International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
REVIEW
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Antiphospholipid antibody testing and standardization K. M. J. DEVREESE
Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Correspondence: Prof. Dr Katrien Devreese, Coagulation Laboratory, Laboratory for Clinical Biology, Ghent University Hospital, De Pintelaan, 185 (2P8), B-9000 Gent, Belgium. Tel.: 00 32 9 332 65 67; Fax: 00 32 332 49 85; E-mail: [email protected] doi:10.1111/ijlh.12234
Received 20 December 2013; accepted for publication 12 March 2014
S U M M A RY
The laboratory criteria that define patients with antiphospholipid syndrome (APS) include lupus anticoagulant (LAC), anticardiolipin antibodies and anti-b2 glycoprotein I antibodies (ab2GPI). All assays show methodological shortcomings and the combination of the three tests, each with different sensitivity and specificity, and hence, differences in clinical utility make the laboratory diagnosis of APS challenging. Consensus guidelines and proposals for antiphospholipid antibodies (aPL) testing have been published in the last 20 years and have led to a substantial improvement. Despite efforts so far, standardization is not reached yet, but progress has been made. On-going efforts to reduce the interlaboratory/interassay variations remain important; even an absolute standardization cannot be feasibly achieved. Taking into account the methodological shortcomings of the means we have available, more detailed guidelines may help in adequate performance of aPL testing. This review will focus on the efforts and achievements in standardization and on the weaknesses and strengths of the current available laboratory methods.
Keywords Antiphospholipid syndrome, anticardiolipin antibodies, anti-b2 glycoprotein I antibodies, lupus anticoagulant, standardization, guidelines
INTRODUCTION The antiphosphospholipid syndrome (APS) is an autoimmune disease characterized by two major elements: the presence of autoantibodies, the so-called antiphospholipid antibodies (aPL) and the occurrence of clinical features defined as thrombosis and pregnancy complications [1]. The diagnosis of APS requires the presence of at least one clinical and one laboratory criterion. The APS classification criteria published in 352
1999 (Sapporo criteria) were revised in 2006 [1, 2]. These consensus classification criteria for definite APS are more strict and specific and have lead to a substantial improvement in APS recognition; however, the diagnosis of APS remains very difficult [1]. The incidence of clinical features associated with APS is high and often determined by other underlying factors. Consequently, the diagnosis of APS relies predominantly on the laboratory results. An accurate laboratory diagnosis is mandatory, as over-diagnosis as
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
well as under-diagnosis has severe clinical implications. The demonstration of aPL in a patient with for instance thrombosis may classify that patient as an APS patient with therapeutical consequences [3]. Otherwise false-negative results of aPL may lead to underestimation of the thrombotic risk. Therefore, we need assays with high diagnostic power, with high sensitivity and high specificity. The current laboratory criteria comprise three types of tests: (i) assays that detect the aPL as inhibitors of coagulation, the lupus anticoagulant (LAC), (ii) immunoassays that detect anticardiolipin antibodies (aCL), and (iii) immunoassays that detect anti-b2glycoprotein I antibodies (ab2GPI). The antibody–antigen complexes compete with the clotting factors for the phospholipids necessary in clotting assays; this so-called LAC activity was one of the first assays to detect the aPL. Equally, Harris et al. [4] showed that also an enzyme-linked immunosorbent assay (ELISA) could detect aPL, the aCL. A third assay, an ELISA measuring antibodies directed against the b2GPI protein, was added after the discovery of this principle cofactor protein for aPL [5]. We need these three types of assays as the aPL are a heterogeneous group of antibodies with overlapping characteristics, although not identical. The assays differ in the antigen toward the antibodies are directed: with tests for LAC, all aPL are detected, including both those binding b2GPI and those binding by other cofactor proteins such as prothrombin. In the immunoassays, antibodies binding toward cardiolipin or directly to the b2GPI protein are detected. The combination of the three tests, each with different sensitivity and specificity and hence differences in clinical utility [6], makes the laboratory diagnosis of APS challenging [7, 8]. But, the serological diagnosis is hampered by a lack of standardization of the assays and a poor correlation with the clinical symptoms [6, 9]. There is a gap between the requirements and the availability of high quality diagnostic tools for the detection of aPL, comparable to the difficulties met in the serological diagnosis of many other autoimmune diseases. Several factors contribute to variability in test results, including pre-, post- and analytical conditions. These factors comprise methodical problems due to the heterogeneity of the autoimmune antibodies, inadequate standardization of assays, differences in local working conditions, discussions or © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
353
limited knowledge on the relevance of the antibodies, difficulties in correct interpretation of the results, lack of large prospective evaluation studies, lack of a link between antibody potency and clinical effect. Furthermore, there is no ‘golden standard’ for neither of the three aPL assays and a large variety of assays are available that differ in setup and production process. That makes the choice of assays and the decision which assay to use very difficult in daily practice. Despite efforts so far, standardization is not reached yet, but progress has been made [9]. Consensus guidelines and proposals for aPL testing have been published in the last 20 years and are a step toward standardization but appear insufficient. New initiatives on guidelines and development of international standard material are on-going. In this article, I give an overview of the efforts and achievements obtained as a result of collaborative work among workers in the field, and the weaknesses and strengths of the current available laboratory methods.
L U P U S A N T I C OAG U L A N T Lupus anticoagulant measures the functional activity of a subgroup of aPL directed against different cofactor proteins, which explains the partial overlap with the aPL detected by solid phase assays. Traditionally, LAC is detected in a three step procedure including screening, mixing and confirmation tests already described in 1995 [10]. In the screening step, the presence of aPL is illustrated, in the mixing step the presence of an inhibitor is indicated and in the confirmation step the phospholipid-dependent character of the antibodies is proved. Occasionally, a fourth step with coagulation factor dosage is necessary to exclude a specific coagulation factor inhibitor (example given an antiFVIII) behaving in the same way as a LAC in the mixing studies. A need to update the guidelines on LAC [1, 10] was driven by unresolved problems resulting in discordances in test results illustrated in external quality control (EQC) exercises. In 2009, the Scientific Standardization Subcommittee (SSC) on LAC/aPL of the International Society of Thrombosis and Haemostasis (ISTH) provided more detailed guidelines how to perform LAC testing [11]. These guidelines have been proved to be a useful step toward the standardization by answering questions on which patients to test,
354
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
blood collection, preparation and storage, choice of assays, interpretation and transmission of results. The technical specifications outlined in the recommendations meant to ameliorate assay standardization and to increase the reproducibility are given in Table 1 [11]. Recently, the Clinical Laboratory Standards Institute (CLSI) has also prepared a document on laboratory testing for LAC not published yet. The CLSI guideline harmonizes closely with the SSC recommendations, but there are some slight differences. The major difference between these two guidelines is that the SSC guidelines are stricter and give stronger recommendations. This is a very concise text of only four pages written by academic people working in the field, the CLSI guideline contains over 80 pages and is written by academic people and representatives of the manufacturers. Also the British Committee on Standards in Hematology recently published guidelines [12]. The purpose of all guidelines is to standardize and harmonize LAC testing and improve the quality of the testing. Although, the 2009 SSC guidelines [11] are a clear improvement some issues remain unclear, and LAC testing remains a challenge for the diagnostic laboratories. LAC detection suffers from many pitfalls resulting in false-positive or false-negative results (Table 2). Variability in the sensitivity and specificity of assays and the lack of a golden standard to compare
with, a lack of consensus for the use of the mixing step, no criteria to classify LAC as strong, moderate or weak, and variation in interpretation of results between laboratories are some of the unsolved problems. As no coagulation test has 100% sensitivity, the 2009 SSC guidelines recommend two assays of different assay principle, the diluted Russell Viper Venom test (dRVVT) and a sensitive activated partial thromboplastin time (aPTT), with silica as an activator because of its sensitivity for LAC [11]. Other tests as kaolin clotting time, diluted prothrombin time and ecarin/textarin tests are no longer recommended to improve harmonization in LAC testing. To decrease the variation between laboratories, the advice is to use commercially available, quality controlled reagents and assays that are robust and highly reproducible; conditions fulfilled for the dRVVT and aPTT. Even with the restriction to these two types of clotting tests to reduce the likelihood of false-positive detection (the more tests are used the larger the chance to false positives), there are a high number of commercial assays suited for LAC detection available. However, they differ in sensitivity and specificity mainly depending on the concentration, composition, and type of phospholipids [13]. Although EQC programs show that the majority of laboratories are able to distinguish
Table 1. SSC guidelines of 2009 for LAC detection: technical specifications [11] Recommendations Blood collection
Test procedure Choice of assays Screening test Mixing test
Confirmation test
0.109 M sodium citrate (9 : 1) Double centrifugation to obtain platelet poor plasma (40 GPL or MPL) of antibodies are included as diagnostic criterion to improve the specificity of the test [1]. Inhouse cutoff values performed with the local reagent/ instruments combination is the best choice [37, 46]. A 99th percentile cutoff value seems more sensitive than the >40 GPL value as defined in the Sydney criteria [1, 56]. However, exception should be considered for purely obstetric APS with low titer aCL or ab2GPI appearing clinically significant but to be confirmed in more studies [57, 58]. Mathematically, to establish a cutoff value by the 99th percentile, at least 120 healthy volunteers are required [16]. Besides, a careful selection of volunteers regarding exclusion and inclusion criteria should be defined, as well as adequate statistical methods [16]. In-house calculated cutoff values may differ largely from those given by the manufacturer [41, 59]. Sensitivity and specificity with in-house cutoff values should be checked in the local patient population with request for aPL testing through a clinical approach regarding the association with thrombotic/pregnancy complications by 2 9 2 contingency tables. To reach a sufficient diagnostic power, the optimal cutoff value should be adapted and can differ from this given by the manufacturer [59]. Transference of a reference interval may form an alternative for laboratories not having the resources to calculate in-house reference values. Applying the CLSI C28-A3 guideline, manufacturer’s cutoffs may be accepted if no more than two out of 20 tested subjects’ values fall outside those of the manufacturer’s
reported reference limits [16]. The specifications of the manufacturer’s reference interval are mandatory with indication of method of calculation and number and demographic characteristics of donors. An effort should be made to establish an interlaboratory cutoff value for users of identical automated systems, applying the same sample type and with comparable demographic characteristics of patients. To improve the interlaboratory variation, it was suggested to report the results semi-quantitatively as negative, low, medium and high positive [60]. However, this is hampered by incomparability of titers due to interassay variation. Even though in the Sydney criteria it is not explicitly mentioned that aCL assays should be b2GPI dependent, it is a mandatory requirement to avoid detection of non-cofactor-related aCL associated with infections or several drugs [1, 55]. aCL solid phase assays are coated with cardiolipin and supplemented by the washing solution or sample diluent with human or bovine b2GPI as a cofactor for binding aPL [44, 47]. Specificity of aCL assays depends on the source and amount of b2GPI. The amount of human b2GPI in the test sample depends on the sample dilution which is much higher in ELISA compared to automated systems. Besides, the buffer may contain animal or no b2GPI (when purified albumin solutions are used). Limited amount of b2GPI may lead to false-negative results; animal b2GPI may lead to false positive results. Human b2GPI should be used as b2GPI source in b2GPI assays. ab2GPI ELISAs detect all antibodies reactive with b2GPI, including nonpathogenic antibodies, phospholipid-independent and low affinity ab2GPI [8]. Specificity of assays measuring directly ab2GPI depends from other variables: purity, density, and formation of the coated b2GPI and the ionic strength. Negatively charged (high binding or gamma-irradiated plates) should be used to mimic the binding of the b2GPI to negatively charged phospholipids and to increase the antigen density [61]. The type of solid phase, antigen concentration, and coating of commercial ELISA’s or automated new platforms cannot be altered; the performance of these assays should be thoroughly validated. The debate about the role of aCL has started some years ago mainly because of lower specificity. However, methodologically correct b2GPI-dependent aCL assays do have diagnostic value with identical sensitivities and © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
specificities compared to ab2GPI assays [42, 62]. The presently commercially available aCL assays coat the solid phase with cardiolipin and b2GPI, which increases the specificity of the assay [44]. Moreover, but not surprising since all these assays use b2GPI antigen, high correlation may be observed between aCL and ab2GPI titers measured by some new automated systems as well as for ELISA [41, 42]. However, this might not be generalized to all test systems and should be validated in other systems. Current criteria recommend increased levels of IgG and IgM aCL and ab2GPI to confirm APS [1]. IgM aPL are less often associated with thrombosis than IgG [63, 64], while for pregnancy morbidity the role of IgM should be further established [57, 58]. The uncertainty about the role of IgM should be further explored. However, the presence of aCL and ab2GPI of the same isotype reinforces the clinical probability of APS [65]. IgA isotype of aCL and ab2GPI were not included in the current laboratory criteria for APS [1] as the IgA isotype appeared to identify a subgroup of patients rather than to have additional clinical relevance [1]. The prevalence of IgA is higher in African Americans compared to other ethnic groups [1, 66]. The clinical relevance of IgA is still under debate with recent studies describing an association with thrombosis [66–69] and studies, indicating that IgA isotype does not improve diagnostic efficiency [67, 70, 71]. At present, there is insufficient published evidence to recommend routine testing for IgA. In analogy with the 2009 SSC guidelines on LAC detection, the SSC completed recommendations to provide additional details and specifications for aCL and ab2GPI detection [72]. The CLSI as well is working on a document. The implementation of an uniform international reference material and consensus guidelines will take some time. Meanwhile we have to work with the currently available assays. In that view, it is important to know the performance characteristics of the assays and to be aware of the shortcomings and interpret the results accordingly [72]. Before implementation of new system, linearity, precision, limit of detection, and lot-to-lot variation should be checked. The decision of singlet or duplicate testing in routine practice depends on the performance characteristics. An inter- and intrarun imprecision of
REVIEW
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Antiphospholipid antibody testing and standardization K. M. J. DEVREESE
Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium Correspondence: Prof. Dr Katrien Devreese, Coagulation Laboratory, Laboratory for Clinical Biology, Ghent University Hospital, De Pintelaan, 185 (2P8), B-9000 Gent, Belgium. Tel.: 00 32 9 332 65 67; Fax: 00 32 332 49 85; E-mail: [email protected] doi:10.1111/ijlh.12234
Received 20 December 2013; accepted for publication 12 March 2014
S U M M A RY
The laboratory criteria that define patients with antiphospholipid syndrome (APS) include lupus anticoagulant (LAC), anticardiolipin antibodies and anti-b2 glycoprotein I antibodies (ab2GPI). All assays show methodological shortcomings and the combination of the three tests, each with different sensitivity and specificity, and hence, differences in clinical utility make the laboratory diagnosis of APS challenging. Consensus guidelines and proposals for antiphospholipid antibodies (aPL) testing have been published in the last 20 years and have led to a substantial improvement. Despite efforts so far, standardization is not reached yet, but progress has been made. On-going efforts to reduce the interlaboratory/interassay variations remain important; even an absolute standardization cannot be feasibly achieved. Taking into account the methodological shortcomings of the means we have available, more detailed guidelines may help in adequate performance of aPL testing. This review will focus on the efforts and achievements in standardization and on the weaknesses and strengths of the current available laboratory methods.
Keywords Antiphospholipid syndrome, anticardiolipin antibodies, anti-b2 glycoprotein I antibodies, lupus anticoagulant, standardization, guidelines
INTRODUCTION The antiphosphospholipid syndrome (APS) is an autoimmune disease characterized by two major elements: the presence of autoantibodies, the so-called antiphospholipid antibodies (aPL) and the occurrence of clinical features defined as thrombosis and pregnancy complications [1]. The diagnosis of APS requires the presence of at least one clinical and one laboratory criterion. The APS classification criteria published in 352
1999 (Sapporo criteria) were revised in 2006 [1, 2]. These consensus classification criteria for definite APS are more strict and specific and have lead to a substantial improvement in APS recognition; however, the diagnosis of APS remains very difficult [1]. The incidence of clinical features associated with APS is high and often determined by other underlying factors. Consequently, the diagnosis of APS relies predominantly on the laboratory results. An accurate laboratory diagnosis is mandatory, as over-diagnosis as
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
well as under-diagnosis has severe clinical implications. The demonstration of aPL in a patient with for instance thrombosis may classify that patient as an APS patient with therapeutical consequences [3]. Otherwise false-negative results of aPL may lead to underestimation of the thrombotic risk. Therefore, we need assays with high diagnostic power, with high sensitivity and high specificity. The current laboratory criteria comprise three types of tests: (i) assays that detect the aPL as inhibitors of coagulation, the lupus anticoagulant (LAC), (ii) immunoassays that detect anticardiolipin antibodies (aCL), and (iii) immunoassays that detect anti-b2glycoprotein I antibodies (ab2GPI). The antibody–antigen complexes compete with the clotting factors for the phospholipids necessary in clotting assays; this so-called LAC activity was one of the first assays to detect the aPL. Equally, Harris et al. [4] showed that also an enzyme-linked immunosorbent assay (ELISA) could detect aPL, the aCL. A third assay, an ELISA measuring antibodies directed against the b2GPI protein, was added after the discovery of this principle cofactor protein for aPL [5]. We need these three types of assays as the aPL are a heterogeneous group of antibodies with overlapping characteristics, although not identical. The assays differ in the antigen toward the antibodies are directed: with tests for LAC, all aPL are detected, including both those binding b2GPI and those binding by other cofactor proteins such as prothrombin. In the immunoassays, antibodies binding toward cardiolipin or directly to the b2GPI protein are detected. The combination of the three tests, each with different sensitivity and specificity and hence differences in clinical utility [6], makes the laboratory diagnosis of APS challenging [7, 8]. But, the serological diagnosis is hampered by a lack of standardization of the assays and a poor correlation with the clinical symptoms [6, 9]. There is a gap between the requirements and the availability of high quality diagnostic tools for the detection of aPL, comparable to the difficulties met in the serological diagnosis of many other autoimmune diseases. Several factors contribute to variability in test results, including pre-, post- and analytical conditions. These factors comprise methodical problems due to the heterogeneity of the autoimmune antibodies, inadequate standardization of assays, differences in local working conditions, discussions or © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
353
limited knowledge on the relevance of the antibodies, difficulties in correct interpretation of the results, lack of large prospective evaluation studies, lack of a link between antibody potency and clinical effect. Furthermore, there is no ‘golden standard’ for neither of the three aPL assays and a large variety of assays are available that differ in setup and production process. That makes the choice of assays and the decision which assay to use very difficult in daily practice. Despite efforts so far, standardization is not reached yet, but progress has been made [9]. Consensus guidelines and proposals for aPL testing have been published in the last 20 years and are a step toward standardization but appear insufficient. New initiatives on guidelines and development of international standard material are on-going. In this article, I give an overview of the efforts and achievements obtained as a result of collaborative work among workers in the field, and the weaknesses and strengths of the current available laboratory methods.
L U P U S A N T I C OAG U L A N T Lupus anticoagulant measures the functional activity of a subgroup of aPL directed against different cofactor proteins, which explains the partial overlap with the aPL detected by solid phase assays. Traditionally, LAC is detected in a three step procedure including screening, mixing and confirmation tests already described in 1995 [10]. In the screening step, the presence of aPL is illustrated, in the mixing step the presence of an inhibitor is indicated and in the confirmation step the phospholipid-dependent character of the antibodies is proved. Occasionally, a fourth step with coagulation factor dosage is necessary to exclude a specific coagulation factor inhibitor (example given an antiFVIII) behaving in the same way as a LAC in the mixing studies. A need to update the guidelines on LAC [1, 10] was driven by unresolved problems resulting in discordances in test results illustrated in external quality control (EQC) exercises. In 2009, the Scientific Standardization Subcommittee (SSC) on LAC/aPL of the International Society of Thrombosis and Haemostasis (ISTH) provided more detailed guidelines how to perform LAC testing [11]. These guidelines have been proved to be a useful step toward the standardization by answering questions on which patients to test,
354
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
blood collection, preparation and storage, choice of assays, interpretation and transmission of results. The technical specifications outlined in the recommendations meant to ameliorate assay standardization and to increase the reproducibility are given in Table 1 [11]. Recently, the Clinical Laboratory Standards Institute (CLSI) has also prepared a document on laboratory testing for LAC not published yet. The CLSI guideline harmonizes closely with the SSC recommendations, but there are some slight differences. The major difference between these two guidelines is that the SSC guidelines are stricter and give stronger recommendations. This is a very concise text of only four pages written by academic people working in the field, the CLSI guideline contains over 80 pages and is written by academic people and representatives of the manufacturers. Also the British Committee on Standards in Hematology recently published guidelines [12]. The purpose of all guidelines is to standardize and harmonize LAC testing and improve the quality of the testing. Although, the 2009 SSC guidelines [11] are a clear improvement some issues remain unclear, and LAC testing remains a challenge for the diagnostic laboratories. LAC detection suffers from many pitfalls resulting in false-positive or false-negative results (Table 2). Variability in the sensitivity and specificity of assays and the lack of a golden standard to compare
with, a lack of consensus for the use of the mixing step, no criteria to classify LAC as strong, moderate or weak, and variation in interpretation of results between laboratories are some of the unsolved problems. As no coagulation test has 100% sensitivity, the 2009 SSC guidelines recommend two assays of different assay principle, the diluted Russell Viper Venom test (dRVVT) and a sensitive activated partial thromboplastin time (aPTT), with silica as an activator because of its sensitivity for LAC [11]. Other tests as kaolin clotting time, diluted prothrombin time and ecarin/textarin tests are no longer recommended to improve harmonization in LAC testing. To decrease the variation between laboratories, the advice is to use commercially available, quality controlled reagents and assays that are robust and highly reproducible; conditions fulfilled for the dRVVT and aPTT. Even with the restriction to these two types of clotting tests to reduce the likelihood of false-positive detection (the more tests are used the larger the chance to false positives), there are a high number of commercial assays suited for LAC detection available. However, they differ in sensitivity and specificity mainly depending on the concentration, composition, and type of phospholipids [13]. Although EQC programs show that the majority of laboratories are able to distinguish
Table 1. SSC guidelines of 2009 for LAC detection: technical specifications [11] Recommendations Blood collection
Test procedure Choice of assays Screening test Mixing test
Confirmation test
0.109 M sodium citrate (9 : 1) Double centrifugation to obtain platelet poor plasma (40 GPL or MPL) of antibodies are included as diagnostic criterion to improve the specificity of the test [1]. Inhouse cutoff values performed with the local reagent/ instruments combination is the best choice [37, 46]. A 99th percentile cutoff value seems more sensitive than the >40 GPL value as defined in the Sydney criteria [1, 56]. However, exception should be considered for purely obstetric APS with low titer aCL or ab2GPI appearing clinically significant but to be confirmed in more studies [57, 58]. Mathematically, to establish a cutoff value by the 99th percentile, at least 120 healthy volunteers are required [16]. Besides, a careful selection of volunteers regarding exclusion and inclusion criteria should be defined, as well as adequate statistical methods [16]. In-house calculated cutoff values may differ largely from those given by the manufacturer [41, 59]. Sensitivity and specificity with in-house cutoff values should be checked in the local patient population with request for aPL testing through a clinical approach regarding the association with thrombotic/pregnancy complications by 2 9 2 contingency tables. To reach a sufficient diagnostic power, the optimal cutoff value should be adapted and can differ from this given by the manufacturer [59]. Transference of a reference interval may form an alternative for laboratories not having the resources to calculate in-house reference values. Applying the CLSI C28-A3 guideline, manufacturer’s cutoffs may be accepted if no more than two out of 20 tested subjects’ values fall outside those of the manufacturer’s
reported reference limits [16]. The specifications of the manufacturer’s reference interval are mandatory with indication of method of calculation and number and demographic characteristics of donors. An effort should be made to establish an interlaboratory cutoff value for users of identical automated systems, applying the same sample type and with comparable demographic characteristics of patients. To improve the interlaboratory variation, it was suggested to report the results semi-quantitatively as negative, low, medium and high positive [60]. However, this is hampered by incomparability of titers due to interassay variation. Even though in the Sydney criteria it is not explicitly mentioned that aCL assays should be b2GPI dependent, it is a mandatory requirement to avoid detection of non-cofactor-related aCL associated with infections or several drugs [1, 55]. aCL solid phase assays are coated with cardiolipin and supplemented by the washing solution or sample diluent with human or bovine b2GPI as a cofactor for binding aPL [44, 47]. Specificity of aCL assays depends on the source and amount of b2GPI. The amount of human b2GPI in the test sample depends on the sample dilution which is much higher in ELISA compared to automated systems. Besides, the buffer may contain animal or no b2GPI (when purified albumin solutions are used). Limited amount of b2GPI may lead to false-negative results; animal b2GPI may lead to false positive results. Human b2GPI should be used as b2GPI source in b2GPI assays. ab2GPI ELISAs detect all antibodies reactive with b2GPI, including nonpathogenic antibodies, phospholipid-independent and low affinity ab2GPI [8]. Specificity of assays measuring directly ab2GPI depends from other variables: purity, density, and formation of the coated b2GPI and the ionic strength. Negatively charged (high binding or gamma-irradiated plates) should be used to mimic the binding of the b2GPI to negatively charged phospholipids and to increase the antigen density [61]. The type of solid phase, antigen concentration, and coating of commercial ELISA’s or automated new platforms cannot be altered; the performance of these assays should be thoroughly validated. The debate about the role of aCL has started some years ago mainly because of lower specificity. However, methodologically correct b2GPI-dependent aCL assays do have diagnostic value with identical sensitivities and © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 352–363
K. M. J. DEVREESE | ANTIPHOSPHOLIPID ANTIBODIES AND STANDARDIZATION
specificities compared to ab2GPI assays [42, 62]. The presently commercially available aCL assays coat the solid phase with cardiolipin and b2GPI, which increases the specificity of the assay [44]. Moreover, but not surprising since all these assays use b2GPI antigen, high correlation may be observed between aCL and ab2GPI titers measured by some new automated systems as well as for ELISA [41, 42]. However, this might not be generalized to all test systems and should be validated in other systems. Current criteria recommend increased levels of IgG and IgM aCL and ab2GPI to confirm APS [1]. IgM aPL are less often associated with thrombosis than IgG [63, 64], while for pregnancy morbidity the role of IgM should be further established [57, 58]. The uncertainty about the role of IgM should be further explored. However, the presence of aCL and ab2GPI of the same isotype reinforces the clinical probability of APS [65]. IgA isotype of aCL and ab2GPI were not included in the current laboratory criteria for APS [1] as the IgA isotype appeared to identify a subgroup of patients rather than to have additional clinical relevance [1]. The prevalence of IgA is higher in African Americans compared to other ethnic groups [1, 66]. The clinical relevance of IgA is still under debate with recent studies describing an association with thrombosis [66–69] and studies, indicating that IgA isotype does not improve diagnostic efficiency [67, 70, 71]. At present, there is insufficient published evidence to recommend routine testing for IgA. In analogy with the 2009 SSC guidelines on LAC detection, the SSC completed recommendations to provide additional details and specifications for aCL and ab2GPI detection [72]. The CLSI as well is working on a document. The implementation of an uniform international reference material and consensus guidelines will take some time. Meanwhile we have to work with the currently available assays. In that view, it is important to know the performance characteristics of the assays and to be aware of the shortcomings and interpret the results accordingly [72]. Before implementation of new system, linearity, precision, limit of detection, and lot-to-lot variation should be checked. The decision of singlet or duplicate testing in routine practice depends on the performance characteristics. An inter- and intrarun imprecision of
