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The Use of Citrated Whole Blood in Thromboelastography

2000; Lippincott Williams & Wilkins; Volume: 90; Issue: 5 Linguagem: Inglês

10.1097/00000539-200005000-00015

1526-7598

V A Bowbrick, Dimitri P. Mikhailidis, Gerard Stansby,

Blood properties and coagulation

Hartet developed thromboelastography in 1948 (1). This technique is used to evaluate the viscoelastic properties of blood during coagulation. The Thrombelastograph® Coagulation Analyzer 3000 (TEG®; Haemoscope, Skokie, IL) can be used to assess the interaction of platelets with the protein coagulation cascade from the time of the initial platelet-fibrin interaction, through platelet aggregation, fibrin cross linkage to eventual clot lysis. Four main variables of clot formation can be measured: reaction time (r), coagulation time (k), angle (α) and maximum amplitude (MA) (2). "Fresh" native whole blood is usually used but must be placed in the TEG® no longer than 6 min after venepuncture, ideally at the 4-min stage. Therefore, if delay between venepuncture and starting the profile cannot be avoided, an alternative to "fresh" native whole blood must be sought. Citrated whole blood can be stored at room temperature or at 4°C but must be recalcified before insertion of the pin in the cup. However, to use citrated whole blood, results need to be comparable with those obtained with "fresh" native whole blood. The aim of this study was to establish if "fresh" native whole blood and recalcified citrated whole blood produce comparable results. We also evaluated how long citrated whole blood can be stored and still provide meaningful TEG® data. Method Local ethical committee approval was obtained. After informed consent was given, eight healthy controls were studied. These were defined as subjects who did not meet any of the criteria that have been shown to modify platelet function (3). Blood was taken by clean peripheral venepuncture from the antecubital fossa using a 21-guage butterfly and double syringe technique with a light tourniquet to avoid stasis or platelet activation. Part of the blood was transferred to two polypropylene tubes containing 0.5 mL of 3.2% (0.105 M) sodium citrate (1 part citrate to 9 parts blood dilution) and gently inverted three times. An aliquot (360 μL) of native whole blood that was therefore not anticoagulated was pipetted into the warmed cuvette (37°C) of the computerized TEG® within 4 min of venepuncture. Citrated whole blood was either stored at room temperature or at 4°C. The blood stored at room temperature was analyzed at 30 and 120 min after venepuncture. That stored at 4°C was analyzed at 45 and 150 min inclusive of 15 min at room temperature to rewarm before being placed in the TEG®. An aliquot (340 μL) was placed in the warmed cuvette in the TEG® and recalcified with 0.2 M calcium chloride (20 μL) at the above mentioned time points. The results were presented as median and range so as not to assume that they are normally distributed. The results for the citrated whole blood were compared with the "fresh" native whole blood by using the Mann-Whitney U-test (P < 0.05 considered significant). We also established how many of our readings were outside the limits as described by Bland and Altman (4). Results Blood was taken from eight healthy volunteers (two female and six male) with a median age of 30 yr (range 24–35). The r, k, α, and MA variables were measured for all samples. The median values of these results are shown in Table 1.Table 1: Median Values of TEG® Variables with Range in ParenthesesThere was no significant difference for the results of citrated whole blood kept at room temperature for 30 min or for that kept at 4°C for 45 and 150 min in comparison with native whole blood. However, by storing citrated whole blood at room temperature for 120 min, there was a difference in the k and α values obtained that was significant (P = 0.0207 for both). The r and MA variables were shown to be acceptable with no significant difference to the "fresh" native whole blood. None of the readings when comparing "fresh" native whole blood with citrated whole blood disagreed with those described by Bland and Altman (4). This analysis also showed that the levels of agreement were narrowest for the r and MA values and that with citrated whole blood there was a trend toward a shorter k value and larger α value. Discussion Thromboelastography is a useful measure of coagulation and fibrinolysis leading to a comprehensive and rapid assessment of coagulopathy, which helps in limiting or directing the appropriate use of blood products (5,6). Its use is limited by the number of channels on any one analyzer and by the fact that "fresh" native whole blood must be used within six minutes of venepuncture. It is therefore useful to have a method that allows for the storage of blood while providing results as reliable as the "gold standard" of "fresh" native whole blood. An alternative test for rapid assessment of hemostasis is the Sonoclot® analyzer (Sienco Inc, Morrison, CO), but its results are influenced by the storage of blood in citrate (7). Our results show that thromboelastography using citrated whole blood is reproducible when the sample is stored at room temperature for 30 minutes or at 4°C for 45 and 150 minutes. In citrated whole blood, the k values showed a tendency to be shorter and the α values to be larger than with "fresh" native whole blood. This would indicate a faster rate of clot formation, but this was only statistically significant in citrated whole blood stored at room temperature for 120 minutes. Results obtained after 120 minutes at room temperature cannot, therefore, be regarded as reliable as there was a significant difference in the k and α values obtained. This may be a reflection of platelet activation after this period of time in the citrate. In this context, there is some evidence of enhanced platelet responses to adenosine diphosphate at 27°C (8). Excessive cooling of platelets can induce aggregation and irreversible platelet damage (9). Therefore, as there was no significant change in the values obtained after storage at 4°C, it is possible that the platelets were not significantly contributing to the TEG® profile. In support of this conclusion, Oshita et al. (10) showed that a significant change in k and MA variables only occurred when the platelet count was considerably lower (< 66 × 103/μl). These findings imply that thromboelastography may not provide a comprehensive and sensitive reflection of impaired platelet function. This study is limited by the small sample size and that it does not include patients with abnormal hemostasis (e.g., patients on drugs that affect hemostasis and both congenital or acquired clotting/fibrinolytic defects). Therefore, caution must be exercised before recommending the use of recalcified citrated blood in these clinical settings. This possibility remains to be evaluated, and we hope that the methodology described in this brief report will trigger such studies. This work will confirm or refute the validity of this approach in various clinical conditions, which include a hemostatic defect. In conclusion, in healthy controls, citrated whole blood may be used in the TEG® when stored at room temperature for 30 minutes or at 4°C up to 150 minutes, giving results that do not differ significantly from those obtained with "fresh" native whole blood. This finding may have practical value in experimental studies involving thromboelastography although before accepting its clinical application further studies are required. In addition, cooling the blood to 4°C will cause irreversible damage to the platelets, but there was no significant change in the TEG® profiles seen in the blood stored at this temperature. This could indicate that thromboelastography is not a sensitive method of assessing platelet function.