Quality Assurance and Quality Control of Thromboelastography and Rotational Thromboelastometry: The UK NEQAS for Blood Coagulation Experience

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1 Quality Assurance and Quality Control of Thromboelastography and Rotational Thromboelastometry: The UK NEQAS for Blood Coagulation Experience Dianne P. Kitchen, F.I.B.M.S., 1 Steve Kitchen, Ph.D., 1 Ian Jennings, Ph.D., 1 Tim Woods, M.B.A., 1 and Isobel Walker, M.D. 1 ABSTRACT Global hemostasis devices are currently being employed in operating rooms to assess the bleeding risk and outcomes for patients undergoing surgery. Two devices currently available are the TEG (Thromboelastograph; Haemoscope Corp., Niles, IL) and the ROTEM (Rotation Thromboelastometer; Pentapharm GmbH, Munich, Germany). Both measure the speed of clot formation, the strength of the clot when formed, and clot fibrinolysis kinetics. The two devices use different parameters so no cross comparisons of results can be made. The devices are usually operated by a member of the operating team and not a laboratory scientist; thus their testing and performance is generally not laboratory controlled, despite quality control being required to ensure reliable results. The UK National External Quality Assessment Scheme (NEQAS) for Blood Coagulation has undertaken a series of exercises evaluating the provision of External Quality Assessment (EQA) material for these devices. A series of four studies have taken place using lyophilized plasmas as the test material. Up to 18 TEG users and 10 ROTEM users have been involved in testing two samples per study, for a total of eight samples tested. The samples were normal plasmas, factor VIII or XI deficient samples, or normal plasmas spiked with heparin. The precision of the tests varied greatly for both devices, with coefficients of variances ranging from 7.1 to 39.9% for TEG and 7.0 to 83.6% for ROTEM. Some centers returned results that were sufficiently different from those obtained by other participants to predict alterations in patient management decisions. Our data indicate that regular EQA/proficiency testing is needed for these devices. KEYWORDS: Thromboelastography, rotational thromboelastometry, quality control, external quality assessment. Thrombelastography (thromboelastograph [TEG]; Haemoscope Corp., Niles, IL) and rotational thromboelastometry (Rotation Thromboelastometer [ROTEM]; Pentapharm GmbH, Munich, Germany) are similar technologies used for measuring global hemostasis, usually but not exclusively in the operating room. As such, 1 UK NEQAS Blood Coagulation, Sheffield, United Kingdom. Address for correspondence and reprint requests: Dianne Kitchen, F.I.B.M.S., Specialist Scientific Lead for Point of Care Programmes, UK NEQAS for Blood Coagulation, Rutledge Mews, 3 Southbourne Road, Sheffield S10 2QN, United Kingdom ( dianne. kitchen@coageqa.org.uk). Global Hemostasis: New Approaches to Patient Diagnosis and Treatment Monitoring; Guest Editor, Maha Othman, M.D., Ph.D. Semin Thromb Hemost 2010;36: Copyright # 2010 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) DOI: ISSN

2 758 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 36, NUMBER these techniques can be considered point-of-care or near patient test and are frequently performed by nonlaboratory scientists. TEG and ROTEM tests can provide a rapid assessment of platelet function, fibrin clot formation, and clot dissolution by fibrinolysis. These global hemostasis tests may indicate that intraoperative or postoperative bleeding is associated with a deficiency of blood coagulation factors, platelet defects, or fibrinolytic problems or else is the result of a surgical problem, and the results may be used to direct therapy (e.g., replacement therapy, fibrinolytic inhibitors, or further surgery). Indeed, randomized controlled trials in cardiac surgery have demonstrated a reduction in transfusion needs if hemostasis is monitored using algorithms incorporating thrombelastography/thromboelastometry. 1 3 TEG and ROTEM, although performing similar tests, use different parameters and therefore cannot be compared directly. Quality control of these devices is required to ensure reliable results. UK National External Quality Assessment Scheme (NEQAS) for Blood Coagulation has undertaken a series of exercises evaluating the provision of external quality assessment (EQA) material for these devices. TEG AND ROTEM DEVICES Both the TEG and ROTEM produce graphical traces on a chart that represent and monitor the clot as it forms, as well as any clot lysis that occurs during this time. 4 Both maintain the blood sample at 378C during monitoring. Each has a metal pin that sits bathed in the blood sample contained within a small cup. The TEG holds the pin in a fixed position while the cup is rotated back and forth. The pin is attached to a torsion wire. As the blood clots, the tension increases, detected by the pin that transmits this to a computer, which produces a trace that can be stored electronically and printed. The ROTEM differs in that the cup containing blood is fixed and the central pin is rotated. Clotting of the blood impedes the rotation of the pin. This is monitored optically, and data are similarly transmitted to a computer where a trace can be stored or printed. Both the TEG and the ROTEM have the capacity to detect the presence of heparin in a sample. The TEG system uses a plain cup for an initial test; if it is abnormal, a heparinase cup can be used. If the test corrects to normal with the heparinase cup, this indicates that heparin was present. For the ROTEM, an initial test may use the INTEM reagents. If it indicates that heparin may be present, it can be confirmed using a HEPTEM test. ROTEM can also perform EXTEM and FIBTEM tests using different reagents. QUALITY CONTROL Internal Quality Control Quality control (QC) of any test is extremely important in ensuring that precise and accurate results are produced. Good QC can be achieved by the use of both internal quality control (IQC) and EQA. IQC tests should be performed regularly with all the results recorded and acted on. IQC material is generally purchased from the manufacturers, who supply a target range for the material. Both TEG and ROTEM technologies have available IQC samples, which should be tested according to a standard operating procedure (SOP). IQC results should have the following data recorded: date of test, batch of reagents and test cups, batch of IQC, acceptable range, and operator identity. If an IQC result is outside the stated acceptable range, a second sample should be tested. If the second sample result is again outside the acceptable range, patient sample testing should be stopped until investigation and action resolves the issue. External Quality Assessment EQA is provided by an external organization, often a national agency. For example, the main EQA provider in the United Kingdom is UK NEQAS and in the United States is the College of American Pathologists. EQA samples are sent to participating centers that perform the test and return the results. Analysis of the results is undertaken by the EQA provider. The performance of each center is assessed, and an individual report is issued to each showing their results and their performance in relation to that of other participating centers. Thus EQA is a retrospective form of QC, in which the target results are not available at the time of testing. EQA exercises are usually distributed according to a fixed timetable (e.g., two to four surveys per year) and provide snapshots of a center s performance at these times. IQC is a more frequent and ongoing process that checks daily variation and permits early detection of test problems that could affect patient results and thus indicate appropriate action to be taken. UK NEQAS programs compare each center s results against the median of the results obtained for the same sample tested by all participating centers. A peer group median is used where there are 10 users of a method. IQC compares an individual center s results to a preset range provided by the manufacturer. STANDARD OPERATING PROCEDURES It is essential that all operators of TEG or ROTEM have SOPs to guide them as to when to perform IQC and EQA, how to perform these tests, and what to do if the

3 QUALITY ASSURANCE AND CONTROL OF THROMBOELASTOGRAPHY/KITCHEN ET AL 759 QC is outside of the set limit for IQC or acceptable EQA performance. The QC testing should be performed by the person that routinely undertakes patient testing. For example, it is not appropriate for a perfusionist to perform all the patients tests but then for a member of the laboratory team to perform the QC material tests. PILOT EXTERNAL QUALITY ASSESSMENT SURVEYS Few published data are available on the precision of TEG or ROTEM tests or the variability of results between centers. Sørensen and coworkers reported on the precision of testing using conventional and modified ROTEM parameters in a single center. 5 They observed coefficients of variance (CVs) of 5 to 17.4% for replicate clot time (CT) and clot formation time (CFT) measurements compared with 3.75 to 9.9% for tests performed using modified software they had developed. The CV for replicate maximum clot firmness (MCF) measurements was 3%. Chitlur and Lusher reported precision on both the TEG and ROTEM with CVs of between 6% and 60% for the different parameters with the lowest CV seen for clotting time (R)/CT and maximum amplitude (MA)/MCF. 6 UK NEQAS performed a series of exercises in which the same sample was analyzed in different centers, as reported later in this article. EXTERNAL QUALITY ASSESSMENT SAMPLES Both the TEG and the ROTEM devices are designed for analysis of whole blood, but a suitably stable whole blood sample was not available and lyophilized plasma samples were used. To date, four pilot EQA surveys have been performed, each consisting of two lyophilized plasma samples. The samples were either deficient in factor (F)VIII (one sample) or FXI (two samples), normal plasma (three samples), or normal plasma spiked with unfractionated heparin (two samples). EXTERNAL QUALITY ASSESSMENT EXERCISE PROCEDURE Eighteen centers using the TEG device and 10 centers using the ROTEM device were sent samples by mail together with instructions and a result sheet. They were asked to perform tests and return results within a period of 3 weeks. The pattern of testing varied among centers. Lyophilized plasma samples were provided together with the premeasured volume of distilled water for reconstitution and Pasteur pipettes. Instructions indicated to reconstitute the samples with the provided diluent and to wait 10 minutes prior to testing. For the TEG users the instructions stated that the samples should be treated in the same manner as citrated venous samples with the addition of kaolin activator and calcium as provided by the manufacturer. For the ROTEM users the sample was to be reconstituted with the provided diluent and then tested with INTEM, EXTEM, or HEPTEM reagents. EXTERNAL QUALITY ASSESSMENT SURVEY RESULTS Both TEG and ROTEM measure the CFT and the MCF as well as other parameters. With no platelets or red blood cells present in the lyophilized samples, the MCF measured in both devices was greatly affected. The TEG and ROTEM record different parameters. Therefore results must be analyzed separately. TEG Results TEG devices measure the clot formation and can also measure the fibrinolysis of the clot. However, for the purposes of this exercise UK NEQAS was concerned with clot formation only. The following parameters were requested: R (reaction time): The time in minutes taken for initial fibrin formation to occur K (clotting time): The time in minutes to reach a specific level of clot strength (clot width of 20 mm) Angle: The angle in degrees of the slope of the graph that measures the speed of fibrin build-up and crosslinking MA (maximum amplitude): The maximum clot width measures in millimeters and represents the strength of the clot and is a measure of platelet function Although results were recorded for the parameters R, K, Angle, and MA, only data for R and MA are shown in Table 1. Centers used plain cups and/or heparinase cups as appropriate for the sample distributed. If a plain cup result was prolonged (indicating the potential presence of heparin), a heparinase cup test could be performed. For the first pilot (a spiked heparin sample and a normal plasma sample), the instruction sheet provided guidance on whether to test both plain and heparinase cups, but for the remaining samples the decision to test with heparinase cup was made locally by each center. Samples of FVIII-deficient <1.0 IU/dL or FXI deficient <1.0 IU/dL showed no clotting for both the plain and heparinase cups. For the two samples that were normal plasma spiked with unfractionated heparin, the plain cups showed no clotting but the heparinase cups yielded normalized results, indicating the presence of heparin in the sample. CVs ranged from 7.1 to 39.9%. Centers were also asked to give an interpretation of either normal or abnormal for the R time for each of the samples. Table 2 shows the results of the interpretations.

4 760 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 36, NUMBER Table 1 Thromboelastrograph Results Sample and Test Performed Parameter Median CV Range of Results 1: Spiked heparin sample Plain cup n ¼ 12 No clot formed Heparinased cup n ¼ 12 R min MA mm : Normal sample Plain cup n ¼ 12 R min MA mm : Factor XI deficient Plain cup n ¼ 13 No clot formed Heparinased cup n ¼ 13 No clot formed 4: Normal sample Plain cup n ¼ 13 R min MA mm Heparinased cup n ¼ 11 R min MA mm : Spiked heparin sample Plain cup n ¼ 17 No clot formed Heparinased cup n ¼ 17 R min MA mm : Factor VIII deficient Plain cup n ¼ 17 No clot formed Heparinased cup n ¼ 17 No clot formed 7: Factor XI deficient Plain cup n ¼ 18 No clot formed Heparinased cup n ¼ 18 No clot formed 8: Normal sample Plain cup n ¼ 18 R min MA mm Heparinased cup n ¼ 14 R min MA mm CV, coefficient of variance; R, reaction time; MA, maximum amplitude. ROTEM Results The ROTEM device, similarly to the TEG, measures lot formation and can also measure clot fibrinolysis. However, for the purposes of this exercise, UK NEQAS was again concerned with clot formation only. The following parameters were requested: CT (clotting time): The time taken for a clot starting to form measured in seconds CFT (clot formation time): The time for the clot to reach a width of 20 mm measured in seconds Angle (degrees): A measure of the slope of the curve once clotting has begun Table 2 Interpretations of Reaction Times Performed on the Thromboelastograph Analyzers Sample Sample Type Plain Cup Heparinase Cup 1 Spiked heparin 11/11 abnormal 8/11 normal 3/11 abnormal 2 Normal 8/11 normal 3/11 abnormal Not tested 3 FXI deficient 7/7 abnormal 6/6 abnormal 4 Normal 7/8 normal 1/8 abnormal 5/6 normal 1/6 abnormal 5 Spiked heparin 12/12 abnormal 10/11 abnormal 1/11 normal 6 FVIII deficient 11/11 abnormal 10/10 abnormal 7 FXI deficient 15/15 abnormal 15/15 abnormal 8 Normal 12/12 abnormal 8/8 abnormal

5 QUALITY ASSURANCE AND CONTROL OF THROMBOELASTOGRAPHY/KITCHEN ET AL 761 Table 3 Rotation Thromboelastometer Results Sample and Test Performed Parameter Median CV Range of Results 1: Spiked heparin sample ITEM n ¼ 10 CT sec MCF mm HEPTEM n ¼ 8 CT sec MCF mm : Normal sample ITEM n ¼ 9 CT sec MCF mm EXTEM n ¼ 9 CT sec 48 *121.1 (18.7) MCF mm : Factor XI deficient ITEM n ¼ 8 CT sec MCF mm HEPTEM n ¼ 6 CT sec MCF mm EXTEM n ¼ 8 CT sec 48 *84.6 (18.5) MCF mm : Normal sample ITEM n ¼ 8 CT sec MCF mm EXTEM n ¼ 6 CT sec 42.5 *119.4 (83.6) MCF mm : Spiked heparin sample ITEM n ¼ 10 CT sec MCF mm HEPTEM n ¼ 6 CT sec MCF mm EXTEM n ¼ 10 CT sec 37.5 *229.7 (16.9) MCF mm : Factor VIII deficient ITEM n ¼ 10 CT sec MCF mm HEPTEM n ¼ 6 CT sec MCF mm EXTEM n ¼ 10 CT sec 43 *204 (25.8) MCF mm : Factor XI deficient ITEM n ¼ 6 CT sec MCF mm HEPTEM n ¼ 4 CT sec MCF mm EXTEM n ¼ 8 CT sec MCF mm : Normal sample ITEM n ¼ 7 CT sec MCF mm HEPTEM n ¼ 4 CT sec MCF mm EXTEM n ¼ 8 CT sec 40.5 *106 (11.6) MCF mm *One outlying result with recalculated result with outlier in brackets. CV, coefficient of variance; CT, clot time; MCF, maximum clot firmness.

6 762 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 36, NUMBER Table 4 Interpretations of Clot Times Performed on the Rotation Thromboelastometer Analyzers Sample Sample Type INTEM HEPTEM EXTEM 1 Spiked heparin 9/9 abnormal 6/7 normal 1/7 abnormal Not tested 2 Normal 8/8 normal Not tested 6/8 normal 2/8 abnormal 3 FXI deficient 6/6 abnormal 3/3 abnormal 5/6 normal 1/6 abnormal 4 Normal 5/6 normal 1/6 abnormal Not tested 3/5 normal 2/5 abnormal 5 Spiked heparin 9/9 abnormal 4/4 abnormal 7/9 normal 2/9 abnormal 6 FVIII deficient 9/9 abnormal 5/5 abnormal 8/9 normal 1/9 abnormal 7 FXI deficient 8/8 abnormal 6/6 abnormal 6/7 normal 1/7 abnormal 8 Normal 7/8 normal 1/8 abnormal 3/3 normal 6/8 normal 2/8 abnormal F, factor. MCF (maximum clot firmness): A measure of the maximum size of the clot measured in millimeters. Although results were recorded for the parameters CT, CFT, Angle, and MCF, only data for CT and MCF are shown in Table 3. For pilot 1, centers were asked to test INTEM and HEPTEM for sample 1 (a spiked heparin sample) and INTEM and EXTEM for sample 2 (a normal plasma sample). For samples 3 (FXI deficient), 4 (Normal plasma), 7 (FXI deficient), and 8 (normal plasma), centers were asked to perform tests as appropriate, and for samples 5 (spiked heparin sample) and 6 (FVIII deficient sample), INTEM, EXTEM, and HEPTEM tests were requested. The FXI-deficient (<1.0 IU/dL) sample showed anormalctresultforextemandaprolongedct result for INTEM and HEPTEM, indicating heparin was not present. The FVIII-deficient (<1.0 IU/dL) sample showed a similar pattern of results. The heparin-spiked samples showed a prolongation of CT results with INTEM tests that corrected with the HEPTEM test, indicating the presence of heparin in the samples. CVs ranged from 0.6% to 229.7%. The higher CVs were seen with the CT for the EXTEM test and the very low CVs were from the Angle measurement. The EXTEM test for all samples except sample 7 gave high CVs, which were due to the presence of an outlying result. One center testing with the ROTEM accounted for all the outlying results for this test (they failed to enter a result for sample 7). Centers were also asked to give an interpretation of either normal or abnormal for the CT time for each of the samples. Table 4 shows the results of the interpretations. DISCUSSION The TEG and ROTEM devices are used to measure global hemostasis 4 and often have a direct impact on patient therapy. It is essential that staff using these devices can be assured of their reliability. Provision of EQA material for the TEG and ROTEM is difficult because the material usually used for EQA purposes is lyophilized plasma, whereas routinely whole blood samples are analyzed. Reference ranges are also for whole blood and generally not available for plasma samples. TEG and ROTEM measurements are also influenced by platelets, which are not present in lyophilized samples. Participants in both the TEG and ROTEM groups successfully identified that heparin was present in the heparin-spiked samples. This is important in view of the use of such devices in patients undergoing cardiac surgery 7 where heparin is widely used. In our surveys both TEG and ROTEM groups found prolonged abnormal results not due to heparin in samples deficient in FVIII or FXI. Again, this is important because many patients with such defects would be predicted to have serious bleeding complications during and after surgery. The normal samples did not give the prolonged times seen with the deficient or the spiked heparin samples, although not all users considered these to be normal in all cases. On several occasions the results obtained in one or more centers were different from those obtained in most of the centers to an extent that would be predicted to influence patient management. For example, one center reported a gross CT abnormality (360 seconds) in EXTEM testing (Table 3) on a sample from a normal subject (median CT: 42 seconds). If this were a genuine patient sample, clotting factor deficiency might incorrectly be suspected where none was present. Our data therefore suggest a need for regular EQA of thetypereportedheretoensurethesafeuseofthese devices. CONCLUDING REMARKS The data provided from our EQA surveys show that it is possible to provide a limited EQA process for these devices, with some parameters successfully measured on lyophilized plasma samples. Further development of EQA material may enable assessment of other parameters with TEG and ROTEM devices.

7 QUALITY ASSURANCE AND CONTROL OF THROMBOELASTOGRAPHY/KITCHEN ET AL 763 REFERENCES 1. Sorensen ER, Lorme TB, Heath D. Thromboelastography: a means to transfusion reduction. Nurs Manage 2005;36(5):27 33; quiz Ronald A, Dunning J. Can the use of thromboelastography predict and decrease bleeding and blood and blood product requirements in adult patients undergoing cardiac surgery? Interact Cardiovasc Thorac Surg 2005;4(5): Ak K, Isbir CS, Tetik S, et al. Thromboelastography-based transfusion algorithm reduces blood product use after elective CABG: a prospective randomized study. J Card Surg 2009; 24(4): Luddington RJ. Thrombelastography/thromboelastometry. Clin Lab Haematol 2005;27(2): Sørensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost 2003;1(3): Chitlur M, Lusher J. Standardization of thromboelastography: values and challenges. Semin Thromb Hemost 2010;36(7): Spiess BD. Coagulation monitoring in the perioperative period. Acta Anaesthesiol Scand Suppl 1996;109: 77 79