Abstract

Background

The development of advanced surgical procedures requires novel and early diagnostic techniques. One of the prime difficulties that need to be overcome is an abnormality in the coagulation system. The blood clot formation and fibrinolysis processes are very complicated and important perioperatively. However, most of the major surgeries, such as liver transplantation, use thromboelastography (TEG) for detection of coagulation abnormalities, even though TEG is not actually an ideal option. Therefore, we compared the sensitivity and specificity of the platelet function analyzer (PFA-100^®) and thromboelastogram (TEG^®) in predicting platelet dys- function and bleeding risk.

Methods

Human blood samples were drawn from healthy volunteers for this study. Levobupivacaine and CGS21680 have antiplatelet effects which were used as the detection target. The platelet counts before comparison, platelet aggregation, the closure time of PFA-100^®, and the parameters of TEG^® were examined for data analysis.

Results

Platelet aggregations were suppressed by all levobupivacaine doses (10 mg/ mL, 50 mg/mL, 200 mg/mL) and CGS21680 (100 nM, 500 nM, 1 mM) in a dose-dependent manner. Levobupivacaine and CGS21680 at maximal test doses produced no significant alteration in any parameter in the TEG^® assay. In the samples measured with PFA-100^®, both levobupivacaine and CGS21680 at maximal test doses significantly prolonged the closure time in the PFA-100^® assay.

Conclusion

We conclude that PFA-100^® offers a higher sensitivity and specificity than TEG^® in detection of platelet dysfunction.

Keywords

platelet function tests; thromboelastography;

1. Introduction

Early detection and appropriate correction of coag-ulation abnormalities are critical events in patients either under critical care or during surgery. Several modalities have been developed for diagnostic pur-poses, such as platelet aggregometry/whole blood aggregometry,  thromboelastography  (TEG)  and  a  platelet function analyzer (PFA-100^®). Whole blood impedance aggregometry and turbidometric plate-let aggregometry remain the current de facto “gold standard” tests for platelet function in the labora-tory because of their ability to measure glycoprotein (GP) IIb/IIIa (integrin α_IIbβ₃)-related platelet aggre-gation,  but,  unfortunately,  platelet  aggregometry  per se has several limitations in clinical practice, including a relatively large sample volume, poor re-producibility, requirement of delicate sample prep-aration,  lengthy  assay  time,  skillful  handling  and  relatively high cost. Consequently, options such as TEG and PFA-100^® for platelet function testing were designed to cope with the needs of bedside care and to  overcome  the  disadvantages  described  above.  Therefore, the rationale for the usefulness of PFA-100^® and TEG in clinical application requires further evaluation.

The Thromboelastography Coagulation Analyzer (TEG^®) was designed for recording and evaluation of the whole process of clot formation and fibrino-lysis.¹ It has been suggested that TEG^® may provide valuable  information  about  coagulation  in  assess-ment of clinical conditions, such as postoperative hemorrhage and/or thrombosis during and following cardiovascular  surgery,  organ  transplantation  and  trauma.^2,3  The  PFA-100^®  analyzer  is  designed  to  mimic  the  platelet-associated  coagulation  under  shear stress, and is used to analyze the function of platelets in primary hemostasis. In detection of the closure time, the sampled blood passes through a collagen  membrane  channel,  coated  with  ADP  or  epinephrine, in which platelets are activated, aggre-gated, and finally form clots to occlude the blood flow.^4,5 The closure time indicates platelet reactiv-ity under different dynamic conditions for the pur-pose of distinguishing normal from abnormal platelet function. It has been demonstrated that PFA-100^® might operate as a fast screening tool for detection of platelet dysfunction.⁶

The aim of this study was to compare the two methodologies, TEG^®  and  PFA-100^®,  in  diagnosing  platelet-associated coagulation deficiency in clini-cal  practice  by  comparing  the  sensitivity  of  TEG^® with that of PFA-100^® in detection of CGS21680 or levobupivacaine-mediated  mild  antiplatelet  func-tion in blood of healthy volunteers. Both CGS21680 (adenosine  A2A  receptor  agonist)  and  levobupi-vacaine  (local  anesthetic)  are  weak  antiplatelet agents,²  which  have  been  well-studied  and  docu-mented in our laboratory (manuscript submitted), and were used for induction of platelet-associated coagulation abnormalities. We intended in this study to  compare  the  sensitivity  of  TEG^®  with  that  of  PFA-100^® in detection of a mild coagulation disor-der to select a faster and more reliable method to predict the risk of perioperative bleeding.

2. Methods

2.1. Blood sampling

With the approval of the Institutional Review Board of  Chang  Gung  Memorial  Hospital  and  written  in-formed consent from volunteers, the study was car-ried out in blood of 28 healthy subjects who did not take  any  medication  for  at  least  2  weeks  before  the study and were free from any blood disorder. From each volunteer 20 mL venous blood was drawn and put into a plastic tube containing 3.2% (0.105 M) sodium citrate and then mixed gently to avoid plate-let activation. A total of 560 mL of sampled blood was used in this study. The samples were allowed to stand at room temperature for 5 minutes and sub-sequently complete blood counts were then deter-mined by Hemavet 950 (Drew Scientific Inc., Shilling Way, Dallas, TX, USA) before the study.

2.2. Chemicals

All the chemicals used in these experiments were of the highest purity. CGS21680 was purchased from Sigma-Aldrich  Co.  (St  Louis,  MO,  USA).  Levobupi-vacaine  was  obtained  from  Abbott  Laboratories  (Abbott Park, IL, USA). Collagen/epinephrine (CEPI) and collagen/ADP (CADP) cartridges were purchased from Dade-Behring (Miami, FL, USA).

2.3. Platelet function PFA-100® studies

The PFA-100^® analyzer (Dade Behring) is intended for the study of blood coagulation by means of simula-tion of clot formation under the dynamics of a dam-aged living microcirculation, such as shear stress, which has been illustrated in detail by Kundu et al,⁵ to analyze the function of platelets in primary he-mostasis. Citrated blood samples were first left at room  temperature  for  10  minutes  and  then  incu-bated with either 50 or 200 μg/mL levobupivacaine, 1 μM  CGS21680,  or  vehicle  only  for  3  minutes  be-fore analysis. Samples were then transferred to two kinds of disposable cartridges coated with CADP or CEPI. While platelets were activated, blood plugs were formed and blood flow was occluded. The clo-sure times determined were the CADP closure time (CADP-CT)  and  the  CEPI  closure  time  (CEPI-CT),  which indicated platelet reactivity under different dynamic conditions for the purpose of distinguishing normal from abnormal platelet function and identi-fying possible aspirin-like effects. The maximal de-tection limit of the closure time was 300 seconds. When the time exceeded the limit, it was counted as 300 seconds.

2.4. TEG hemostasis analyzer studies

The  blood  samples  subjected  to  analysis  by  the  PFA-100^® analyzer were also cross-checked by the Thromboelastography  Coagulation  Analyzer  5000  (TEG^®; Haemoscope Corp., Niles, IL, USA) for com-parison of the whole process of clot formation and fibrinolysis.⁷ Therefore, TEG^® presented a complete picture of the balance or imbalance between clot formation and dissolution. In brief, citrated whole blood  (340  μL)  was  pretreated  with  50  μg/mL  or  200 μg/mL levobupivacaine, 1 μM CGS21680 or ve-hicle for 3 minutes and then samples were trans-ferred  to  a  kaolin  tube.  After  gentle  mixing,  the  samples were pipetted into a prewarmed cuvette containing 20 μL CaCl₂ (0.2 M) for automatic evalu-ation of clot formation and dissolution. Parameters indicating the mechanical properties were obtained after software analysis as follows: the clotting time (R value) represents the latency before initial fibrin formation. The clot kinetics could be characterized by the kinetic time (K value) and alpha angle which describes  the  speed  of  a  specific  strength  of  clot  formation  and  the  rapidity  of  fibrin  network  for-mation (clot strengthening). The maximum strength of clot formation (MA value) corresponds to the dy-namic binding properties between fibrin and plate-let via GP IIb/IIIa. The clot stability is measured by the amplitude reduction 30 minutes after MA.

2.5. Statistical analysis

All  experimental  results  are  expressed  as  mean  ± standard  deviation.  Statistical  analysis  was  per-formed  with  one  way  analysis  of  variance.  The  variance  within  each  group  was  checked  by  the  F-test  and  the  differences  between  groups  were  further checked by Dunnett’s multiple comparison test.   Statistical   significance   was   indicated   by   p < 0.05.

3. Results

3.1. Both levobupivacaine and CGS21680 inhibited ADP-induced platelet aggregation in whole blood samples in a dose-dependent manner

In  order  to  establish  the  basis  of  comparison  be-tween  TEG^®  and  PFA-100^®,  we  first  defined  the  suppressive effects and dosage range of levobupi-vacaine and CGS21680 in ADP-induced platelet ag-gregation.  Whole  blood  samples  were  incubated  with either levobupivacaine or CGS21680 at differ-ent doses and the relevant suppression of platelet aggregation  was  then  measured  by  whole  blood  aggregometer. As shown in Figure 1, platelet aggre-gation  represented  by  blood  impedance  was  sup-pressed  by  levobupivacaine  (10  μg/mL,  50  μg/mL, 200 μg/mL) (Figure 1A) or CGS21680 (100 nM, 500 nM, 1 μM) (Figure 1B) in a dose-dependent manner. The maximal inhibitory effects were observed with an increase in concentration of both levobupivacaine (50 μg/mL to 200 μg/mL) and CGS21680 (500 nM to 1 μM).  These  results  demonstrated  that  levobupi-vacaine and CGS21680 had a direct inhibitory effect on platelet aggregation, and also provided a basis to evaluate the sensitivity of TEG^® and PFA-100^® in detection of a coagulation abnormality.

Download full-size image

Figure 1 Assessment of the anti-aggregation effects of levobupivacaine and CGS21680 in ADP-induced whole blood aggregation. Graphs show the ADP (10 μM)-induced aggregation assay by whole blood impedance aggregometry. (A) The ADP-induced whole blood aggregation was suppressed by levobupivacaine in a dose-dependent manner (10 μg/mL to 200 μg/mL). (B) Similar patterns of anti-aggregation effects were also observed with CGS21680 (100 nM to 1 μM) treatment.

3.2. Neither levobupivacaine nor CGS21680 at maximal test doses had any significant effect on any parameter in the TEG® assay

We  first  examined  the  sensitivity  of  TEG^®  in  de-tecting the coagulation abnormality by treating the blood samples with various doses of levobupivacaine or  CGS21680  (Figures  2A−C  and  2E−G),  and  each  dose had been shown to inhibit platelet aggregation in whole blood aggregometry as described above. Both  the  levobupivacaine  and  CGS21680-treated  samples revealed no significant parametric altera-tions at different dosages, and the K value, R value, alpha angle and MA were recorded and compared with the control samples (Figures 3A−D). Interest-ingly,  increasing  K  values,  although  not  sufficient  to be significant, were observed in samples treated with high dose levobupivacaine (200 μg/mL) (Figure 3B).  However,  this  slight  and  inconclusive  result  indicated  that  TEG^®  cannot  detect  the  subtle  co-agulation abnormality mediated by both levobupi-vacaine and CGS21680.

Download full-size image

Figure 2 Representative thromboelastography (TEG®) profile of blood coagulation with levobupivacaine and CGS21680 treatment. Merged data (D) showed that there was no difference observed in samples treated with CGS21680 at different dosages (B, C) compared with the control group (A, E). Similarly, no significant difference in each parameter with low dose levobupivacaine (50 μg/mL) was observed (E, F). However, a slight alteration in reac-tion time (R) and coagulation time (K) was found with a high dose of levobupivacaine (200 μg/mL; G, H) but with no statistical significance (p > 0.05). MA = maximum strength of clot formation; alpha angle = speed of a specific strength of clot formation.

Download full-size image

Figure 3 Effects of various concentrations of levobupivacaine and CGS21680 on TEG® parameters R, K, α, and MA values in citrated whole blood. Statistical analysis of each TEG® parameter affected by CGS21680 and levobupi-vacaine revealed that neither levobupivacaine nor CGS21680 significantly altered R, K, α and MA values in TEG® experiments (A−D) (p > 0.05).

3.3. Both levobupivacaine and CGS21680 at maximal test doses significantly prolonged the closure time in the PFA-100® assay

We further examined the sensitivity of PFA-100^® in predicting the coagulation abnormality by treating the  blood  samples  with  various  concentrations  of  levobupivacaine (10 μg/mL, 50 μg/mL, 200 μg/mL) or CGS21680 (100 nM, 500 nM, 1 μM) which had been proven to inhibit platelet aggregation in whole blood aggregometry, whereas the PFA-100^® simulated the in vivo microcirculation with shear stress thus as-sessing the dynamic hemostasis represented by clo-sure time of the column. The baseline closure times of control healthy donor blood were 154.3 ± 27.1  sec-onds for CADP and 85.1 ± 7.9 seconds for CEPI car-tridges (Table 1). Levobupivacaine-treated samples revealed little prolongation of CADP-CT. However, a  significant  increase  in  CEPI-CT  was  observed  in samples treated with levobupivacaine at 200 μg/mL (Figure 4A), indicating that PFA-100^® was sensitive in detecting the levobupivacaine-mediated aspirin-like effect in this experimental setting. Furthermore, our data also demonstrated that CGS21680 (1 μM)-treated samples significantly prolonged the CADP-CT and CEPI-CT, as shown in Figures 4A and 4B and were consistent with our previous aggregation tests, sug-gesting that PFA-100^® was able to detect the subtle CGS21680-mediated coagulation abnormality.

Download full-size image

Download full-size image

Figure 4 Effect of levobupivacaine and CGS21680 in the platelet function analyzer (PFA-100®) experiments. The closure times (CT) in PFA-100® analysis were measured by collagen/ADP (ADP-CT) and collagen/epinephrine (EPI-CT) cartridge. The cut-off point for closure time was set at 300 seconds by the manufacturers. Any closure time greater than 300 seconds was recorded as 300 seconds. Data are shown as mean ± standard deviation. Our data showed that CGS21680 (1 μM) and levobupivacaine (200 μg/mL) significantly prolonged both ADP-CT and EPI-CT. *p < 0.05 when compared with baseline control.

4. Discussion

We compared the sensitivity of TEG^® and PFA-100^® to  determine  which  of  the  two  was  the  faster  screening tool for detection of a blood coagulation abnormality. For this purpose, two mild anticoag-ulants,  levobupivacaine  and  CGS21680,  were  em-ployed to induce a prolonged coagulation time in ex vivo blood samples collected from healthy volun-teers. We first demonstrated the antiplatelet prop-erties  of  both  levobupivacaine  and  CGS21680  by  whole blood platelet aggregometry in order to en-sure  the  feasibility  of  the  experimental  model  in  prolonged coagulation blood samples. In addition, we evaluated the responsiveness of both TEG^® and PFA-100^® to the blood samples treated with either levobupivacaine or CGS21680 at doses able to induce antiplatelet  aggregation.  Our  results  showed  that  PFA-100^® had higher sensitivity in detection of the blood  coagulation  abnormality  than  TEG^®  in  both  levobupivacaine and CGS21680-treated blood sam-ples, suggesting that PFA-100^® could be much better than TEG^® in the screening of platelet-associated bleeding disorders.

Some previous clinical reports were not consis-tent with our results. A study of platelets from 172 healthy parturients at term showed that there was no significant correlation between PFA-100^® closure time and platelet count, or between PFA-100^® clo-sure time and TEG MA. However, TEG MA revealed a good correlation with platelet count. Therefore the authors in that study concluded that the TEG was a better tool to evaluate coagulation in parturients with thrombocytopenia.⁸ Unfortunately, there was no  solid  evidence  to  validate  the  correlation  be-tween coagulation and thrombocytopenia in these patients. Other evidence also indicated that PFA-100® was relatively insensitive in detecting muco-cutaneous hemorrhages.⁹ Nonetheless, we found no indication  that  TEG^®  had  higher  sensitivity  in  the  same experimental setting. Furthermore, TEG has been suggested as having better predictivity than activated coagulation time in post-cardiopulmonary bypass (CPB) bleeding,¹⁰ and a reduction in hemo-static factor transfusion was achieved by heparinase-modified TEG.¹¹

However, the predictivity of TEG during CPB pro-cedures has also been shown to be controversial.¹² In uremic patients, skin bleeding time (SBT) is the well-established  predictor  of  bleeding  in  clinical  practice. Study of the correlation between SBT and other laboratory methods revealed that both TEG and PFA-100^® were poor predictors of SBT.¹³ Inter-estingly, evidence indicated that there was no cor-relation between postoperative chest drain and TEG variables, and thus failed to predict postoperative hemorrhage in cardiac patients.¹⁴ Moreover, using PFA-100^® for testing the platelet function in patients with cardiovascular disease and predicting the risk of myocardial damage appeared to have excellent positive results, in which patients with hypersensi-tivity to ADP (shorter CADP-CT < 90 seconds) were shown to have a higher risk for both recurrent events and death even with effective aspirin therapy.¹⁵
Our  statements  that  PFA-100^®  is  a  better  tool  for clinically relevant detection of platelet inhibi-tion could be further supported by a recent report in which PFA-100^® showed a high sensitivity of 84% in detection of diclofenac-induced anticoagulation with no false positive result.¹⁶ Although the sensi-tivity of PFA-100^® was lower than the 94% sensitivity of  whole  blood  impedance  aggregometry  demon-strated  in  the  same  report,  the  results  from  TEG  were even more unreliable. These results not only helped form the logistics of our experimental de-sign from impedance aggregometry to PFA-100^® and TEG®, but also justified the point of our argument that PFA-100^® is superior to TEG^® in detection of a mild  blood  coagulation  abnormality.  Further  evi-dence  in  support  of  our  conclusion  was  based  on  other recent studies of hemostatic function in pre-eclampsia, in which an increased severity of preec-lampsia was associated with a prolonged length of CADP-CT. In contrast, the TEG MA in severe preec-lampsia remained within the 95% reference interval of that in normal pregnancy.¹⁷

One  might  argue  that,  as  in  previous  reports,  TEG is much more convenient in clinical point-of-care  algorithms  to  decrease  blood  loss  and  blood  component transfusion perioperatively. In practice, it takes about 30−60 minutes to finish a complete TEG analysis to provide a set of arbitrary parameters of  coagulation  calculated  from  the  artificial  clot  formation by stirring; however, it takes only 5 min-utes to finish a PFA-100^® CADP-CT cartridge test by mimicking the platelet aggregation in the microcir-culation under shear stress. Interestingly, a study of near-patient assessment in management of postop-erative  bleeding  in  cardiac  surgery  demonstrated  that both TEG and PFA-100^® could foretell a similar medium blood loss,¹⁸ suggesting that PFA-100^® was as  good  as  TEG  in  management  of  perioperative  bleeding. The inference of this study directly chal-lenges the earlier reports by their emphasis on the superiority  of  TEG  in  prediction  of  postoperative  bleeding and our results disclosed that PFA-100^® had higher sensitivity than TEG. Taken together, PFA-100^® has  higher  sensitivity  in  detection  of  a  platelet-associated blood coagulation abnormality and is as good as TEG in prediction of perioperative bleeding.

Although PFA-100^® is very powerful in monitor-ing GP IIb/IIIa-related coagulation abnormalities,^19,20 it failed to observe the correlation of closure time with  the  incidence  of  subsequent  major  morbidi-ties.²¹  Because  of  the  use  of  an  ADP  cartridge  as  the major testing tool and with regard to P₂Y₁ and P₂Y₁₂  purinoceptor  pharmacology,  PFA-100^®  is  not  recommended for monitoring the situation with an ADP receptor antagonist, such as clopidogrel.²²

In conclusion, we failed to identify the correlation between TEG^® parameters and platelet aggregation in our experimental setting. TEG^® parameters did not reveal any abnormality in mild antiplatelet-induced coagulation. In contrast, PFA-100^® successfully pro-vided a reliable closure time in detecting the mild platelet dysfunction. Therefore, we conclude that TEG^® could be useful in providing reliable informa-tion  about  hemostatic  disturbances  in  a  platelet-independent situation. However, PFA-100^® is indeed capable of serving as a platelet analyzer and could be successfully used as a good screening device in clinical practice, and is not secondary to TEG^® use. Our data also supported that PFA-100^® may serve as a better point-of-care means for detection of a platelet-associated blood coagulation abnormality.

Acknowledgments

This  study  was  partly  supported  by  Chang  Gung  Memorial  Hospital  research  grants  (CMRPG33136,  CMRPG360331-2), and partly by a National Science Council  grant  (94-2314-B-182A-084).  The  authors  would like to thank those who were willing to be healthy volunteers to help accomplish these studies.