Quality in routine coagulation testing

Giuseppe Lippi
MD
Associate Professor of Clinical Biochemistry
University of Verona
Italy

Massimo Franchini
MD
Senior Assistant of Transfusional Medicine
Azienda Hospital
Verona, Italy

Haemostasis means a delicate balance between anti- and prothrombotic factors that promote the blood being in a fluid state within vascular compartments and the formation of blood clots. Rapid and accurate investigation of haemostasis disturbances, either of haemorrhagic or thrombotic nature, can be carried out through the reliable use of laboratory resources, which include first- and second-line testing.(1,2) First-line testing consists of routine coagulation assays, including activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen, which reflect the global function of the coagulation pathway. Individual factors can be tested separately, but usually only at specialised coagulation laboratories. APTT is a global coagulation test that includes all steps, from the activation of factor XII to the formation of soluble fibrin. A prolonged APTT usually indicates an increased bleeding tendency (eg, acquired or congenital haemophilia). However, APTT may also be prolonged by the presence of in-vivo or ex-vivo anticoagulants, such as lupus anticoagulants and heparin. The PT (according to Quick) measures the activity of coagulation factors VII, X, V, II and fibrinogen, and it may be prolonged by deficiencies of one or more of these factors. The PT is recalculated according to the international normalised ratio (INR), which makes it possible to compare test results from laboratories with different assay kits. In several countries, PT is also used for monitoring anticoagulant therapy with antivitamin K drugs. PT may be prolonged by vitamin K deficiency and liver failure. The fibrinogen assay is an additional screening test. Fibrinogen levels are usually increased in inflammatory states, whereas low levels can be a sign of increased consumption, for example in patients with disseminated intravascular coagulation (DIC), liver cirrhosis, general fibrinolysis or, less frequently, those who are carriers of congenital fibrinogen deficiencies.

Quality in routine coagulation testing
Quality of coagulation testing, as in other areas of in-vitro diagnostics, is widely assumed rather than being assured or guaranteed, especially for several pre- and postanalytical processes carried out outside the laboratory or that escape from its supervision.(3) However, due to the elevated analytical sensitivity of the assay methodology and the adverse clinical consequences attached to a spurious test result, standardisation of each phase of the testing process in this area cannot be overlooked. Conventionally, the overall testing process develops through Lundberg’s brain-to-brain turnaround time loop, which encompasses preanalytical, analytical and postanalytical phases.(4) Although quality assurance in each phase is essential to produce reliable and clinically useful results, the greatest part of the errors is actually generated during the manually intensive activities of the preanalytical phase, including collection, handling and processing of the specimens.(5) The quality of the diagnostic specimens covers a variety of aspects, including the choice of appropriate containers and anticoagulants, definition of the optimal sample size and stability of the analyte in the sample matrix. Therefore, most unsuitable specimens for coagulation testing can be traced to missing or wrong identification, haemolysis, clotting, inappropriate containers and inadequate filling.(6) As in all healthcare settings, the appropriate identification of the specimen is a challenging endeavour. Within the 2007 National Patient Safety Goals, recently issued by the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), major emphasis was placed on improving patient identification, a major goal which would apply to all accredited healthcare organisations and those seeking accreditation.(7) Although patient misidentification often goes undetected, it may represent up to 2.6% of all laboratory errors, and it is associated with the worst clinical outcome due to misdiagnosis and mishandled therapy. Several solutions were proposed to prevent this hazard, including the use of two patient identifiers before collecting blood samples and adoption of wristbands with unique barcoded patient identifiers. Haemolysed specimens are a frequent occurrence in the coagulation laboratory (nearly 60% of unsuitable specimens are rejected for this reason) and are traditionally detected by the presence of a pink-to-red hue after centrifugation.

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Haemolysis causes serious problems in coagulation assays, as it may produce false elevations in ­several intracellular parameters, spurious decrease of others and interference with some test ­methodologies, especially spectrophotometric assays.(8) The rational management of haemolytic ­samples is crucial and encompasses a simple flowchart based on haemolysis detection and quantification, ­analysis of the interference on test results and recollection of new samples. Rapid feedback with the ­clinics is also essential to rule out in-vivo haemolysis, a serious threat to the patient’s health. Unnecessary­ venous ­stasis, as generated by prolonged tourniquet placement, may seriously impact laboratory testing. The tourniquet should ideally only be applied if strictly necessary, and removed when the needle is in the vein.

However, venous stasis is often maintained throughout extended or challenging venipunctures, ­producing haemoconcentration and spurious results in several coagulation assays.(9) This adverse circumstance can be prevented or minimised by nonapplication or early release of the tourniquet, standardisation of external pressure, use of easy-to-apply re/de-inflatable devices and standardised sequences of tubes. The collection of inappropriate containers for testing is an additional source of ­concern, accounting for as high as one-sixth of unsuitable specimens.(6) It is recommended that anticoagulant-containing blood collection tubes for coagulation testing be filled to the proper level, usually to complete the vacuum volume, and gently inverted several times to allow effective mixing. Inappropriate tube filling variably affects coagulation test results, depending on the nature of the vial, the concentration of the anticoagulant in the specimen and the reagents’ sensitivity.

Clinical Laboratory Standards Institute
The Clinical Laboratory Standards Institute (CLSI) currently recommends that coagulation samples be discarded if the evacuated tube contains less than 90% of the expected fill volume.(10) Specimen ­stability is critical in most coagulation tests. However, either plasma or whole-blood samples can be accepted for most routine and second-line coagulation testing 6–12 hours from collection, when stored at room temperature or refrigerated.(3) The current CLSI guidelines recommend centrifuge tubes for ­routine coagulation testing at 1,500 g for no less than 15 minutes at room temperature.(10) However, even whole- blood specimen centrifugation with slight temperature biases may not generate significant ­analytical or clinical variations.(11)

Conclusion
Poor control and standardisation of each phase of the overall testing process still plague the reliability of coagulation testing.(3) Assay interferences from improper collection and handling of primary tubes represent significant challenges as they are barely detectable by ongoing quality-control or proficiency testing programmes. Strict monitoring of outcomes through implementation of performance indicators is pivotal, along with a rigorous educational policy to inform operators with blood collection
responsibilities on this unwanted variability.(12)

A reliable solution to this problem would be the adoption of standardised and easy-to-understand policies for collecting samples, implementation of information technology (barcoded wristbands, ­computerised order entry systems and expert systems based on autovalidation, as well as delta ­checking and other similar algorithms) and error detection systems that would enable either redesign or reorganisation of activities in a less hazardous model, with decreased complexity and fewer error-prone steps. Good laboratory practice and compliance with new accreditation or ­certification programmes, such as the International Organization for Standardization 15189 standard expressly developed for medical laboratories,(14) would lead to enormous organisational and economic benefits for the laboratory and the entire healthcare system.

References

1. Lippi G, et al. Clin Chem Lab Med 2007;45:2-12.
2. Franchini M, et al. Crit Rev Clin Lab Sci 2006;43:249-90.
3. Lippi G, et al. Blood Coagul Fibrinolysis 2006;17:513-9.
4. Lundberg GD. Clin Chim Acta 1999;280:3-11.
5. Lippi G, et al. Clin Chem Lab Med 2006;44:358-65.
6. Lippi G, et al. Clin Chem 2006;52:1442-3.
7. Joint Commission on Accreditation of Healthcare Organization. 2007 Laboratory Services National Patient Safety Goals. www.jointcommission.org/PatientSafetyNationalPatientSafety Goals/07_lab_npsgs.htm
8. Lippi G, et al. Arch Pathol Lab Med 2006;130:181-4.
9. Lippi G, et al. Blood Coagul Fibrinolysis 2005;16:453-8.
10. Clinical and Laboratory Standards Institute. Collection, transport, and processing of blood specimens for coagulation testing and general performance of coagulation assays. Approved guideline H21-A4, 3rd ed. Wayne (PA): National Committee for Clinical Laboratory Standards; 2003.
11. Lippi G, et al. Clin Chem 2006;52:537-8.
12. Lippi G, et al. Clin Lab 2006;52:457-62.
13. Lippi G, et al. Clin Lab 2006;52:217-30.
14. Wiwanitkit V. Blood Coagul Fibrinolysis 2004;15:613-7.