Quality issues in laboratory haemostasis

Abstract

Tests performed in coagulation laboratories play an essential role in the diagnosis and management of patients with familial and acquired bleeding and thrombotic disorders. It is therefore apparent that the generated results should be accurate, reliable and reproducible. This is particularly important in respect of tests undertaken to diagnose, or to exclude, a possible familial disorder. In recent years, there has been a marked increase in the workload and scope of the coagulation laboratory and this has been accompanied by the introduction of increasingly complicated automated equipment employing a variety of different techniques. It is apparent that the potential for error is considerable and for this reason it is essential that the performance of laboratories be monitored through its participation in an external quality assessment (EQA) programme. External quality assessment compares the results obtained from different laboratories on the same sample. It thus provides information in respect of the accuracy of results produced by individual participating laboratories. Participants in an EQA programme receive plasma samples from their EQA provider with instructions to perform specific tests using their usual method. It is important that the EQA samples are not passed on to senior laboratory staff but are tested by the same staff who normally perform the requested test or assay. The results are then returned to the EQA provider for detailed analysis. In most EQA programmes, the distributed samples are lyophilized. It is clearly extremely important that EQA providers ensure that results on the reconstituted lyophilized plasma are similar to those obtained on the native plasma. The assignment of target values for the distributed plasmas is an essential component of any EQA programme and for haemostasis; the overall consensus value is frequently adopted as the target value for EQA participants. However, this approach is not without its problems, particularly in the larger programmes where the number of different reagents, instruments and calibrants is extremely large with major differences being observed in the results obtained with different combinations. For this reason, it is more appropriate to assess results against the peer group median result. An assessment against the overall median is only considered appropriate where the number of laboratories deploying a single method with identical reagents is too small (<10) for statistical analysis or when different methods are known to give the same results. The assessment of laboratory performance by EQA providers is of utmost importance as it enables laboratories to compare their results against those obtained from other laboratories using the same reagents and the same methodology. The identification of unsuspected analytical problems allows laboratories to adjust their procedures and/or change their reagents or their calibrants. It is important for a laboratory to monitor it’s performance over time. Results that are consistently above or below the peer group median are informative in that they may indicate either a systematic or a calibration error; information that is not apparent from a single assessment. A laboratory that obtains persistently poor results in an EQA programme should examine its internal quality control (IQC) records over the same time period. This will assist in distinguishing between imprecise and inaccurate results. When sequential EQA assay results fluctuate above and below the target values then assay imprecision is the most likely explanation and IQC results will be variable with low precision. EQA results that are consistently higher or lower than those of the corresponding peer group usually indicate high precision in the assay. In this instance the consistent IQC results indicate that the method is precise whereas the EQA results demonstrate that they are inaccurate. Thus, when EQA results are consistently higher or lower than the corresponding target values, all of the components of the assay system should be investigated and replaced where necessary. Another possibility is wide fluctuation of EQA results both above and below the target values. This indicates that the method is poorly controlled and it is likely that the corresponding IQC results will show a high degree of imprecision. In this instance consideration should be given to the possibility of variable performance of a particular analyser or else reagent instability. In addition to their role in identifying poor laboratory performance, the larger EQA programmes are able to identify problems relating to instruments, reagents and calibrants. This is achieved by comparing results obtained by peer group analysis. For example, the UK NEQAS programme was able to demonstrate that the use of different commercial reference plasmas was associated with significantly different FVIII:C and FV:C assay results [Preston FE, Kitchen S, Srivastava A. External Quality Assessment in Hemostasis: its importance and significance. In: Kitchen S, Olson JD, Preston FE (eds). Quality in laboratory hemostasis and thrombosis. Wiley-Blackwell 2009: 51–62]. To conclude, a comprehensive EQA programme serves a number of important functions. It identifies and assists laboratories whose performance is unsatisfactory and thus serves to improve laboratory performance and therefore patient care. It also assists laboratories in their choice of reagents and instrument-reagent combinations by identifying unsatisfactory reagents, reference materials and methods. Bleeding and thrombotic disorders are major healthcare problems, worldwide [Lippi G, Favaloro EJ, Franchini M. Laboratory diagnostics and therapy in thrombosis and hemostasis: from bedside to bench to bedside. Semin Thromb Hemost 2009; 35: 3–8]. The approach to screening, diagnosis and monitoring of most disturbances of the haemostatic balance encompasses an appropriate and a discretionary use of laboratory diagnostics [Favaloro EJ, Lippi G, Franchini M. Laboratory diagnostics in thrombosis and hemostasis: the past, the present, and the future. Semin Thromb Hemost 2008; 34: 579–83]. Nevertheless, total quality in coagulation testing is an essential condition for producing clinically reliable and usable data. Several problems in the total testing process occur for events outside the direct control or jurisdiction of the laboratories performing the tests. Although there is perception that the preanalitical variability can adversely impact on results of first- and second-line coagulation testing, only a few studies have addressed this issue in detail. As such, the vast majority of problems, extraordinarily magnified over the traditional laboratory diagnostic for the peculiarity of the tests performed, have been related to inappropriateness test request, lack of knowledge of biological and circadian variability, collection of samples at the wrong time, misidentification, use of inappropriate procedures for sample collection (venous stasis, type of device, needle gauge), collection of unsuitable sample for quantity (incorrect blood to anticoagulant ratio) or quality (inappropriate sample matrix, haemolytic and lipaemic specimens, contamination from infusion routes), improper management of the sample after collection (inappropriate mixing, unrestrained procedures for transportation, spinning, storage, freezing and thawing) and other miscellanea variables [Lippi G, Franchini M, Montagnana M, Salvagno GL, Poli G, Guidi GC. Quality and reliability of routine coagulation testing: can we trust that sample? Blood Coagul Fibrinolysis 2006; 17: 513–9/Favaloro EJ. Pre-analytical variables in coagulation testing. Blood Coag Fibrinolysis 2007; 18: 86–89/Favaloro EJ, Lippi G, Adcock DM. Preanalytical and postanalytical variables: the leading causes of diagnostic error in hemostasis? Semin Thromb Hemost 2008; 34: 612–34] (Fig. 1). All these variables can seriously affect the integrity of the sample and impact on the reliability of the tests performed, thereby producing clinical and economical consequences that might adversely impact on laboratory organization and patient outcome. As such, unsuitable samples might account for up to 5.5% of all the specimens received for routine and specialized coagulation in a clinical laboratory. The highest frequency is traditionally observed for samples refereed from paediatric departments. The problems more frequently encountered have been identified with samples missing after a doctor’s order (49.3%), haemolysis (19.5%), clotting (14.2%) and inappropriate volume (13.7%). In particular, specimens missing seem to prevail in the intensive care unit, surgical and clinical departments, whereas clotted and haemolysed specimens are those most frequently recorded from paediatric and emergency departments respectively [Salvagno GL, Lippi G, Bassi A, Poli G, Guidi GC. Prevalence and type of pre-analytical problems for inpatients samples in coagulation laboratory. J Eval Clin Pract 2008; 14: 351–3]. Extra-analytical problems in laboratory haemostasis. An appropriate process of sample collection is essential for laboratory diagnostics, especially in those areas of testing where preanalytical variables might critically impact on the reliability of test result and patient outcome, such as haemostasis. Phlebotomy suffers from a high degree of preanalytical variability. Besides identification errors, which occur in almost every area of laboratory testing and are associated with the worst clinical outcomes (misdiagnosis and administration of inappropriate therapy) [Lippi G, Blanckaert N, Bonini P, Green S, Kitchen S, Palicka V, et al. Causes, consequences, detection, and prevention of identification errors in laboratory diagnostics. Clin Chem Lab Med 2009; 47: 143–53], most of the problems arise from cumbersome venipunctures, caused by shortages of skilled staff and overloaded phlebotomists. As such, several preanalytical variables in this process might led to unsuitable samples for testing (e.g., haemolytic, clotted, insufficient samples), and include blood collection from wrong sites (e.g., varicose veins, veins of arm or hand from the side of a mastectomy), using unsuitable disposals (i.e., infusive devices, cannulae, butterfly needle devices) [Lippi G, Guidi GC. Effect of specimen collection on routine coagulation assays and D-dimer measurement. Clin Chem 2004; 50: 2150–2], after a prolonged venous stasis caused by the tourniquet [Lippi G, Salvagno GL, Montagnana M, Guidi GC. Short-term venous stasis influences routine coagulation testing. Blood Coagul Fibrinolysis 2005; 16: 453–8] and by using an inappropriate procedure. The use of inappropriate container (i.e., a wrong blood tube) is another major source of concern for coagulation testing, along with inappropriate filling and mixing of the tubes. In particular, underfilling of blood tubes for the classical coagulation tests might significantly alter test results and, as such, the current H21-A5 Clinical Laboratory Standards Institute (CLSI) guideline recommends that coagulation samples should be discarded if the evacuated tube contains <90% of the expected fill volume [Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar RA, Szamosi DI, Warunek DJ. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays: Approved Guideline, 5th edn. Wayne, PA: Clinical Laboratory Standards Institute, CLSI document H21-A5; 2008]. At variance with the previous indications, there is clear evidence that no clinically meaningful differences in results of testing can be observed between the first and the second blood tubes collected sequentially, so that the necessity for drawing a discard tube before that used for coagulation testing is circumstantial at best. Upon collection, blood samples should be properly mixed (e.g., gently inverted four to six times) to allow effective mixing between blood and anticoagulants, and without producing haemolysis, clotting or platelet activation [Lippi G, Salvagno GL, Montagnana M, Guidi GC. Influence of primary sample mixing on routine coagulation testing. Blood Coagul Fibrinolysis 2007; 18: 709–11]. To ensure quality of coagulation testing once a specimen has been collected properly, it is important to adhere to standard recommendations for processing, transportation and storage. Using plasma on spun-down cells at room temperature, add-on tests for routine coagulation testing can be performed within an 8-h period, obtaining results similar to what would be obtained from testing unstored specimens [Neofotistos D, Oropeza M, Ts’ao CH. Stability of plasma for add-on PT and APTT tests. Am J Clin Pathol 1998; 109:758–63], except for activated partial thromboplastin time (APTT) as measured on samples of patients receiving unfractionated heparin therapy [Adcock D, Kressin D, Marlar RA. The effect of time and temperature variables on routine coagulation tests. Blood Coagul Fibrinolysis 1998; 9: 463–70]. The growing trend towards consolidation of laboratory diagnostics in large, centralized facilities networked with peripheral phlebotomy services further enhances the implications of sample stability and transportation. This implies that whole blood samples rather than separated plasma samples may arrive to the central laboratory from varying distances, under variable storage and transportation modes. As such, we have recently established that a maximum of 6-h storage of uncentrifuged specimens at either room temperature or 4°C is allowed to maintain test results within the analytical quality specifications for desirable bias [Salvagno GL, Lippi G, Montagnana M, Franchini M, Poli G, Guidi GC. Influence of temperature and time before centrifugation of specimens for routine coagulation testing. Int J Lab Hematol 2009; 31: 462–7]. Sample transportation should also proceed as per current recommendations, that is non-refrigerated, at room (ambient) temperature, in the shortest possible time. For in-hospital transport, the novel generation of pneumatic tube systems is a valuable option, as it reduces turnaround times and labour, without introducing preanalytical errors for analysis of routine haematology, coagulation parameters and platelet function [Wallin O, Söderberg J, Grankvist K, Jonsson PA, Hultdin J. Preanalytical effects of pneumatic tube transport on routine haematology, coagulation parameters, platelet function and global coagulation. Clin Chem Lab Med 2008; 46: 1443–9]. Freeze-thawing of stored specimens can also produce the degradation of some labile factors, especially factor V (FV) and factor FVIII (FVIII). Accordingly, a low FVIII, for example, obtained from a referral laboratory might be simply artefactual because of inappropriate freeze-thaw cycles. When using frozen specimens, it might also be important to warm the sample to 37°C for at least 10 min before testing, to ensure reversal of any cryoprecipitate formed during freezing, especially when analysing FVIII and von Willebrand factor (VWF) [Favaloro EJ, Nair SC, Forsyth CJ. Collection and transport of samples for laboratory testing in von Willebrand’s disease (VWD): Time for a reappraisal? Thromb Haemost 2001; 86: 1589–90/Favaloro EJ, Soltani S, McDonald J. Potential laboratory misdiagnosis of haemophilia and von Willebrand disorder due to cold activation of blood samples for testing. Am J Clin Pathol 2004; 122: 686–92]. Inappropriate samples are also difficult to be detected when received as secondary plasma aliquots. As such, we have recently developed two simple, quick and inexpensive algorithms to help in the differential identification of citrated plasma vs. other samples with 100% sensitivity and specificity, should there be suspicion of inappropriate collection. The former algorithm is based on the sequential measurement of potassium, calcium and sodium, the latter on potassium and sodium [Lippi G, Salvagno GL, Adcock DM, Gelati M, Guidi GC, Favaloro EJ. Right or wrong sample received for coagulation testing? Tentative algorithms for detection of an incorrect type of sample. Int J Lab Hematol 2009 Feb 7. (Epub ahead of print)]. As regards centrifugation, the CLSI guideline contains specific recommendation to centrifuge capped specimens at 1500 g for no <15 min at room temperature [CLSI]. Basically, the centrifugation time is inversely associated with residual blood cell elements in plasma, especially platelets. Nevertheless, statistically significant variations from the reference 15-min centrifuge specimens were observed for fibrinogen in samples centrifuged for 5 min at most and for the APTT in samples centrifuged for 2 min at most. Meaningful biases related to the desirable bias were observed for fibrinogen in samples centrifuged for 2 min at most, and for the APTT in samples centrifuged for 1 min at most. According to these experimental conditions, a 5–10 min centrifuge time at 1500 g may be thereby still for primary tubes collected for routine coagulation testing [Lippi G, Salvagno GL, Montagnana M, Guidi GC. Influence of the centrifuge time of primary plasma tubes on routine coagulation testing. Blood Coagul Fibrinolysis 2007; 18: Results of recent also that whole blood specimen centrifugation at different than that are not likely to significant analytical or clinical biases [Lippi G, Salvagno GL, Montagnana M, Poli G, Guidi GC. Influence of centrifuge temperature on routine coagulation testing. Clin Chem 2006; The use of g is not usually because they might platelet activation and For some it might be to use plasma, to inappropriate testing that may occur when using plasma. The use of plasma is no [Favaloro EJ, Lippi G, Adcock DM. Preanalytical and postanalytical variables: the leading causes of diagnostic error in hemostasis? Semin Thromb Hemost 2008; 34: but not it was recently that a plasma might occur in primary tubes for coagulation testing, thereby introducing a bias in and to time values and higher fibrinogen in the lower than in the of a primary collection As such, it has been that plasma should be separated from the after centrifugation and mixed before analysis or [Lippi G, Salvagno GL, Bassi A, Montagnana M, Poli G, Guidi GC. of citrated plasma in primary collection tubes for routine coagulation testing. Blood Coagul Fibrinolysis 2008; and Srivastava A of an increasingly important role in the management of patients and their with bleeding disorders a in the number of laboratories that tests for haemostatic disorders and in the of to these laboratories on IQC and EQA and testing programmes for quality to maintain the quality and integrity of their In such as in the testing for the diagnosis of haemophilia and is by the external quality assessment and participation in such programmes is a for laboratory programmes also in most developed in and and However, except for from a few specific a significant in knowledge the of testing in laboratories the To we a laboratories in both the and developed in A was developed of which the first was to the laboratory the and and their the type and number of testing services for diagnosis of haemophilia A and haemophilia and bleeding disorders. The of the on laboratory and in during the analytical and postanalytical of testing. 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It is possible that laboratories performing such as analysis have been in their to this of the quality in the laboratories wide variability. Although the majority of laboratories for and this was not to the laboratory was controlled and diagnostic laboratory were stored in a area in of laboratories. reagents were used during testing in of or for and or for in calibration of used in testing such as the was performed in of laboratories. A similar number an for diagnosis on the diagnosis were also not of laboratories to and only of the a of clinical evidence quality were more in laboratories from This is further in the that only 100% in developed of laboratories in in any of testing External quality for tests are in To we have an for analysis of disorders for laboratories in are laboratories in the programme. obtained with from patients with haemophilia A, haemophilia and thrombotic disorders are for analysis along with clinical EQA have been For haemophilia A, analysis was most used with only laboratory performing direct analysis using and Assessment of haemophilia was performed by only of laboratories and is performed by All laboratories in testing use for for this are with a accuracy of over A performance is to all participants. that for tests can be established in In that quality have not diagnostic testing laboratories consistently the and in in This the for the and of participation in laboratory programmes, EQA programmes that are and specific for of testing. To increase participation in participation in such programmes should be would to all the laboratory who in this Molecular J The and of Institute for and All of also to for their help with collection and analysis.