Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis (2012)

Publications known to the writing group were supplemented with additional papers identified by searching Medline/PubMed for publications in the last 12 years using keywords: coagulation assays, amidolytic assays, haemostasis assays, and thrombophilia testing, in core clinical journals and English language. Additional relevant articles were identified by screening reference lists and by publications known to the writing group. The writing group produced the draft guideline which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, BCSH and the British Society for Haematology committee and comments incorporated where appropriate. The ‘GRADE’ system does not apply (because clinical trials are not available to support the ‘best practice’ recommendations that have been generated) and was therefore not used. This guideline is intended to help clinical laboratories perform valid and reliable assays of pro- and anticoagulant factors in blood. Guidance on lupus anticoagulant (LA) testing, fibrinogen assays, D-dimer assays, platelet function tests, von Willebrand factor assays and anticoagulant monitoring is available in other BCSH and UKHCDO documents (www.bcshguidelines.com; www.ukhcdo.org/UKHCDOguidelines.htm). Clinical guidelines on thrombophilia, haemophilia and other bleeding disorders are also available from these bodies and should be used in conjunction with these laboratory guidelines. Global assays of coagulation such as thrombelastography or thrombin generation will not be considered here. The requirements for blood collection, handling and storage have been discussed in detail elsewhere 1-3, but key points are summarized as recommendations in Table 1. Particular attention is drawn to the correct filling of tubes containing the appropriate anticoagulant and the effects of high haematocrit. Many tests used to assess haemostasis can be influenced by medication and this must be taken into account. Coagulation screening tests such as prothrombin time (PT) and activated partial thromboplastin time (APTT) may provide useful information (particularly when full clinical details are not available) which can help to avoid problems in the interpretation of coagulation assay results (e.g. anticoagulant effects or the presence of coagulopathy). Haemolysis can affect some tests of haemostasis, either because of the presence of thromboplastic substances or interference of haemoglobin pigment with photo-optical systems. For example, the APTT can be shortened and antithrombin (AT) decreased by in vitro haemolysis 4. Where in vitro haemolysis has occurred, such samples should not be tested. Gross icterus and lipaemia may also affect analysers, by interfering with optical absorbance or impeding light transmittance. Rheumatoid factor and anti-mouse antibodies may produce erroneous results in immunoassays 5, 6. Whenever possible, normal reference ranges should be established locally with an appropriate number of healthy normal subjects; apparently normal patients should be excluded, as they may be subject to stress which influences certain haemostasis proteins. A minimum number of 120 subjects have been recommended for establishing reference intervals 7. Some authors suggest that a close approximation can be obtained by testing at least 30 normal subjects 8, but with groups <50, the values defining the reference interval have a confidence interval wider than the standard deviation of the observations 9. The reference interval may vary for different reagent and analyser combinations and for some analytes, age, sex and blood group should be considered in the interpretation. Specific and separate intervals are required for paediatric populations 10, 11. Coagulation end-point assays are bioassays used for estimating the concentration of individual blood coagulation factors within a test sample. The concentration is determined relative to a standard or reference preparation (that has itself been calibrated against a standard), by assessing the degree to which dilutions of those preparations (i.e. the standard and the test) correct the coagulation time of a substrate plasma specifically deficient in the factor, in a suitable test system (e.g. PT or APTT). A linear portion of the dose response curve should be determined for each set of reagents by testing a wide range of normal plasma dilutions (e.g. serial doubling dilutions 1/2–1/1024, plus a buffer blank to determine the maximal coagulation time). At least two dilution sets should be made independently to avoid systematic error. This process produces a classical sigmoid dose–response curve with a central linear component (Figure 1a). The differences between plasma dilutions in the series selected for an assay should be as large as possible (i.e. twofold, threefold, fourfold, etc. dilutions), to enable the assay system to discriminate between potency values. However, the coagulation times must remain within the linear portion of the dose–response curve. The selection of dilutions must also be balanced for their ability to allow the determination of relative potencies over a wide range of concentrations. Coagulation assay data may be analysed by the parallel-line method 13, where the dose–response curve of serially diluted test plasma is plotted against the corresponding dilutions of a standard (or reference preparation). With coagulation times on the Y-axis and log concentration (or dilution) on the X-axis, the line of best fit is constructed through the data points of the standard and then the best parallel straight line is constructed for the test sample (Figure 1b). In some cases, coagulation times may need to be log transformed to achieve linearity. This process allows not only quantification of relative potency, but also reveals any evidence of inhibition (e.g. because of the presence of antibodies to coagulation factors, LA, unfractionated heparin (UFH), direct factor Xa (FXa) or thrombin inhibitor), or test sample activation (e.g. because of poor venesection, or the presence of activated coagulation factors) (Figure 2). Determining the relative potency from a single dilution of test sample can give misleading results because linearity and parallelism cannot be determined. Parallelism of the test and standard results is essential for validation of an assay. Although originally assessed subjectively from manually plotted graphs, computer programs 14-16 improved the reliability and accuracy of bioassays. The programs assess variance ratios and statistically significant deviation from linearity (if a minimum of three dilutions of test sample and standard are used) or parallelism (if a minimum of two dilutions are tested). By testing the reference preparation at the beginning and end of the run, the effect of temporal drift during the assay can be assessed and removed from the potency estimate. These programs have been incorporated into modern, automated coagulation analysers so that a calibration curve is processed, assessed for linearity and then subsequently either validated or rejected by the operator. If accepted, the curve is saved and used to determine the relative potencies of test samples. Validation of a linear calibration curve is usually achieved by measuring the correlation coefficient (r), which should generally be 0.998–1.000. Deviation from the usual r-value for a given assay may indicate contamination or deterioration of reagents, such as pH drift in buffer or reagent. A few coagulation analysers perform a statistical evaluation of linearity and parallelism of the calibrator and test sample(s). From the regression model, a slope ratio (calibrator slope/test sample slope) can be calculated (to estimate parallelism) and this should normally be in the range 0.9–1.1; when the ratio falls outside this range, it may indicate an activated sample or the presence of an inhibitor. The use of stored calibration curves on analysers is widely practiced and can give acceptable results for a single reagent batch 17, but has not been recommended 18 because test samples and standards should be treated in exactly the same way to avoid analyser or operator errors, the effects of different buffer batches, changes in ambient temperature, and other differences. Synthetic amidolytic (chromogenic) substrates contain three or four amino acids selected to give appropriate specificity for the given coagulation serine protease 19. A chromophore, usually para-nitroaniline (pNA) is cleaved from the C-terminus amide bond by the protease and free pNA exhibits a yellow colour with absorbance at 405–410 nm, which can be detected by most modern coagulation analysers. The absorbance is proportional to the protease concentration (or inversely proportional to the concentration of natural inhibitors such as AT). Assay specificity may be improved by the inclusion of specific protease inhibitors or direct activators. Fluorogenic substrates have also been employed, but are generally less automated, time-consuming and require careful set-up of individual fluorometers. However, fluorescence is less affected by sample turbidity and these assays have greater sensitivity. Amidolytic assays are performed as end-point assays (absorbance is measured after a fixed incubation time), or kinetic assays (the reaction rate is continuously monitored). End-point assays may be unreliable if sample opacity adds to the absorbance. Some analysers record initial absorbance and subtract it from the end-point value, which reduces this problem. When the analyte level is above the upper level of the calibration curve, substrate depletion can occur and causes underestimation. Kinetic assays are recommended for reliability, but they must be designed to operate within the linear portion of the reaction rate curve and if a nonlinear curve is observed, the results will not be reliable (a different plasma dilution may rectify the problem). If serine proteases other than the one being assayed (or proteases complexed to alpha-2-macroglobulin (A2M)) are present in the test sample, they may cleave the substrate and cause erroneous values 20. High bilirubin levels may cause problems in amidolytic assays where there is little dilution of the test sample [e.g. protein C (PC) assays], and this may be corrected by employing a reagent blank. A variety of immunoassays are available for haemostasis proteins, including radial immunodiffusion (RID), immunoelectrophoresis (IEP), ELISA and immunoturbidimetric (ITB) methods. The latter two are generally recommended for their ease of use, precision and sensitivity. They are usually employed to identify type II defects (where a dysfunctional protein is present, i.e. antigen exceeds activity) and to detect molecular variants where activity assays are inappropriate or unavailable. It is important to know the lower limit of detection for most analytes 21. In coagulation assays, an indication is obtained from the buffer blank time. However, to establish an accurate detection limit in coagulation, amidolytic and immunoassays, the minimum dilution of the analyte that reliably gives a value statistically significant from zero may be determined. When clinical samples are tested, results below the detection limit should be reported as being less than this value. World Health Organization (WHO) International Standards (IS) are available from the National Institute for Biological Standards and Control (NIBSC, Potters Bar, Hertfordshire, UK) for most plasma coagulation factors. However, these are only intended for calibrating working standards. The International Society for Thrombosis and Haemostasis/Scientific and Standardization Committee Secondary Coagulation Standard (distributed by NIBSC) has been calibrated against the WHO IS and is available to manufacturers for the calibration of commercial reference preparations. Lyophilized or frozen commercial calibrators are also available with assigned potencies for most coagulation factors, relative to either pooled normal plasma, or the WHO IS. For factors where there is an IS available, potencies should be assigned in IU. When ‘in house’ methods are used in clinical laboratories for the purpose of diagnosis or patient management, they should be validated to the same standard as ‘CE’ marked products to meet the requirements of the EU In Vitro Diagnostic Medical Devices Directive 22. This also applies to the use of commercial assays on analysers for which the product does not have a CE mark. In clinical laboratories, coagulation factor levels are predominantly measured by one-stage assays based on the ability of test samples to correct either the PT (for factors VII, X, V, II), or the APTT (for factors XII, XI, VIII, IX), of a factor-deficient substrate plasma. PT reagents vary in their source of tissue factor (e.g. rabbit brain, human placenta and relipidated recombinant human tissue factor). Human tissue factor reagents usually give the best sensitivity in coagulation factor assays (see Bolton-Maggs et al. 2 for further discussion in relation to diagnosis of rare bleeding disorders). Some of these preparations also contain a heparin neutralizing agent. APTT reagents vary in type of contact activator (e.g. ellagic acid, kaolin, or micronized silica) and phospholipid composition (e.g. rabbit brain cephalin, soy bean phospholipid extract, or synthetic phospholipid mixtures). Some preparations are relatively insensitive to LA 23-25, and these may be advantageous, making the assay more specific. Factor-deficient substrate plasmas are available from congenitally deficient human donors or after specific depletion (chemical inactivation or immunoadsorption) of normal plasma and are available lyophilized or frozen. Wherever possible, the specificity of the deficient plasma should be checked to ensure that there is a single factor deficiency. The deficient plasma should have normal levels of the nondeficient factors and <1 U/dL of the deficient factor. It has been reported that some FVIII immune-depleted plasmas (also deficient in VWF) can give anomalous results in the Nijmegen modification of the Bethesda assay of FVIII inhibitors 26. When cooled reagent positions on coagulometers are used for deficient plasma, the plasmas should be allowed to equilibrate before use to avoid temporal drift. Snake venoms have been used as activators instead of PT and APTT reagents to improve specificity 27, 28 and some forms of factor X deficiency not detected by the conventional assays have been identified using Russell's viper venom as activator 29. Factor VIII:C is most commonly assayed by one-stage APTT-based assay (reviewed by 30), but two-stage coagulation and amidolytic methods are sometimes used. Amidolytic assays are available for many coagulation factors 19, but their use other than for FVIII assay is usually limited to prekallikrein (where one-stage assays have poor sensitivity) and factor X (used for monitoring oral anticoagulant therapy, particularly in patients with LA 31-33. The results obtained by the different types of FVIII assay are concordant in most instances, but in certain situations may differ significantly 34, particularly in DIC and to a lesser extent in pregnancy. In mild and moderate haemophilia A, the assays usually, but not always, yield concordant results 35, 36. There are reports of a between results of one-stage and other assays in to of mild haemophilia A to of haemophilia A have a normal one-stage activity and but a activity determined by the two-stage coagulation or amidolytic assay In these the bleeding are usually in with the two-stage or amidolytic assay and the is with FVIII the between the and The activity is and two-stage coagulation or amidolytic activity is has also been At least some of these are not with a bleeding but have been identified when of an APTT other such with different defects may have a mild bleeding The most widely used for the detection of factor deficiency is the plasma is with thrombin in the presence and in the of a is The are then in acid, or A by UK that the most was thrombin and The test is relatively only deficiency (i.e. and may be Specific assays are required to detect mild deficiency and should be used in assays of are recommended in thrombophilia testing Amidolytic assays are and and are or is with diluted plasma and and then is measured using an amidolytic absorbance being inversely proportional to II influences human assays and therefore these are not recommended thrombin and human or assays are considered specific. function may be decreased to defects in the or with effects In type II level may be when measured by assays, assays may be normal the incubation time of the sample, dilution with the may the sensitivity to type II defects It may be that assays are most single assay can be to detect type II in test plasma is usually to activated by an from venom known as the coagulation time of plasma through of factor or and on assay APTT-based assays are not influenced by the type of contact but the phospholipid composition is as it influences sensitivity to the anticoagulant effect of which in is to the reagent sensitivity to and There is also to the protein of the assay are on the APTT to achieve a linear dose–response curve APTT-based assays may the level in samples with high the of and and may levels measured in coagulation assays of patient plasma in plasma may or the of this and may sensitivity to levels of assays generally testing a single patient plasma testing three dilutions of standard and test plasma so that parallelism can be LA may affect the assay on the reagent phospholipid concentration and Coagulation assays may detect defects that are by amidolytic and of patients have type deficiency antigen and of the have type with levels by coagulation and amidolytic deficiency detected by the coagulation assay is The of to detect a rare by amidolytic assay must be balanced against the specificity and generally precision of coagulation The latter may be required for diagnosis of where a type is using the amidolytic assay The amidolytic assay is recommended for screening for deficiency because the assays are and are more specific than coagulation assays in patient plasma of an activator (see is measured using an amidolytic The substrates have relatively poor being cleaved by other serine proteases (e.g. factor either free or complexed to can be in end-point assays by including a where the activator is by The absorbance value for this blank is from the test absorbance and the level is In kinetic assays, this may be more as it is to determine the of other proteases to the rate of of the this may sometimes be by poor linearity of the reaction should be performed where effects of proteases are for with paediatric and in patients with coagulation or In this a blank must also be performed for and samples. samples and give a high level with substrate depletion that cannot be corrected using a blank. may be over by amidolytic assay in patients because of the presence of which has reaction to and when the In a of patients on oral values were and by coagulation assays based on the APTT and an amidolytic antigen is generally assayed by ELISA or the latter being time-consuming with poor sensitivity and must be in the assay to ensure that and forms at the same rate ELISA is relatively and to levels of assays type and II deficiency and help identify of type II deficiency when the activity within the normal However, there is in between type and II These are on the ability of to as for inactivation of or and This activity may be detected using APTT or coagulation time assays may detect type II defects which are by assay. However, in the many patients were as type II deficiency when to in of by assay In the presence of plasma dilution will not levels can be over or by assay in the presence of If assays are patient samples should be at three dilutions to assess UK have identified marked differences in results between different commercial and this the interpretation of for these assays are therefore not recommended for use and immunoassays of free are for initial A of of plasma is to protein the free as a for inactivation of and antigen can be measured by ELISA and by The assay is time-consuming and because must be from to avoid this assay is therefore not considered suitable for In some types of ELISA assays, antibodies with different to free and and must be from by high plasma dilution (e.g. plasma dilution) as as incubation of plasma dilution with In certain commercial methods using antibodies with the same for free and such are not The free antigen level activity in plasma and can be measured by ELISA or ELISA for free either specific antibodies or to free from plasma. antibodies can be used in the detection of the It has been reported that in of diluted test sample to the or incubation can in of free in deficient dilutions should be incubation times must not the validated and incubation must be assays of free either two antibodies specific for free or with to free then in the presence of this These assays are designed for specific coagulation analysers, but may be for methods are and with The of methods is that they are to perform and deficiency is by levels of and free antigen as as II deficiency is by deficiency of in type deficiency is but free antigen and activity are of free was to be at deficiency in subjects with defects than of It has been reported that anti-mouse antibodies and the factor in plasma can in significant of analyte in ELISA and the and information in these guidelines is to be and accurate at the time of to the the British Society for Haematology the any for the of these guidelines. authors to the for papers and of the and the Task Force at the time of writing this guideline A