International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
REVIEW
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Current insights into the laboratory diagnosis of HIT € T. BAKCHOUL, H. Z OLLNER, A. GREINACHER
Department of Immunology and Transfusion Medicine, Universit€atsmedizin Greifswald, Greifswald, Germany Correspondence: Tamam Bakchoul, Department of Immunology and Transfusion Medicine, Universit€atsmedizin Greifswald, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany. Tel.: +49 3834 865458; Fax: +49 3834 865489; E-mail: [email protected] doi:10.1111/ijlh.12236
Received 19 January 2014; accepted for publication 12 March 2014 Keywords Heparin, thrombocytopenia, diagnosis, antibody, HIT
S U M M A RY
Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction and prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After antibody binding to PF4/heparin complexes, HIT antibodies are capable of intravascular platelet activation by cross-linking Fc gamma receptor IIa (FccRIIa) on the platelet surface leading to a platelet count decrease and/or thrombosis. In contrast to most other immune-mediated disorders, the currently available laboratory tests for anti-PF4/heparin antibodies show a high sensitivity also for clinically irrelevant antibodies. This makes the diagnosis of HIT challenging and bears the risk to substantially overdiagnose HIT. The strength of the antigen assays for HIT is in ruling out HIT when the test is negative. Functional assays have a higher specificity for clinically relevant antibodies, but they are restricted to specialized laboratories. Currently, a Bayesian approach combining the clinical likelihood estimation for HIT with laboratory tests is the most appropriate approach to diagnose HIT. In this review, we give an overview on currently available diagnostic procedures and discuss their limitations.
INTRODUCTION Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse drug reaction that occurs after exposure to unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), or other polyanions such as hypersulfated chondroitin sulfate [1]. The in vivo formation of highly immunogenic multimolecular complexes consisting of the cationic protein platelet factor 4 (PF4) and negatively charged polyanions results in antibody formation in many heparin-exposed patients [2, 3]. Only a minority of these patients, however, develop clinical HIT
296
characterized by a fall in platelet count occurring typically between days 5 and 14 after start of heparin therapy often complicated by additional thromboembolic events [4]. Antibodies against PF4/heparin complexes are responsible for the prothrombotic nature of HIT due to their capability to induce platelet activation by cross-linking platelet Fcc receptor IIa (CD32a). This leads to the release of procoagulant platelet-derived microparticles, which provide the catalytic surface for excess thrombin generation [5, 6]. Thrombin generation is one of the clinically most important characteristics of HIT. Therefore, when HIT is strongly
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
297
suspected, not only heparin should be stopped, but more importantly, alternative nonheparin anticoagulant therapy is required to prevent new thromboembolic complications. However, as alternative anticoagulants are only approved for the niche indication HIT, many physicians have limited experience in handling these drugs. This increases the risk for both bleeding due to overdosing and thrombotic events due to underdosing. Therefore, reliable diagnosis of ‘true’ HIT is essential for safe and cost-effective patient management. Two approaches can help the treating physician to rule out HIT: (i) systematic assessment of the clinical presentation using scoring models that determine the pretest probability of HIT and (ii) in vitro demonstration of anti-PF4/heparin antibodies. Characteristics of current laboratory tests for the detection of heparindependent antibodies and the interpretation of their results in the clinical context are the focus of this review.
bypass (CPB) surgery [9]. This score evaluates three independent parameters: platelet count pattern, time from CPB to the first day HIT was suspected, and CPB duration. In a retrospective study, the score showed a good NPV, but this needs to be confirmed in a prospective study. Another scoring system is the HIT expert probability (HEP) score. HEP relies on eight clinical and laboratory features to identify the risk of having HIT. In the first evaluation study, the HEP score had higher inter-observer agreement compared to the 4Ts score [10]. Additionally, a simple score to exclude HIT in patient receiving heparin was suggested by Messmore et al. [11]. This system is designed to arrive at low (0) or possible (1) probability scores depending on the presence or absence of typical HIT manifestation. Both of these novel scores require more validation in larger prospective studies before firm conclusion can be drawn on their performance characteristics.
D I AG N O S T I C PAT H WAYS
Laboratory tests
Evaluating the pretest risk of HIT Application of a scoring system, which helps to systematically check the presence of the typical HIT features, allows even the nonexpert to estimate the probability that HIT is the cause for the clinical presentation of the patient. The most thoroughly evaluated clinical scoring system is the 4Ts score (Thrombocytopenia, Timing, Thrombosis, and oTher causes for thrombocytopenia) [7]. In a recent systematic review and meta-analysis of the predictive values of the 4Ts score, it was shown that a low probability score (≤3) is associated with a high negative predictive value (NPV) for HIT (99.8%) [8]. The positive predictive value (PPV) of the model was, however, limited. The PPV of intermediate (4-5) or high (>6) probability scores was 14% and 64%, respectively [8]. Thus, the 4Ts score is helpful in identifying patients who are unlikely to have HIT. In case of a 4Ts score below 4, the cause for thrombocytopenia is most probably not HIT. At a higher 4Ts score, HIT should be considered and further laboratory investigations should be initiated. A simple early diagnostic score system has been suggested for patients undergoing cardiopulmonary © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
As HIT can often not be excluded on clinical grounds, laboratory testing for heparin-dependent antibodies is a mainstay of the diagnosis. Two different classes of assays are available: immunoassays, which detect binding of anti-PF4/heparin antibodies, and functional laboratory assays, which investigate the capability of antibodies in the patient serum/plasma to activate platelets in the presence of heparin.
Immunoassays Two main types of immunoassays are available to detect anti-PF4/heparin antibodies: enzyme-linked immunosorbent assays (ELISAs) and particle-based immunoassays. In ELISAs, the target antigen (PF4/polyanion complex) is bound onto a solid phase, for example microtiter plate wells, into which patient’s serum or plasma is added. After incubating and washing, an enzymelabeled secondary antibody is added and an enzyme– substrate reaction producing a color change is used to detect anti-PF4/heparin antibodies binding in a semiquantitative fashion. The intensity of color change is proportional to the amount of bound antibodies and measured as optical density (OD).
298
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
At least three ELISAs are commercially available. One assay (from Diagnostica Stago, Asni eres sur Seine, France) utilizes recombinant PF4 bound to heparin [12]. Another assay (from GTI Inc., Waukesha, WI, USA) makes use of purified PF4 (obtained from outdated platelets) bound to the nonheparin polyanion, polyvinyl sulfonate [13]. Another more recent assay (Zymutest HIA, from HYPHEN BioMed, Neuville-Sur-Oise, France) uses platelet lysate (source of PF4 and, possibly, other heparin-binding proteins) to bind to surface-immobilized heparin. Although ELISAs have excellent NPV to rule out HIT, their specificity is lower (40–80%, depending in part on the assay), accounting for the high rate of false positives. Another drawback of most immunoassays is that they cannot detect antibodies directed against heparin-dependent antigens other than PF4, with the exception of those which employ platelet lysate. Some of the platelet-activating antibodies against other chemokines such as interleukin-8 (IL-8) or neutrophil-activating peptide 2 (NAP-2) may induce symptoms resembling clinical HIT [14, 15]. Particle-based immunoassays detect anti-PF4/heparin antibodies through the agglutination of particles bearing PF4 or PF4/heparin complexes. One particlebased immunoassay is known as the particle gel immunoassay (PaGIA), manufactured by DiaMed (Division of Bio-Rad Laboratories, Cressier sur Morat, Switzerland). This assay uses PF4/heparin complexes bound to red, high-density polystyrene beads. After addition of patient serum or plasma, the anti-PF4/ heparin antibodies bind to the antigen-coated beads. Upon centrifugation, the agglutinated beads (indicating the presence of anti-PF4/heparin antibodies) do not migrate through the sephacryl gel, whereas nonagglutinated beads (indicating absence of antibodies) pass through the gel [16]. Test results are then evaluated visually. Unlike ELISAs, particle-based assays are relatively nonquantitative, and they do not distinguish between antibody classes. The major advantage of the particle immunoassays is the rapid turnaround time (15 min). To date, few prospective studies have compared the performance of PaGIA to ELISA in predicting HIT. Two studies reported, however, on falsenegative results obtained by this assay [17, 18]. Another rapid assay is the ‘HealthTEST Heparin/ Platelet Factor 4 Antibody Assay’ (Akers Biosciences, Inc., Thorofare, NJ, USA). Here, a membrane filter is
used to separate nonagglutinated blue-colored particles from agglutinated ones. Hence, a blue color in the result well indicates a negative test result, whereas no color indicates a positive result. This assay fared poorly in comparative studies [19]. Lateral-flow technology was also used to develop a point-of-care test for anti-PF4/heparin antibodies [20]. The lateral-flow immunoassay for the detection of HIT antibodies (LFI-HIT) is based on the principle of capillary action which induces a flow of the test sample along a solid phase, Figure 1. Patient’s serum and PF4 in complex with a polyanion are added to the sample pad. This test system is designed to investigate one patient sample and can be performed within 85% that the sample contains platelet-activating antibodies [23].
Functional assays The clinically most relevant antibodies against PF4/ heparin complexes are those which have the © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
(a)
299
(b)
Figure 1. The lateral-flow immunoassay for detection of HIT antibodies (LFI-HIT). (a) Schematic illustration of the LFI-HIT test. In the positive sample (test line), a goat anti-human IgG capture antibody printed onto the membrane retains human HIT-IgG antibodies bound to platelet factor 4/polyanion complexes attached to gold nanoparticles. The control line indicates that the test has worked properly by binding gold nanoparticles. (b) Two test cassettes of the lateral-flow immunoassay for the detection of HIT antibodies. Ten minutes after application of the sample and reagent, the result (test line-T) can be read.
capability to activate platelets. This can be investigated using functional assays. All functional assays are based on assessing the activation of platelets of normal donors in the presence of patient sera/plasma and heparin. Two groups of functional assays can be distinguished: (i) assays using whole blood or platelet-rich plasma (PRP) and (ii) assays using washed platelets. The readout for all assays is platelet activation. The specificity of all functional assays can be increased by inhibition of platelet activation by adding heparin in excess (100 IU/mL; proving the heparin dependency) and by showing Fc c receptor IIa-dependent activation using a blocking monoclonal antibody (clone IV.3).
aggregometry). Blood from a selected donor is collected in hirudin tubes instead of citrate. UFH is then added (0.5 or 100 IU/mL final concentration), and the suspension is incubated with patient’s citrated platelet-poor plasma (PPP) or heat-inactivated serum. Changes in impedance are then recorded over a 15min period. Under controlled conditions, using the same selected high-responder donor, this assay was shown to have similar sensitivity as serotonin release assay (SRA) [24]. The possibility to perform this assay using whole blood sample avoids the time-consuming preparation of PRP or washed platelets.
Platelet-rich Whole blood assay. In this assay, platelet-activating antibodies are detected using whole blood impedance analyser (Multiplateâ, multiple electrode platelet © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
plasma. In this assay, platelet aggregometry is performed with citrated PRP isolated from one selected donor. Platelet activation (aggregation) is investigated in the presence of the
300
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
patient’s plasma or serum and heparin in two concentrations (0.5 and 100 IU/mL). Although the performance of this assay is easier than washed platelets assays, its sensitivity was shown to vary largely depend on the selected donor and to be inferior to SRA [25].
Washed platelet assays. Most commonly, the heparininduced platelet activation (HIPA) assay and the SRA [26, 27] are used. Both assays employ washed platelets and detect their activation by heparin-dependent antibodies by visually observing formation of platelet aggregates or release of serotonin, respectively, and have similar operating characteristics. In the HIPA assay (Figure 2a), washed platelets from four healthy unselected donors are used. Washed platelets are incubated with the patient’s serum in the presence of buffer (negative control), therapeutic heparin (0.2 anti-FXa/mL, reviparin), or supratherapeutic heparin (100 IU/mL, UFH) in a round bottom microtiter plate. Magnetic stirrers are used as source of shear force, and formation of platelet aggregates is visually determined every 5 min. The test is considered positive if there is platelet aggregation in therapeutic heparin concentrations with the platelet suspensions of at least two of four donors, but not in supratherapeutic heparin concentration, within 30 min. Test results in HIPA are quantitated depending on the lag time to platelet activation. In the SRA (Figure 2b), platelets obtained from a donor previously shown to be reactive to a panel of anti-PF4/heparin antibodies are used. The donor PRP is incubated with 14C-serotonin. Platelets take up the radiolabeled serotonin and store it in their dense granules. Platelets are then washed and incubated with patient’s serum and heparin in flat-bottomed microtiter wells in duplicate on a plate shaker. After incubation for 60 min and centrifugation, supernatants of each reaction mixture are collected and their radioactivity is measured. Test results are expressed as percentage of serotonin release compared to a maximum after detergent-induced platelet lysis (100%). The test is considered positive if there is >20% release at therapeutic heparin levels and
REVIEW
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Current insights into the laboratory diagnosis of HIT € T. BAKCHOUL, H. Z OLLNER, A. GREINACHER
Department of Immunology and Transfusion Medicine, Universit€atsmedizin Greifswald, Greifswald, Germany Correspondence: Tamam Bakchoul, Department of Immunology and Transfusion Medicine, Universit€atsmedizin Greifswald, Ferdinand-Sauerbruch-Straße, 17475 Greifswald, Germany. Tel.: +49 3834 865458; Fax: +49 3834 865489; E-mail: [email protected] doi:10.1111/ijlh.12236
Received 19 January 2014; accepted for publication 12 March 2014 Keywords Heparin, thrombocytopenia, diagnosis, antibody, HIT
S U M M A RY
Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction and prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After antibody binding to PF4/heparin complexes, HIT antibodies are capable of intravascular platelet activation by cross-linking Fc gamma receptor IIa (FccRIIa) on the platelet surface leading to a platelet count decrease and/or thrombosis. In contrast to most other immune-mediated disorders, the currently available laboratory tests for anti-PF4/heparin antibodies show a high sensitivity also for clinically irrelevant antibodies. This makes the diagnosis of HIT challenging and bears the risk to substantially overdiagnose HIT. The strength of the antigen assays for HIT is in ruling out HIT when the test is negative. Functional assays have a higher specificity for clinically relevant antibodies, but they are restricted to specialized laboratories. Currently, a Bayesian approach combining the clinical likelihood estimation for HIT with laboratory tests is the most appropriate approach to diagnose HIT. In this review, we give an overview on currently available diagnostic procedures and discuss their limitations.
INTRODUCTION Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse drug reaction that occurs after exposure to unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), or other polyanions such as hypersulfated chondroitin sulfate [1]. The in vivo formation of highly immunogenic multimolecular complexes consisting of the cationic protein platelet factor 4 (PF4) and negatively charged polyanions results in antibody formation in many heparin-exposed patients [2, 3]. Only a minority of these patients, however, develop clinical HIT
296
characterized by a fall in platelet count occurring typically between days 5 and 14 after start of heparin therapy often complicated by additional thromboembolic events [4]. Antibodies against PF4/heparin complexes are responsible for the prothrombotic nature of HIT due to their capability to induce platelet activation by cross-linking platelet Fcc receptor IIa (CD32a). This leads to the release of procoagulant platelet-derived microparticles, which provide the catalytic surface for excess thrombin generation [5, 6]. Thrombin generation is one of the clinically most important characteristics of HIT. Therefore, when HIT is strongly
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
297
suspected, not only heparin should be stopped, but more importantly, alternative nonheparin anticoagulant therapy is required to prevent new thromboembolic complications. However, as alternative anticoagulants are only approved for the niche indication HIT, many physicians have limited experience in handling these drugs. This increases the risk for both bleeding due to overdosing and thrombotic events due to underdosing. Therefore, reliable diagnosis of ‘true’ HIT is essential for safe and cost-effective patient management. Two approaches can help the treating physician to rule out HIT: (i) systematic assessment of the clinical presentation using scoring models that determine the pretest probability of HIT and (ii) in vitro demonstration of anti-PF4/heparin antibodies. Characteristics of current laboratory tests for the detection of heparindependent antibodies and the interpretation of their results in the clinical context are the focus of this review.
bypass (CPB) surgery [9]. This score evaluates three independent parameters: platelet count pattern, time from CPB to the first day HIT was suspected, and CPB duration. In a retrospective study, the score showed a good NPV, but this needs to be confirmed in a prospective study. Another scoring system is the HIT expert probability (HEP) score. HEP relies on eight clinical and laboratory features to identify the risk of having HIT. In the first evaluation study, the HEP score had higher inter-observer agreement compared to the 4Ts score [10]. Additionally, a simple score to exclude HIT in patient receiving heparin was suggested by Messmore et al. [11]. This system is designed to arrive at low (0) or possible (1) probability scores depending on the presence or absence of typical HIT manifestation. Both of these novel scores require more validation in larger prospective studies before firm conclusion can be drawn on their performance characteristics.
D I AG N O S T I C PAT H WAYS
Laboratory tests
Evaluating the pretest risk of HIT Application of a scoring system, which helps to systematically check the presence of the typical HIT features, allows even the nonexpert to estimate the probability that HIT is the cause for the clinical presentation of the patient. The most thoroughly evaluated clinical scoring system is the 4Ts score (Thrombocytopenia, Timing, Thrombosis, and oTher causes for thrombocytopenia) [7]. In a recent systematic review and meta-analysis of the predictive values of the 4Ts score, it was shown that a low probability score (≤3) is associated with a high negative predictive value (NPV) for HIT (99.8%) [8]. The positive predictive value (PPV) of the model was, however, limited. The PPV of intermediate (4-5) or high (>6) probability scores was 14% and 64%, respectively [8]. Thus, the 4Ts score is helpful in identifying patients who are unlikely to have HIT. In case of a 4Ts score below 4, the cause for thrombocytopenia is most probably not HIT. At a higher 4Ts score, HIT should be considered and further laboratory investigations should be initiated. A simple early diagnostic score system has been suggested for patients undergoing cardiopulmonary © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
As HIT can often not be excluded on clinical grounds, laboratory testing for heparin-dependent antibodies is a mainstay of the diagnosis. Two different classes of assays are available: immunoassays, which detect binding of anti-PF4/heparin antibodies, and functional laboratory assays, which investigate the capability of antibodies in the patient serum/plasma to activate platelets in the presence of heparin.
Immunoassays Two main types of immunoassays are available to detect anti-PF4/heparin antibodies: enzyme-linked immunosorbent assays (ELISAs) and particle-based immunoassays. In ELISAs, the target antigen (PF4/polyanion complex) is bound onto a solid phase, for example microtiter plate wells, into which patient’s serum or plasma is added. After incubating and washing, an enzymelabeled secondary antibody is added and an enzyme– substrate reaction producing a color change is used to detect anti-PF4/heparin antibodies binding in a semiquantitative fashion. The intensity of color change is proportional to the amount of bound antibodies and measured as optical density (OD).
298
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
At least three ELISAs are commercially available. One assay (from Diagnostica Stago, Asni eres sur Seine, France) utilizes recombinant PF4 bound to heparin [12]. Another assay (from GTI Inc., Waukesha, WI, USA) makes use of purified PF4 (obtained from outdated platelets) bound to the nonheparin polyanion, polyvinyl sulfonate [13]. Another more recent assay (Zymutest HIA, from HYPHEN BioMed, Neuville-Sur-Oise, France) uses platelet lysate (source of PF4 and, possibly, other heparin-binding proteins) to bind to surface-immobilized heparin. Although ELISAs have excellent NPV to rule out HIT, their specificity is lower (40–80%, depending in part on the assay), accounting for the high rate of false positives. Another drawback of most immunoassays is that they cannot detect antibodies directed against heparin-dependent antigens other than PF4, with the exception of those which employ platelet lysate. Some of the platelet-activating antibodies against other chemokines such as interleukin-8 (IL-8) or neutrophil-activating peptide 2 (NAP-2) may induce symptoms resembling clinical HIT [14, 15]. Particle-based immunoassays detect anti-PF4/heparin antibodies through the agglutination of particles bearing PF4 or PF4/heparin complexes. One particlebased immunoassay is known as the particle gel immunoassay (PaGIA), manufactured by DiaMed (Division of Bio-Rad Laboratories, Cressier sur Morat, Switzerland). This assay uses PF4/heparin complexes bound to red, high-density polystyrene beads. After addition of patient serum or plasma, the anti-PF4/ heparin antibodies bind to the antigen-coated beads. Upon centrifugation, the agglutinated beads (indicating the presence of anti-PF4/heparin antibodies) do not migrate through the sephacryl gel, whereas nonagglutinated beads (indicating absence of antibodies) pass through the gel [16]. Test results are then evaluated visually. Unlike ELISAs, particle-based assays are relatively nonquantitative, and they do not distinguish between antibody classes. The major advantage of the particle immunoassays is the rapid turnaround time (15 min). To date, few prospective studies have compared the performance of PaGIA to ELISA in predicting HIT. Two studies reported, however, on falsenegative results obtained by this assay [17, 18]. Another rapid assay is the ‘HealthTEST Heparin/ Platelet Factor 4 Antibody Assay’ (Akers Biosciences, Inc., Thorofare, NJ, USA). Here, a membrane filter is
used to separate nonagglutinated blue-colored particles from agglutinated ones. Hence, a blue color in the result well indicates a negative test result, whereas no color indicates a positive result. This assay fared poorly in comparative studies [19]. Lateral-flow technology was also used to develop a point-of-care test for anti-PF4/heparin antibodies [20]. The lateral-flow immunoassay for the detection of HIT antibodies (LFI-HIT) is based on the principle of capillary action which induces a flow of the test sample along a solid phase, Figure 1. Patient’s serum and PF4 in complex with a polyanion are added to the sample pad. This test system is designed to investigate one patient sample and can be performed within 85% that the sample contains platelet-activating antibodies [23].
Functional assays The clinically most relevant antibodies against PF4/ heparin complexes are those which have the © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
(a)
299
(b)
Figure 1. The lateral-flow immunoassay for detection of HIT antibodies (LFI-HIT). (a) Schematic illustration of the LFI-HIT test. In the positive sample (test line), a goat anti-human IgG capture antibody printed onto the membrane retains human HIT-IgG antibodies bound to platelet factor 4/polyanion complexes attached to gold nanoparticles. The control line indicates that the test has worked properly by binding gold nanoparticles. (b) Two test cassettes of the lateral-flow immunoassay for the detection of HIT antibodies. Ten minutes after application of the sample and reagent, the result (test line-T) can be read.
capability to activate platelets. This can be investigated using functional assays. All functional assays are based on assessing the activation of platelets of normal donors in the presence of patient sera/plasma and heparin. Two groups of functional assays can be distinguished: (i) assays using whole blood or platelet-rich plasma (PRP) and (ii) assays using washed platelets. The readout for all assays is platelet activation. The specificity of all functional assays can be increased by inhibition of platelet activation by adding heparin in excess (100 IU/mL; proving the heparin dependency) and by showing Fc c receptor IIa-dependent activation using a blocking monoclonal antibody (clone IV.3).
aggregometry). Blood from a selected donor is collected in hirudin tubes instead of citrate. UFH is then added (0.5 or 100 IU/mL final concentration), and the suspension is incubated with patient’s citrated platelet-poor plasma (PPP) or heat-inactivated serum. Changes in impedance are then recorded over a 15min period. Under controlled conditions, using the same selected high-responder donor, this assay was shown to have similar sensitivity as serotonin release assay (SRA) [24]. The possibility to perform this assay using whole blood sample avoids the time-consuming preparation of PRP or washed platelets.
Platelet-rich Whole blood assay. In this assay, platelet-activating antibodies are detected using whole blood impedance analyser (Multiplateâ, multiple electrode platelet © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 296–305
plasma. In this assay, platelet aggregometry is performed with citrated PRP isolated from one selected donor. Platelet activation (aggregation) is investigated in the presence of the
300
€ T. BAKCHOUL, H. ZOLLNER, AND A. GREINACHER | LABORATORY DIAGNOSIS OF HIT
patient’s plasma or serum and heparin in two concentrations (0.5 and 100 IU/mL). Although the performance of this assay is easier than washed platelets assays, its sensitivity was shown to vary largely depend on the selected donor and to be inferior to SRA [25].
Washed platelet assays. Most commonly, the heparininduced platelet activation (HIPA) assay and the SRA [26, 27] are used. Both assays employ washed platelets and detect their activation by heparin-dependent antibodies by visually observing formation of platelet aggregates or release of serotonin, respectively, and have similar operating characteristics. In the HIPA assay (Figure 2a), washed platelets from four healthy unselected donors are used. Washed platelets are incubated with the patient’s serum in the presence of buffer (negative control), therapeutic heparin (0.2 anti-FXa/mL, reviparin), or supratherapeutic heparin (100 IU/mL, UFH) in a round bottom microtiter plate. Magnetic stirrers are used as source of shear force, and formation of platelet aggregates is visually determined every 5 min. The test is considered positive if there is platelet aggregation in therapeutic heparin concentrations with the platelet suspensions of at least two of four donors, but not in supratherapeutic heparin concentration, within 30 min. Test results in HIPA are quantitated depending on the lag time to platelet activation. In the SRA (Figure 2b), platelets obtained from a donor previously shown to be reactive to a panel of anti-PF4/heparin antibodies are used. The donor PRP is incubated with 14C-serotonin. Platelets take up the radiolabeled serotonin and store it in their dense granules. Platelets are then washed and incubated with patient’s serum and heparin in flat-bottomed microtiter wells in duplicate on a plate shaker. After incubation for 60 min and centrifugation, supernatants of each reaction mixture are collected and their radioactivity is measured. Test results are expressed as percentage of serotonin release compared to a maximum after detergent-induced platelet lysis (100%). The test is considered positive if there is >20% release at therapeutic heparin levels and
