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 unecessary.     

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.     

The Role of Thrombin in ADP-Induced Platelet Aggregation and Release: a Critical Evaluation

S ummary . The role of thrombin in ADP-induced aggregation and release in vitro was critically examined. The addition of heparin or hirudin to citrated platelet rich plasma did not prevent aggregation or release. The addition of citrate to heparinized plasma restored secondary aggregation and release. Hirudin did not prevent irreversible aggregation. These results are incompatible with the hypothesis that thrombin is a primary and necessary mediator of platelet aggregation (Ardlie & Han, 1974; Han & Ardlie, 1974a, b, c). This hypothesis is based in part on the assumption that EDTA enhances the elution of clotting factors from platelets; we found no enhanced elution of factors II, V, X, VIII, IX or XI when platelets were washed in 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.     

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.     

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.     

The mechanism of heparin-induced platelet aggregation

When heparin was added to platelet-rich plasma, mild but irreversible platelet aggregation was demonstrated. This platelet response was not accompanied by release of alpha-granules and dense body constituents, nor by prostaglandin biosynthesis. It did, however, require metabolic energy and divalent cations as metabolic inhibitors (anti-mycin A and 6-deoxyglucose) and EDTA blocked the reaction. Bernard-Soulier syndrome platelets, which lack glycoprotein (GP) Ib, but not Glanzmann's Thrombasthenia platelets, which lack GP IIb/IIIa, were aggregated by heparin. Monoclonal antibody (mAb) against GP IIb/IIIa, but not mAb against GP Ib, strongly inhibited the reaction. These combined results suggest the participation of GP IIb/IIIa but not GP Ib in heparin-induced platelet aggregation. Fibrinogen was a cofactor in the reaction as gel-filtered platelets were unreactive to heparin but addition of fibrinogen restored their reactivity. Antithrombin III and fibronectin inhibited platelet response to heparin, suggesting that these proteins may protect platelets from aggregation by heparin.     

Effective estimation of correct platelet counts in pseudothrombocytopenia using an alternative anticoagulant based on magnesium salt

Pseudothrombocytopenia remains a challenge in the haematological laboratory. The pre‐analytical problem that platelets tend to easily aggregate in vitro, giving rise to lower platelet counts, has been known since ethylenediamine‐tetra acetic acid EDTA and automated platelet counting procedures were introduced in the haematological laboratory. Different approaches to avoid the time and temperature dependent in vitro aggregation of platelets in the presence of EDTA were tested, but none of them proved optimal for routine purposes. Patients with unexpectedly low platelet counts or flagged for suspected aggregates, were selected and smears were examined for platelet aggregates. In these cases patients were asked to consent to the drawing of an additional sample of blood anti‐coagulated with a magnesium additive. Magnesium was used in the beginning of the last century as anticoagulant for microscopic platelet counts. Using this approach, we documented 44 patients with pseudothrombocytopenia. In all cases, platelet counts were markedly higher in samples anti‐coagulated with the magnesium containing anticoagulant when compared to EDTA‐anticoagulated blood samples. We conclude that in patients with known or suspected pseudothrombocytopenia the magnesium‐anticoagulant blood samples may be recommended for platelet counting.     

Substantial reduction of platelet adhesion by heparin-coated stents

Although optimized antiplatelet medication has improved the clinical outcome after coronary stenting, vessel occlusion and restenosis still remain a relevant clinical problem. Platelets play a key role in this process. Therefore, the authors compared the platelet adhesion on different stent surface modifications (electropolished without coating or coated with carbon, carbon and additional heparin, silicon carbide, or heparin alone) to investigate their role in reducing platelet adhesion. All stents and additional stainless steel plates were incubated in heparinized whole blood with radiolabeled platelets. After washing the stents and plates four times, radioactivity caused by the adhesion of radiolabeled platelets was measured. The adhesion of radiolabeled platelets, compared to uncoated, electropolished stents, was reduced through silicon carbide coating to 58.6%, by carbon coating with additional heparin to 32.9%, and heparin coating alone to 7.7%. Stent coating with heparin is the most effective among the examined coatings in reducing platelet adhesion in vitro.     

Pseudo grey platelet syndrome--grey platelets due to degranulation in blood collected into EDTA

We have studied a woman with a history of mild bruising and bleeding, with a normal platelet count and normal clotting factors, who had platelets that appeared grey when stained and viewed under the microscope. Unlike the grey platelet syndrome, the abnormality was only evident when blood had been collected into EDTA and not when citrate or heparin was used as anticoagulant. This 'pseudo grey platelet syndrome' was associated with platelet dense body and alpha granule secretion with no aggregation and occurred on removal of extracellular Ca2+. We discovered that a plasma factor was responsible which could be an immunoglobulin but which is clearly different from the EDTA-sensitive antibodies which cause platelet aggregation and agglutination. We were not able to demonstrate a relationship between the mild bleeding tendency and the in vitro abnormality.     

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.     

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.     

Differing platelet aggregating effects by two tumor cell lines: Absence of role for platelet-derived ADP

Two different mechanisms of aggregation of heparinized human platelet-rich plasma have been identified with two tumor cell lines: In neither case are these mechanisms dependent on platelet-derived ADP. U87MG cells from a glioblastoma line of human origin caused a single irreversible wave of aggregation simultaneously with the onset of platelet secretion, and this was inhibited by heparin and hirudin but not by apyrase or phospholipase D. In contrast, Hut 20 cells from an undifferentiated tumor cell line of murine origin gave an initial reversible wave followed by a second irreversible wave, which then led to secretion. The first wave of platelet aggregation was unaffected by heparin or hirudin but was inhibited by apyrase, and the second wave was inhibited by phospholipase D. Citrate caused irreversible inhibition with either cell line, and aggregation did not occur with gel filtered platelets. These results suggest that platelet aggregation by the Hut 20 line is initially dependent on ADP released from the tumor cells, whereas aggregation induced by the U87MG line is dependent on a procoagulant activity of the tumor cell surface.     

Complications of heparin administration in normal individuals

The incidence, severity, and pathogenic mechanism of heparin-associated thrombocytopenia with both bovine and porcine heparin administration were studied in forty normal males randomized to one of four treatment groups: beef lung heparin #1, beef lung heparin #2, porcine gut heparin, and saline control. All of the subjects receiving heparin developed a reversible increase in serum transaminases (SGOT, SGPT). However, other measurements of liver function were normal. Thirty-three percent of these heparinized normals had decreased platelet counts. The incidence of platelet count decrease was similar for both bovine and porcine heparins, but 4 of the 20 normals receiving bovine heparin had platelet counts less than 150,000/microliters. Immune pathogenesis was investigated by analyzing plasma from the volunteers for both platelet antibody and immune complexes. None of the men had increased platelet-associated IgG. Among the ten subjects with decreased platelet counts, IgG immune complexes were detected in three and C1q in seven. The heparin-associated thrombocytopenia appears not to be mediated by a platelet antibody. More probably it reflects a direct effect of the heparin on platelets.     

Effect of heparin on platelet aggregation

The effect of heparin on platelet aggregation was systematically examined on platelets in plasma (PRP), as well as on gel-filtered, washed, and formaldehyde-fixed platelets. Results indicate that, although heparin causes a mild potentiation of platelet aggregation in the PRP systems, a significant inhibitory activity is observed when heparin is added to isolated platelets. This inhibitory activity appears to be specific and not related to the impurities in the heparin preparations, as heparinase, as well as protamine, effectively neutralizes the heparin-mediated inhibitory activity on platelet aggregation. Although heparin-mediated inhibitory activity can be demonstrated in the presence of a number of different agonists (ADP, arachidonic acid, thrombin, Ionophore A23187, epinephrine, and ristocetin), the most pronounced inhibition is seen in the presence of ristocetin. Further studies show that heparin enhances thromboxane generation in isolated platelets. Platelets pretreated with heparin, however, fail to respond to preformed thromboxane. These findings suggest that, in addition to the potentiation of thromboxane production in platelets, heparin may also attribute some change(s) to the platelet(s)/platelet membrane, which interferes with their ability to respond to the agonists of platelet aggregation. This antiaggregatory activity of heparin was found to be inhibited by a factor(s) present in plasma but not in serum.     

'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.     

探讨药物对EDTA依赖性血小板聚集的抑制和解离作用

目的:探讨丁胺卡那霉素、氟化钠在体外对EDTA依赖性血小板聚集的抑制和解离作用,为临床EDTA依赖性假性血小板减少症患者提供更好的解决方案。方法:应用血液分析仪检测添加了氟化钠或不加药物的EDTA抗凝血中血小板的数目,对比它们在不同时段的变化。结果:即时或1h内加入药物,2种药物对EDTA依赖性血小板聚集抑制作用效果相近,均能显著地抑制或解离EDTA依赖性血小板聚集;但1h后加入药物,2种药物对EDTA依赖性血小板聚集的解离作用微弱。结论:1h内加入丁胺卡那霉素、氟化钠对EDTA依赖性血小板聚集有很好的抑制或解离作用,1h后加入丁胺卡那霉素、氟化钠对EDTA依赖性血小板聚集的解离作用不佳。     

Different platelet specificities of heparin-dependent platelet aggregating factors in heparin-associated immune thrombocytopenia

Delayed onset heparin-associated thrombocytopenia (HAT) is thought to be a result of formation of antiplatelet antibodies which cause platelet aggregation in the presence of heparin. Platelet aggregation in response to serum from patients with HAT has been studied in platelet-rich plasma (PRP) from a panel of normal blood donors. Heparin-dependent aggregation with any HAT serum occurred in PRP from only some donors. PRP from the non-responding donors did, however, aggregate in the presence of heparin with other HAT sera. The same patterns of aggregation or lack of response to HAT sera were seen in washed platelet suspensions. Heparin (0.06-2 U/ml) did not cause aggregation in the presence of normal serum with PRP from these donors. However, in PRP from four of the 17 individuals studied, heparin (0.25-1 U/ml) alone caused rapid platelet aggregation and some HAT sera heated at 56 degrees C caused platelet aggregation without added heparin. Sub-aggregating concentrations of adrenaline could replace heparin in promoting aggregation by heated HAT sera in PRP of the other donors. HAT IgG showed the same platelet specificities as the serum in causing either heparin- or adrenaline-dependent aggregation. Thus in HAT, antibodies are directed towards different platelet antigens which are expressed differently in different individuals. Platelet activation by heparin and adrenaline either exposes these antigens or causes aggregation of antibody-coated platelets.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Measurement of platelet aggregation in diabetics using the new electronic platelet aggregometer

Platelet function has been studied in diabetic subjects using a new electronic platelet aggregometer which enables platelet aggregation to be studied in whole blood. This may be a more physiological approach to the assessment of platelet behaviour as centrifugation is avoided and platelets are studied in the presence of other blood elements which may be important modulators of platelet function in vivo. Twenty insulin-dependent diabetic subjects were studied along with 20 age and sex-matched controls. Platelet aggregation to collagen (1 microgram/ml) and arachidonic acid (1 mM) was significantly increased in the diabetic group. In addition the sensitivity of diabetic platelets to the antiaggregatory effects of prostacyclin was significantly reduced. A significant inverse correlation was found between platelet sensitivity to prostacyclin and glycosylated haemoglobin concentration in the diabetic group. It is unlikely that the platelet abnormalities in this diabetic group are due to underlying vascular disease as none of the patients had evidence of diabetic complications. These findings may have important implications for the development of vascular disease in diabetics.