research paper
Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert® HIT) for the diagnosis of heparin-induced thrombocytopenia
Dorothee Leroux,1 Nathalie Hezard,2 Aurelien Lebreton,3 Anne Bauters,4 Pierre Suchon,5 Emmanuel de Maistre,6 Christine Biron,7 Marie-Genevieve Huisse,8 Catherine Ternisien,9 Sophie Voisin,10 Yves Gruel1 and Claire Pouplard1 1
Haemostasis Laboratory and UMR CNRS 7292,
H^opital Trousseau and Universite Francßois Rabelais Tours, Tours, 2Haemostasis Laboratory, H^opital Robert Debre, Reims, 3Haemostasis Laboratory, CHU Estaing, Clermont Ferrand, 4
Haemostasis Laboratory, CHRU, Lille, 5Haemo-
stasis Laboratory, H^opital de la Timone, Marseille, 6Haemostasis Laboratory, H^opital du Bocage, Dijon, 7Haemostasis Laboratory, H^opital Saint Eloi, Montpellier, 8Haemostasis Laboratory, H^opital Bichat, Paris, 9Haemostasis Laboratory, H^opital Hotel Dieu, Nantes, and
10
Haemostasis
Laboratory, H^opital Rangueil, Toulouse, France Received 9 January 2014; accepted for publication 19 March 2014
Abstract A rapid lateral flow immunoassay (LFIA) (STic Expert HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 287%, 617% and 96% of patients, respectively. Definite HIT was diagnosed in 40 patients (120%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 09). The negative predictive value of LFIA with plasma samples was 996% with a negative likelihood ratio (LR) of 003, and was comparable to those of the particle gel immunoassay (H/PF4PaGIA) performed in 124 cases. Positive predictive value and positive LR were 444% and 587, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 112% to 04% when the LFIA result was negative and increased to 425% when it was positive. In conclusion, the STic Expert HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT. Keywords: heparin-induced thrombocytopenia, Immunoassay, diagnosis, Bayes’ theorem.
Correspondence: Claire Pouplard, Department of Haematology-Haemostasis. H^ opital Trousseau, CHU de Tours. 37044 Tours, France. E-mail: [email protected]
Introduction Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, mainly due to IgG antibodies that recognize multimolecular complexes of platelet factor 4 (PF4) and heparin. These complexes bind to platelet FccRIIA receptors, inducing strong cell activation with the release of procoagulant platelet-derived microparticles, which contribute to the formation of venous and arterial thromboses that are the clinical hallmarks of HIT. The diagnosis of HIT is difficult in practice because most patients present several causes of thrombocytopenia. However, HIT is today recognized as a clinicopathological syndrome and its diagnosis is based on the combination of a compatible clinical picture and the biological detection of First published online 12 May 2014 doi: 10.1111/bjh.12939
platelet activating anti-PF4/H antibodies (Warkentin et al, 2011). Clinical scoring systems (4Ts [magnitude of thrombocytopenia, timing of thrombocytopenia with respect to heparin exposure, thrombosis or other sequelae of HIT, likelihood of other causes of thrombocytopenia] and HEP [HIT Expert Probability]) have been proposed to assess the pre-test likelihood of HIT (Lo et al, 2006; Cuker et al, 2010), and are now widely used in combination with biological assays. Moreover, a large number of assays are currently available to diagnose HIT, and these can be divided in two major categories: (i) antigen assays that detect the presence of HIT PF4-dependent antibodies and (ii) functional assays that provide evidence that these antibodies are able to induce platelet activation in the presence of heparin. Functional assays, such ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 774–782
STic Expert HIT for the Diagnosis of HIT as the platelet aggregation test and serotonin release assay (SRA), were the first reported and they are specific for the diagnosis of HIT, but require fresh human platelets and strict quality controls. In addition, they are time-consuming and no commercial quality control is available, which is contrary to the recommendations of the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (Warkentin et al, 2011). Amiral et al (1992) demonstrated that PF4/H complexes are the main antigens recognized by HIT antibodies, and specific immunoassays then became rapidly available. The first generation of such assays used H/PF4 or Polyvinyl-Sulfonate/PF4 complexes as antigen targets and detected all classes of antibodies (IgG, IgA and IgM). The second generation of enzyme-linked immunosorbent assays (ELISAs) detected IgG HIT antibodies only, resulting in increased specificity and positive predictive value (PPV) compared with polyspecific assays. Two new fully automated quantitative chemiluminescence assays have also been developed recently, but their wide use is prevented by the fact they require special laboratory equipment (Legnani et al, 2010). Moreover, most immunoassays are preferentially tailored for testing series rather than single patient samples. A rapid particle gel immunoassay for the detection of HIT antibodies in emergent conditions that provides a result in less than 1 h after blood sampling was developed more than 10 years ago (H/PF4-PaGIA, Biorad, Marne La Coquette, France). This test detects IgG, IgA and IgM antibodies to PF4/H complexes, and therefore has relatively low specificity and PPV (Pouplard et al, 2007; Nellen et al, 2012). An IgG-specific rapid lateral flow immunoassay (LFIA) was developed more recently for the detection of HIT antibodies (Sachs et al, 2011), with levels of effectiveness suggesting a high potential for reducing both the risk of misdiagnosis and the cost in patients with suspected HIT. The aim of this multicentre prospective study was therefore to evaluate the effectiveness of this rapid assay in combination with the clinical pre-test 4Ts score in a large prospective multicentre cohort of patients with suspected HIT. In addition, we evaluated inter-reader reproducibility and compared our results to those obtained with the particle gel immunoassay.
Patients and methods This prospective study was undertaken between February and October 2012, and included 380 consecutive patients with suspected HIT treated in ten different French centres. Fortysix patients were excluded because clinical information and/ or plasma/serum samples were not available.
Patients All patients included in the study had been treated with unfractionated heparin (UFH) and/or low-molecular ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 774–782
weight-heparin (LMWH) and were suspected by their clinicians of having developed HIT, with platelet counts of
Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert® HIT) for the diagnosis of heparin-induced thrombocytopenia
Dorothee Leroux,1 Nathalie Hezard,2 Aurelien Lebreton,3 Anne Bauters,4 Pierre Suchon,5 Emmanuel de Maistre,6 Christine Biron,7 Marie-Genevieve Huisse,8 Catherine Ternisien,9 Sophie Voisin,10 Yves Gruel1 and Claire Pouplard1 1
Haemostasis Laboratory and UMR CNRS 7292,
H^opital Trousseau and Universite Francßois Rabelais Tours, Tours, 2Haemostasis Laboratory, H^opital Robert Debre, Reims, 3Haemostasis Laboratory, CHU Estaing, Clermont Ferrand, 4
Haemostasis Laboratory, CHRU, Lille, 5Haemo-
stasis Laboratory, H^opital de la Timone, Marseille, 6Haemostasis Laboratory, H^opital du Bocage, Dijon, 7Haemostasis Laboratory, H^opital Saint Eloi, Montpellier, 8Haemostasis Laboratory, H^opital Bichat, Paris, 9Haemostasis Laboratory, H^opital Hotel Dieu, Nantes, and
10
Haemostasis
Laboratory, H^opital Rangueil, Toulouse, France Received 9 January 2014; accepted for publication 19 March 2014
Abstract A rapid lateral flow immunoassay (LFIA) (STic Expert HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 287%, 617% and 96% of patients, respectively. Definite HIT was diagnosed in 40 patients (120%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 09). The negative predictive value of LFIA with plasma samples was 996% with a negative likelihood ratio (LR) of 003, and was comparable to those of the particle gel immunoassay (H/PF4PaGIA) performed in 124 cases. Positive predictive value and positive LR were 444% and 587, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 112% to 04% when the LFIA result was negative and increased to 425% when it was positive. In conclusion, the STic Expert HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT. Keywords: heparin-induced thrombocytopenia, Immunoassay, diagnosis, Bayes’ theorem.
Correspondence: Claire Pouplard, Department of Haematology-Haemostasis. H^ opital Trousseau, CHU de Tours. 37044 Tours, France. E-mail: [email protected]
Introduction Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin treatment, mainly due to IgG antibodies that recognize multimolecular complexes of platelet factor 4 (PF4) and heparin. These complexes bind to platelet FccRIIA receptors, inducing strong cell activation with the release of procoagulant platelet-derived microparticles, which contribute to the formation of venous and arterial thromboses that are the clinical hallmarks of HIT. The diagnosis of HIT is difficult in practice because most patients present several causes of thrombocytopenia. However, HIT is today recognized as a clinicopathological syndrome and its diagnosis is based on the combination of a compatible clinical picture and the biological detection of First published online 12 May 2014 doi: 10.1111/bjh.12939
platelet activating anti-PF4/H antibodies (Warkentin et al, 2011). Clinical scoring systems (4Ts [magnitude of thrombocytopenia, timing of thrombocytopenia with respect to heparin exposure, thrombosis or other sequelae of HIT, likelihood of other causes of thrombocytopenia] and HEP [HIT Expert Probability]) have been proposed to assess the pre-test likelihood of HIT (Lo et al, 2006; Cuker et al, 2010), and are now widely used in combination with biological assays. Moreover, a large number of assays are currently available to diagnose HIT, and these can be divided in two major categories: (i) antigen assays that detect the presence of HIT PF4-dependent antibodies and (ii) functional assays that provide evidence that these antibodies are able to induce platelet activation in the presence of heparin. Functional assays, such ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 774–782
STic Expert HIT for the Diagnosis of HIT as the platelet aggregation test and serotonin release assay (SRA), were the first reported and they are specific for the diagnosis of HIT, but require fresh human platelets and strict quality controls. In addition, they are time-consuming and no commercial quality control is available, which is contrary to the recommendations of the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (Warkentin et al, 2011). Amiral et al (1992) demonstrated that PF4/H complexes are the main antigens recognized by HIT antibodies, and specific immunoassays then became rapidly available. The first generation of such assays used H/PF4 or Polyvinyl-Sulfonate/PF4 complexes as antigen targets and detected all classes of antibodies (IgG, IgA and IgM). The second generation of enzyme-linked immunosorbent assays (ELISAs) detected IgG HIT antibodies only, resulting in increased specificity and positive predictive value (PPV) compared with polyspecific assays. Two new fully automated quantitative chemiluminescence assays have also been developed recently, but their wide use is prevented by the fact they require special laboratory equipment (Legnani et al, 2010). Moreover, most immunoassays are preferentially tailored for testing series rather than single patient samples. A rapid particle gel immunoassay for the detection of HIT antibodies in emergent conditions that provides a result in less than 1 h after blood sampling was developed more than 10 years ago (H/PF4-PaGIA, Biorad, Marne La Coquette, France). This test detects IgG, IgA and IgM antibodies to PF4/H complexes, and therefore has relatively low specificity and PPV (Pouplard et al, 2007; Nellen et al, 2012). An IgG-specific rapid lateral flow immunoassay (LFIA) was developed more recently for the detection of HIT antibodies (Sachs et al, 2011), with levels of effectiveness suggesting a high potential for reducing both the risk of misdiagnosis and the cost in patients with suspected HIT. The aim of this multicentre prospective study was therefore to evaluate the effectiveness of this rapid assay in combination with the clinical pre-test 4Ts score in a large prospective multicentre cohort of patients with suspected HIT. In addition, we evaluated inter-reader reproducibility and compared our results to those obtained with the particle gel immunoassay.
Patients and methods This prospective study was undertaken between February and October 2012, and included 380 consecutive patients with suspected HIT treated in ten different French centres. Fortysix patients were excluded because clinical information and/ or plasma/serum samples were not available.
Patients All patients included in the study had been treated with unfractionated heparin (UFH) and/or low-molecular ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 166, 774–782
weight-heparin (LMWH) and were suspected by their clinicians of having developed HIT, with platelet counts of
