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Anesth Analg 2004;99:1440-1444 © 2004 International Anesthesia Research Society doi: 10.1213/01.ANE.0000134805.30532.59
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Abstract |
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In this study, we characterized clot morphology with a scanning electron microscope (SEM) at time points corresponding to the commonly used thrombelastography (TEG®) variables, illustrating the correlation of the physical clot formation with TEG® tracings. The first channel of the TEG® analyzer was used to obtain the tracings of clot formation, while the sub-samples for the SEM were obtained from the second TEG® channel. Different types of samples were examined, including whole blood, abciximab-treated whole blood, platelet-rich plasma (PRP), and abciximab-treated PRP. The SEM images were obtained at reaction time, different amplitudes (5–30 mm), maximum amplitude (MA), and at amplitude 60 min after MA. In the whole blood, coarse fibrin and activated platelets were observed at reaction time and fibrin strands progressively became more solid and intertwined at amplitude 10 mm and thereafter. Red blood cells were surrounded with fibrin strands at amplitude 30 mm and were tightly packed by fibrin strands at MA. In abciximab-treated whole blood, red blood cell shape was maintained at MA. The process of fibrin formation and platelet activation was also examined in PRP. Abciximab did not block platelet shape change, although the blockage of fibrin binding to platelets was shown on the TEG® analyzer. In summary, we have shown structural changes of the forming clot in relation to TEG® variables.
IMPLICATIONS: We have shown structural changes of the forming clot in relation to thrombelastography variables using scanning electron microscopy. Clot structure was examined in the whole blood and platelet-rich plasma in the presence and the absence of abciximab.
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Introduction |
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Thromboelastography (TEG®) is a bedside monitor of coagulation (1,2). Since its inception by Hartert in 1948, multiple variables have been used to describe different phases of clotting, but there is a paucity of data regarding the correlation between the TEG® tracing and the clot morphology. The TEG® instrument traces the viscoelasticity of the forming clot between a suspended pin and a cuvette wall; therefore, the result may be affected by various plasma and cellular components of coagulation. We hypothesized that the scanning electron microscopy (SEM) would allow us to determine whether the TEG® analyzer output would reflect structural changes of the forming clot. Furthermore, we questioned whether different sample preparations (whole blood [WB] or plasma) or modifications (abciximab addition) would change the TEG® tracing and clot morphology to the same extent. We aimed to characterize the clot morphology with SEM at time points corresponding to commonly used TEG® variables.
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Methods |
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The study was approved by the IRB of Saitama Medical School. After written informed consent was obtained, blood samples were collected from six healthy volunteers with no history of aspirin ingestion or other medications that might interfere with coagulation over the preceding 2 wk. The blood sample (10 mL) was obtained from an antecubital vein using a two-syringe technique to avoid tissue factor contamination and was immediately mixed with 3.2% trisodium citrate in a volume ratio of 1:9.
For the preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP), citrated WB samples were centrifuged at 240g for 10 min and at 2500g for 10 min, respectively, at room temperature. Then, PRP was diluted with PPP to adjust the platelet count to 200 x 103/µL. In addition, portions of the WB samples were preincubated with abciximab (ReoPro®; Centocor, Malvern, PA), with a final concentration of 25 µg/mL) for 10 min at room temperature to obtain abciximab-treated WB and PRP.
The two-channel TEG® (Model 3000T; HaemoscopeTM Corp, Niles, IL) analyses were performed using 340 µL recalcified WB or PRP samples with 20 µL of 0.2 M CaCl2. The first channel was used to obtain the TEG® traces and the second channel was used to obtain subsamples for microscopy at various time points. TEG® tracings and subsamples were obtained from the four types of blood samples (n = 3 each): WB, WB with abciximab pretreatment (WB+abciximab), PRP, and PRP with abciximab pretreatment (PRP+abciximab). TEG® analyses and subsampling were performed solely by one investigator (JK) to avoid interoperator variability. For TEG® results in each sample category (WB or PRP), data were compared using paired Student’s t-test. A P value of <0.05 was considered significant.
Morphological evaluation of the clot formation with a SEM (S-510; Hitachi High-Technologies, Tokyo, Japan) was conducted by an experienced technician who was blinded as to the type (WB or PRP) and sequences (time points) of samples. On the basis of the commonly used TEG® variables (Fig. 1, top), the following time points were examined: start of TEG® analysis (baseline); reaction time (R time); amplitudes of 5, 10, 20, or 30 mm (A5, A10, A20, or A30); maximum amplitude (MA), and 60 min after MA.
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Specimens were taken at a midpoint between the pin and cuvette wall, with an approximate depth of 3 mm from the surface of the blood. The subsamples were fixed immediately in 2.5% glutaraldehyde in 0.1M cacodylate buffer-2% sucrose, pH 7.2 for morphological examination by SEM. To assure the consistency of SEM images, at least 8 micrographs were obtained for each sample point and 6 similar images were selected by the aforementioned technician. We subsequently chose one representative image.
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Results |
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The mean ± SD values of TEG® variables obtained from the six subjects are presented in Table 1. The representative traces from TEG® analysis with the WB, WB with abciximab, PRP, and PRP with abciximab are shown in Figure 1.
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At time point R, scattered, coarse fibrin and platelet filopods were already seen (Fig. 2, panel I). Fibrin attachment to the platelet with lamellipods was observed at A10 point (Fig. 2, panel II). At K time (the rate of early clot formation) (A20 point), fibrin strands were more clearly visualized and some red blood cells (RBCs) were bundled among fibrins (Fig. 2, panel III).
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RBCs were surrounded with a thick mesh of fibrin strands at A30 point (Fig. 2, panel IV). Subsequently, RBCs were tightly packed and deformed by fibrins at MA point (Fig. 2, panel V). At 60 min after MA, it seemed that gaps between RBCs were wider than those at MA point, although fewer differences in the alteration of morphology of RBCs and fibrin strands were generally observed (Fig. 2, panel VI).
A discoid platelet was observed at 5 min after the start (Fig. 2, panel VII). At time R, coarse fibrin and platelets with filopods were seen (Fig. 2, panel VIII). At A5 point, extensive platelet activation with filopods was seen, but the network of fibrin strands still appears to be immature (Fig. 2, panel IX). RBC shapes were still maintained at MA point (Fig. 2, panel X).
At baseline, a few platelets showed signs of activation during PRP preparation (Fig. 3, panel I). At time point R, platelets with filopods and scattered fibrin were observed (Fig. 3, panel II). At A5 point, platelets with filopods were connected with fibrin strands (Fig. 3, panel III), and platelets and fibrin formed a large aggregate at A10 point (Fig. 3, panel IV). Beyond A10 point, delineating platelet and fibrin structure was technically impossible as a result of extensive aggregation and clotting.
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Scattered fibrin and a platelet with filopods were seen at time point R (Fig. 3, panel V). Fibrin strands formed a solid and thick mesh at A10 point (Fig. 3, panel VI).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We investigated morphological changes in the developing clot in relation to TEG® variables using a SEM. Platelet activation took place before R time was reached, and intense fibrin mesh was generated after K time was recorded. As shown in the microscope figures for WB samples with or without abciximab, the strength of the clot was reflected in the extent of deformed RBCs.
TEG® is a viscoelastic monitor that measures the strength of fibrin-platelet bonds that link a rotating cup and a suspended pin. TEG® analysis can provide quantitative and qualitative information on clot formation and clot lysis. Multiple variables were proposed to interpret TEG® tracings and to correlate them with clinical coagulation status (1).
For TEG® analysis, R time has been defined as duration from the start of measurement until the amplitude of 2 mm is reached. This onset is generally affected by anticoagulants such as heparin or low molecular weight heparin (3) but not by antiplatelet drugs per se (4). Our data showed that abciximab did not cause significant prolongation of R time (Table 1), and SEM also showed that early coarse fibrin formation is present regardless of platelet inhibition with abciximab (Fig. 2, panel VIII).
When we closely examined platelets using PRP, activated platelets with filopods were well visualized at the R time (Fig. 3, panel II). Extensive platelet aggregation occurred when the amplitude of 10 mm was achieved (Fig. 3, panel IV). The sequence of events was quite similar to the platelet aggregation in PRP stimulated with collagen (5).
K time is defined as the rate of early clot formation (duration from R time until the amplitude 20 mm is achieved). Because K time is shown only after the amplitude reaches 20 mm on computerized TEG® analyses, K time for abciximab-treated WB (mean MA, 16.2 ± 1.0 mm) is not shown in Table 1.
An additional kinetic variable, 
-angle, is closely related to K time. The -angle reflects the rapidity of fibrin buildup and cross-linking, as we clearly demonstrated with serial electron micrographs (Fig. 2, panels II, III, and IV). Our results showed that K time and -angle were both affected by abciximab, which inhibits platelet-fibrin interaction by blocking glycoprotein IIb/IIIa receptors (Table 1). Reduced interaction between platelets and fibrin because of abciximab resulted in a smaller MA, the peak strength of the clot (Table 1 and Fig. 1).
The contribution of platelets to the structural rigidity of the fibrin network was clearly shown in our WB and WB+abciximab micrographs (Fig. 2, panels V and X). The cobblestone appearance of deformed RBCs disappeared when the sample was pretreated with abciximab. The tension between TEG® pin and cup presumably increases as a result of the contraction of platelet cytoskeletal actin fibers (4,6).
Interestingly, we observed platelet filopods and fibrin fiber formation on the electronmicrograph despite abciximab treatment, which clearly decreased TEG®-MA (Fig. 1; Fig. 2, panels IX-X; Fig. 3, panels V-VI). Because platelets are activated by thrombin on the TEG® analyzer, platelet activation and shape change could take place even though binding of platelets to fibrin is blocked by abciximab treatment. The incomplete inhibition of platelets with abciximab is in agreement with Gawaz et al. (7), who demonstrated exposure of unblocked platelet glycoprotein IIb/IIIa receptors after stimulation with a thrombin-receptor activating peptide.
A very small amount of thrombin is needed to cleave fibrinogen and to activate platelets (8). However, a larger amount of thrombin is required to construct a more stable complex of platelet-fibrin polymers (9). This basic principle explains why conventional clotting tests, such as prothrombin time (PT) and partial thromboplastin time (PTT), do not correlate with actual bleeding.
Although a clot forms rather quickly (10–35 seconds) in the presence of a coagulation trigger during PT/PTT tests, the end-point of these tests is the early product of fibrin clots, which may not reflect the strength of the final form of the clot (10).
In addition, PT and PTT are typically performed with added phospholipids (replacement for activated platelets) using PPP, which precludes any estimation of platelet procoagulant activity in an individual patient. As we have shown in our study, clot formation and its strength can be monitored with TEG® using native WB, which may provide advantages over the conventional clotting tests (11,12).
In PRP with a platelet count adjusted to 200 x 103/µL, MA values of TEG® were larger than WB with comparable platelet counts (mean, 246 ± 22 x 103/µL) (Fig. 1). Because the mass of RBCs is replaced with platelet-containing plasma in PRP, the total amount of platelets and fibrinogen in an individual cup (360 µL) is larger in PRP samples than in WB samples. RBCs contribute to coagulation in vitro or in vivo by releasing adenosine diphosphate (a platelet agonist) and by mass effect (margination of platelets to the wall) (13,14); thus additional studies are needed to evaluate this effect on TEG® analysis.
In summary, we have shown the different phases of clot formation in relation to the TEG® variables (R, K, -angle, and MA) with structural changes of the forming clot. Although the analyses of the SEM images were rather descriptive, the current study clearly depicts the thrombin-mediated activation processes of fibrin and platelets on the TEG®. It was shown that abciximab effectively reduces clot strength, but some thrombin-mediated platelet activation still takes place.
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Acknowledgments |
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The authors thank Shinji Itoyama, MD, of the Department of Pathology, Saitama Medical Center, for his support in data collection using the scanning electron microscope.
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Footnotes |
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Presented, in part, at the IARS 77th Clinical & Scientific Congress, New Orleans, Louisiana, March 24, 2003.
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References |
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- Tuman KJ, Spiess BD, McCarthy RJ, Ivankovich AD. Comparison of viscoelastic measures of coagulation after cardiopulmonary bypass. Anesth Analg 1989; 69: 69–75.
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- Oshita K, Az-ma T, Osawa Y, Yuge O. Quantitative measurement of thromboelastography as a function of platelet count. Anesth Analg 1999; 89: 296–9.
[Free Full Text] - Gawaz M, Ruf A, Pogatsa-Murray G, et al. Incomplete inhibition of platelet aggregation and glycoprotein IIb-IIIa receptor blockade by abciximab: importance of internal pool of glycoprotein IIb-IIIa receptors. Thromb Haemost 2000; 83: 915–22.[Web of Science][Medline]
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