity (MFI) of CD62P (1.10 +/- 0.14 vs. 0.94 +/- 0.12 arbitrary units of fluorescence), CD41 (27.3 +/- 6.3 vs. 15.6 +/- 2.1) and PAC-1 (6.21 +/- 1.25 vs. 0.55 +/- 0.12) when compared to unfixed samples. At the same time, MFI of CD42b was reduced from 28.2 +/- 1.6 to 22.6 +/- 2.3 (P<0.01). When fixation was initiated after antibody labelling, we observed less prominent increases in MFI of CD41 (P<0.05) and PAC-1 (P<0.05) while there was no significant difference for CD62P and rather a moderate rise in CD42b than a decrease (P<0.05). Because these alterations cannot be explained by unspecific effects only, it must be concluded that PFA induces a systematic stimulation of platelets. The lowest in vitro platelet activation was found when antibody labelling was started immediately after blood sampling and when samples were analysed within 10 minutes after being stored without fixation of 4 degrees C in the dark.