Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia

Abstract Background Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent “autoimmune” HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs. Objectives To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3). Methods Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method). Results 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin. Conclusion 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.

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Challenges in Detecting Clinically Relevant Heparin-Induced Thrombocytopenia Antibodies

Hämostaseologie ◽

10.1055/a-1223-3329 ◽

2020 ◽

Vol 40 (04) ◽

pp. 472-484

Author(s):

Theodore E. Warkentin

Keyword(s):

Platelet Activation ◽

False Negative ◽

Serotonin Release ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Thrombosis Risk ◽

Activation Assay ◽

Release Assay ◽

Chemiluminescent Immunoassay

AbstractHeparin-induced thrombocytopenia (HIT) is an antibody-mediated hypercoagulable state featuring high thrombosis risk and distinct pathogenesis involving immunoglobulin G-mediated platelet activation. The target of the immune response is a cationic “self” protein, platelet factor 4 (PF4), rendered antigenic by heparin. A key problem is that only a minority of anti-PF4/polyanion antibodies induced by heparin are pathogenic, i.e., capable of causing platelet activation and thereby clinical HIT. Since thrombocytopenia occurs frequently in hospitalized, heparin-treated patients, testing for “HIT antibodies” is common; thus, the problem of distinguishing between pathogenic and nonpathogenic antibodies is important. The central concept is that those antibodies that have platelet-activating properties demonstrable in vitro correlate well with pathogenicity, as shown by platelet activation tests such as the serotonin-release assay (SRA) and heparin-induced platelet activation assay. However, in most circumstances, immunoassays are used for first-line testing, and so it is important for clinicians to appreciate which immunoassay result profiles—in the appropriate clinical context—predict the presence of platelet-activating antibodies (Bayesian analysis). Clinicians with access to rapid, on-demand HIT immunoassays (e.g., particle gel immunoassay, latex immunoturbidimetric assay, chemiluminescent immunoassay) can look beyond simple dichotomous result interpretation (“negative”/“positive”) and incorporate semiquantitative interpretation, where, for example, a strong-positive immunoassay result (or even combination of two immunoassays) points to a greater probability of detecting platelet-activating antibodies, and hence supporting a diagnosis of HIT. Recent recognition of “SRA-negative HIT” has increased the importance of semiquantitative interpretation of immunoassays, given that strong immunoassay reactivity is a potential clue indicating possible HIT despite a (false) negative platelet activation assay.

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Pathophysiology of Heparin-Induced Thrombocytopenia

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1657-pohit ◽

2000 ◽

Vol 124 (11) ◽

pp. 1657-1666 ◽

Cited By ~ 4

Author(s):

Fabrizio Fabris ◽

Sarfraz Ahmad ◽

Giuseppe Cella ◽

Walter P. Jeske ◽

Jeanine M. Walenga ◽

...

Keyword(s):

Platelet Activation ◽

Serotonin Release ◽

Platelet Factor 4 ◽

Thrombin Inhibitors ◽

Platelet Factor ◽

Cellular Activation ◽

Heparin Induced Thrombocytopenia ◽

New Information ◽

Study Selection ◽

Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

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Serologic Results in >1000 Patients With Suspected Heparin-Induced Thrombocytopenia

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029607304721 ◽

2007 ◽

Vol 14 (4) ◽

pp. 410-414 ◽

Cited By ~ 8

Author(s):

Suresh G. Shelat ◽

Anne Tomaski ◽

Eleanor S. Pollak

Keyword(s):

Laboratory Diagnosis ◽

Serotonin Release ◽

Enzyme Linked Immunosorbent Assay ◽

Functional Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Life Threatening ◽

Release Assay ◽

Pathogenic Antibodies

Heparin-induced thrombocytopenia (HIT) can lead to life-threatening and limb-threatening thrombosis. HIT is thought to be initiated by the interaction of pathogenic antibodies toward a complex platelet factor 4 (PF4) and heparin (PF4:H), which can activate platelets and predispose to thrombosis. As such, the laboratory diagnosis of HIT includes antigenic and functional assays to detect antibodies directed at PF4:H complexes. We performed a retrospective analysis of 1017 consecutive samples tested by serotonin-release assay and by enzyme-linked immunosorbent assay (ELISA). Most samples showed no serologic evidence of HIT, whereas 4% to 5% of samples demonstrated both antigenic and functional serological evidence for HIT. Approximately 12% to 18% of samples showed immunologic evidence of anti-PF4:H antibodies but without functional evidence of serotonin release in vitro. Interestingly, a small minority of samples (0.7%) caused serotonin release but were negative in the ELISA. The results are presented using cutoff values established at our hospital and for the ELISA manufacturer. This study provides a pretest probability of the serologic results from an antigenic assay (ELISA) and a functional assay (serotonin-release assay) in patients clinically suspected of having HIT.

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Comparison of an IgG-Specific Enzyme-Linked Immunosorbent Assay Cutoff of 0.4 Versus 0.8 and 1.0 Optical Density Units for Heparin-Induced Thrombocytopenia

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029615606532 ◽

2016 ◽

Vol 23 (3) ◽

pp. 282-286 ◽

Cited By ~ 5

Author(s):

Brianne M. Ritchie ◽

Jean M. Connors ◽

Katelyn W. Sylvester

Keyword(s):

Optical Density ◽

Immunosorbent Assay ◽

Predictive Value ◽

Retrospective Chart Review ◽

Serotonin Release ◽

Enzyme Linked Immunosorbent Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Institutional Review ◽

Sensitivity Specificity

Background: Previous studies have demonstrated optimized diagnostic accuracy in utilizing higher antiheparin–platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA) optical density (OD) thresholds for diagnosing heparin-induced thrombocytopenia (HIT). We describe the incidence of positive serotonin release assay (SRA) results, as well as performance characteristics, for antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units in the diagnosis of HIT at our institution. Methods: Following institutional review board approval, we conducted a single-center retrospective chart review on adult inpatients with a differential diagnosis of HIT evaluated by both antiheparin–PF4 ELISA and SRA from 2012 to 2014. The major endpoints were to assess incidence of positive SRA results, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy at antiheparin–PF4 ELISA values ≥0.4 OD units when compared to values ≥0.8 and ≥1.0 OD units. Clinical characteristics, including demographics, laboratory values, clinical and safety outcomes, length of stay, and mortality, were collected. Results: A total of 140 patients with 140 antiheparin–PF4 ELISA and SRA values were evaluated, of which 23 patients were SRA positive (16.4%) and 117 patients were SRA negative (83.6%). We identified a sensitivity of 91.3% versus 82.6% and 73.9%, specificity of 61.5% versus 87.2% and 91.5%, PPV of 31.8% versus 55.9% and 63.0%, NPV of 97.3% versus 96.2% and 94.7%, and accuracy of 66.4% versus 86.4% and 88.6% at antiheparin–PF4 ELISA thresholds ≥0.4, ≥0.8, and ≥1.0 OD units, respectively. Conclusion: Our study suggests an increased antiheparin–PF4 ELISA threshold of 0.8 or 1.0 OD units enhances specificity, PPV, and accuracy while maintaining NPV with decreased sensitivity.

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Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia

Journal of Clinical Medicine ◽

10.3390/jcm9041226 ◽

2020 ◽

Vol 9 (4) ◽

pp. 1226 ◽

Cited By ~ 6

Author(s):

Brigitte Tardy ◽

Thomas Lecompte ◽

François Mullier ◽

Caroline Vayne ◽

Claire Pouplard

Keyword(s):

Platelet Activation ◽

Platelet Rich Plasma ◽

Serotonin Release ◽

Membrane Expression ◽

Platelet Factor ◽

Strict Adherence ◽

Heparin Induced Thrombocytopenia ◽

Functional Assays ◽

Healthy Donors ◽

Sensitivity Specificity

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances.

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Comparison of Antigen and Activation Assays with a Probability Scoring Model for the Diagnosis of Heparin Induced Thrombocytopenia.

Blood ◽

10.1182/blood.v110.11.3215.3215 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3215-3215

Author(s):

Dimitrios Scarvelis ◽

Gail Rock

Keyword(s):

Laboratory Testing ◽

Laboratory Tests ◽

Serotonin Release ◽

Reference Standard ◽

Platelet Response ◽

Platelet Factor ◽

Platelet Recovery ◽

Heparin Induced Thrombocytopenia ◽

Activation Assay ◽

Heparin Anticoagulation

Background: Heparin induced thrombocytopenia (HIT) is a syndrome characterized by thrombocytopenia and an elevated risk of thrombosis. In the evaluation of patients with suspected HIT, laboratory testing is used to help differentiate between those patients who have HIT and those who do not and therefore do not require non-heparin anticoagulation. Laboratory tests include activation (serotonin release, heparin-induced platelet aggregation, ATP luminescence) and antigen (enzyme-linked immunoabsorbant assay (ELISA)) assays directed against the platelet factor 4-heparin or PF4/PVS complex. A pre-test scoring system (Warkentin 4T’s) has been derived to assess the probability of HIT which can be used in conjunction with laboratory testing in the evaluation for HIT. Objectives: To compare the activation assay used at The Ottawa Hospital (ATP luminescence) with the PF4 enhanced® ELISA and correlate results with clinical data. Methods: Patients undergoing HIT testing were identified. ELISA and activation assays were performed. Charts were reviewed in order to derive a 4T’s score and to assess response to therapeutic non-heparin anticoagulation (when administered). Correlation between ELISA, activation assay, 4T’s score and platelet response to alternative anticoagulation was determined. Sensitivity, specificity, PPV and NPV of laboratory tests or 4T’s scores were calculated depending on the measure chosen as the reference standard to diagnose HIT. Results: 111 patients undergoing HIT testing were evaluated. 41 and 43 were positive by ELISA (OD > 0.4) or activation assay respectively. 12 were positive only by ELISA (mean optical density (OD): 1.27) and 10 were positive by both activation assay and ELISA (mean OD: 2.28). Clinical information was available for 70 patients. 26 of these received therapeutic non-heparin anticoagulation as treatment for suspected HIT. Reference standard= activation assay: ELISA: Sens 95% (95% CI 73–99%), Spec 80% (66–89%), PPV 66% (46–81%), NPV 98% (86–100%). 4T’s (low vs. high score): Sens 85% (54–97%), Spec 95% (83–99%), PPV 85% (54–97%), NPV 95% (83–99%). ELISA OD > 1.5 + high 4T’s score: Sens 61% (36–82%), Spec 100% (56–100%), PPV 100% (68–100%), NPV 50% (24–76%). Reference standard= 4T’s score (high and low score only, excludes intermediate category): ELISA: Sens 85% (53–97%), Spec 77% (61–88%), PPV 52% (30–74%), NPV 94% (79–99%). Activation: Sens 85% (54–97%), Spec 95% (83–99%), PPV 85% (54–97%), NPV 95% (83–99%). Reference standard= “clear response to non-heparin anticoagulation” defined as significant platelet increase within 48 hours of start of therapy and no other explanation for platelet recovery: ELISA: Sens 100% (72–100%), Spec 54% (26–80%), PPV 68% (43–86%), NPV 100% (56–100%). Activation: Sens 85% (54–97%), Spec 69% (39–90%), PPV 73% (45–91%), NPV 82% (48–97%). 4T’s (low vs. high score): Sens 90% (54–99%), Spec 100% (60–100%), PPV 100% (63–100%), NPV 89% (51–99%). ELISA OD > 1.5 + 4T’s (low vs. high score): Sens 89% (51–99%), Spec 100% (56–100%), PPV 100% (60–100%), NPV 88% (47–99%). Conclusions: A high 4T’s score best predicts a clinical response to non-heparin therapeutic anticoagulation when HIT is suspected. ELISA OD > 1.5 does not add additional information in this respect. A negative ELISA and a low 4T’s score have comparable NPVs when an activation assay is the reference standard. Future trials should employ a clinical reference standard such as “clear response to non-heparin anticoagulation” when evaluating the operator characteristics of HIT assays.

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Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Blood ◽

10.1182/blood.v120.21.2189.2189 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2189-2189

Author(s):

Susanne Macher ◽

Nazanin Sareban ◽

Camilla Drexler ◽

Gerhard Lanzer ◽

Katharina Schallmoser

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Laboratory Diagnosis ◽

Heparin Therapy ◽

Enzyme Linked Immunosorbent Assay ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Score System ◽

Single Center Experience

Abstract Abstract 2189 Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA). Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk. Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA. From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive. Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation. Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay. LFI-HIT PaGIA IgG-ELISA OD IgG-ELISA HIPA assay Median (range) Samples n=121 Pos 10 Pos 10 Pos 8 2.366 (0.902-3.000) 7/7 pos Neg 2 0.199 and 0.170 1/2 pos, 1/2 neg Neg 0 - - - - Neg 111 Pos 9 Pos 2 1.318 and 2.019 2/2 pos Neg 7 0.110 (0.054-0.139) 6/6 neg Neg 102 Pos 7 0.436 (0.404-1.463) 1/2 pos, 1/2 neg Neg 95 0.082 (0.013-0.376) Disclosures: No relevant conflicts of interest to declare.

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Spontaneous Heparin-Induced Thrombocytopenia Syndrome: Two New Cases and a Proposal For Defining This Disorder

Blood ◽

10.1182/blood.v122.21.2328.2328 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2328-2328 ◽

Cited By ~ 1

Author(s):

Theodore E. Warkentin ◽

Paul Andrew Basciano ◽

Richard A. Bernstein

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Research Funding ◽

Left Vertebral Artery ◽

Platelet Factor ◽

Shoulder Hemiarthroplasty ◽

Heparin Induced Thrombocytopenia ◽

Activation Assay ◽

Count Recovery ◽

Heparin Exposure

Abstract Introduction Heparin-induced thrombocytopenia (HIT) is a transient, autoimmune-like, prothrombotic disorder caused by heparin-dependent, platelet-activating IgG reactive against platelet factor 4/heparin (PF4/H). There is an emerging literature (Am J Med 2008;121:632-6. J Thromb Haemost 2008;6:1598-1600; Thromb Haemost 2013;109:669-75) pointing to rare instances of “spontaneous” HIT in patients without preceding heparin. We report 2 new cases and propose a definition for this controversial disorder. CASE #1. A 62-y.o. man presented with left middle cerebral artery stroke and thrombocytopenia (platelet count, 65×109/L). There was no previous history of thrombocytopenia, surgery, hospitalization, or heparin exposure. Clot extraction performed with heparin was complicated by further platelet count decline to 27 (nadir) and progressive thrombosis of the carotid artery. Aspirin was started, and the platelets recovered to >150 by day 13. CASE #2. A 54-y.o. female developed right leg swelling, left-upper extremity weakness/paresthesias, and thrombocytopenia (61×109/L) 15 days post-shoulder hemiarthroplasty; no intra-/postoperative heparin had been given. Brain MRI demonstrated acute infarct in the left posterior inferior cerebellar artery territory; angiography showed non-visualization of the left vertebral artery. Ultrasound revealed right lower-limb deep-vein thrombosis. Heparin treatment resulted in further platelet count fall to 37 (nadir). Treatment with argatroban, followed by fondaparinux, was associated with platelet count recovery to >150 by day 39. Methods Testing for HIT antibodies was performed by commercial EIA-IgG/A/M (Immucor GTI Diagnostics), in-house EIA-IgG (McMaster), and serotonin-release assay (SRA). Results Both patients’ sera (obtained before any heparin administration) tested strongly positive for HIT antibodies (Table), including strong platelet activation at 0.1 and 0.3 IU/mL heparin, as well as at 0 U/mL heparin, with no platelet activation at 100 IU/mL heparin: these serological features are characteristic of “delayed-onset HIT” (Ann Intern Med 2001;135:502-6). Antibody reactivity declined markedly by 2 to 4 weeks (including loss of platelet-activating properties at 0 IU/mL heparin), in keeping with the usual transience of HIT antibodies (N Engl J Med 2001;344:1286-92), and paralleling both patients’ platelet count recovery. Discussion These cases further support spontaneous HIT as an unusual explanation for acute arterial stroke and thrombocytopenia. One patient had preceding orthopedic surgery, an event previously reported with spontaneous HIT (Thromb Haemost 2013;109:669-75). The strong serum-dependent platelet activation at 0 IU/mL heparin helps to explain how thrombocytopenia and thrombosis can occur in a patient not receiving heparin. RECOMMENDATION. Based on the serological findings of these and previous cases, we propose that a definitive diagnosis of spontaneous HIT syndrome should be based upon all of the following criteria: thrombocytopenia, thrombosis, lack of proximate heparin exposure, strong-positive PF4-dependent immunoassay(s), and a strong-positive platelet activation assay featuring both heparin-dependent (e.g., high heparin neutralization) and heparin-independent platelet activation (at 0 IU/mL heparin). Disclosures: Warkentin: Pfizer Canada: Honoraria; Paringenix: Consultancy; Immucor GTI Diagnostics: Research Funding; WL Gore: Consultancy; GSK: Research Funding.

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Clinical Features of Heparin-Induced Thrombocytopenia Diagnosed By Lumi-Aggregometry

Blood ◽

10.1182/blood.v124.21.4185.4185 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4185-4185

Author(s):

Elona Turley ◽

Artur J. Szkotak ◽

Irwindeep Sandhu ◽

Cynthia M Wu

Keyword(s):

Platelet Activation ◽

Light Flash ◽

Patient Characteristics ◽

Serotonin Release ◽

Radioactive Isotopes ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Platelet Counts ◽

Dense Granules ◽

Heparin Exposure

Abstract Background: Heparin-induced thrombocytopenia (HIT) is associated with anti-platelet factor-4 (PF4)/heparin antibodies that activate platelets, resulting in thrombocytopenia and a pro-thrombotic state. At our institution antibody-mediated platelet activation is demonstrated by lumi-aggregometry, which is a method previously validated against the gold standard serotonin-release assay (SRA). Lumi-aggregometry does not involve radioactive isotopes, which is its major advantage over the SRA. The clinical course of HIT diagnosed via SRA and ELISA has been previously described, and clinical prediction tools such as the 4-T score were validated using these diagnostic tests. However, the clinical picture of HIT diagnosed by lumi-aggregometry has not been previously described. Aims: The objective of this study is to describe the clinical and laboratory presentation of patients diagnosed with HIT by lumi-aggregometry. Methods: Patients with clinically suspected HIT and quantitative anti-PF4 IgG-specific ELISA OD ≥0.400 (Gen-Probe, San Diego) received confirmatory HIT testing by lumi-aggregometry. Briefly, HIT antibody-induced activation of washed healthy donor platelets was tested at therapeutic (0.1U/mL and 0.5U/mL) and high (100U/mL) porcine heparin concentration. The degree of platelet activation was quantitated luminographically based on the light flash reaction of ATP (released from platelet dense-granules) with luciferin luciferase reagent. A ratio of therapeutic to high heparin luminescence amplitude of >5.0 and platelet aggregation at therapeutic, but not high, concentrations was considered a positive result. The results of assays performed by our regional HIT testing referral laboratory from June 2009 to July 2012 were reviewed to identify patients with positive HIT testing by lumi-aggregometry. Patient records were retrospectively reviewed to obtain predefined data on baseline patient characteristics, heparin exposure, platelet counts, and thrombotic events occurring in the 5 days preceding or the 30 days following the date of positive HIT testing. Results: We identified 43 patients diagnosed with HIT by lumi-aggregometry (median age 68.0, 49% male) while under the care of local academic (46%) or urban community hospitals (37.2% medical; 53.5% surgical; 9.3% intensive care). Median baseline platelet count was 187 (14-349). Median date of platelet drop post-heparin exposure was 6 days (range 3-14) in patients without prior heparin exposure or platelet transfusions (Figure 1). Platelet drop >50% and platelet nadir ≥20x109/L were present in the majority of patients (Table 1). Thrombocytopenia occurred prior to (70.5%) or the same day (23.5%) as thrombosis in 16/17 patients with serial platelet counts who developed HIT-associated thromboembolism. Conclusion: Patients diagnosed with HIT by lumi-aggregometry present with similar findings to those described in SRA-confirmed HIT. These findings lend support to the use of lumi-aggregometry as an accurate diagnostic assay for the clinico-pathologic syndrome of HIT. Figure 1 Figure 1. Table 1. Percentage platelet drop from baseline and platelet nadir Percent platelet drop Platelet nadir >50% 30-50% <30% ≥20 x 109/L 28 3 2 10-19 x 109/L 5 1 0 <10 x 109/L 1 1 0 Disclosures Szkotak: Alexion Pharmaceuticals: Research Funding. Sandhu:Celgene: Honoraria; Jansen: Honoraria; Novartis: Honoraria.

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How I Diagnose and Manage HIT

Hematology ◽

10.1182/asheducation-2011.1.143 ◽

2011 ◽

Vol 2011 (1) ◽

pp. 143-149 ◽

Cited By ~ 90

Author(s):

Theodore E. Warkentin

Keyword(s):

Platelet Activation ◽

Low Cost ◽

Factor Xa ◽

Clinical Situation ◽

Serotonin Release ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Platelet Factor ◽

Activation Assay ◽

Reduce Risk

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays, such as the serotonin-release assay, are superior to PF4-dependent immunoassays in discerning which heparin-induced antibodies are clinically relevant. When HIT is strongly suspected, standard practice includes substituting heparin with an alternative anticoagulant; the 2 US-approved agents are the direct thrombin inhibitors (DTIs) lepirudin and argatroban, which are “niche” agents used only to manage HIT. However, only ∼ 10% of patients who undergo serological investigation for HIT actually have this diagnosis. Indeed, depending on the clinical setting, only 10%-50% of patients with positive PF4-dependent immunoassays have platelet-activating antibodies. Therefore, overdiagnosis of HIT can be minimized by insisting that a positive platelet activation assay be required for definitive diagnosis of HIT. For these reasons, a management strategy that considers the real possibility of non-HIT thrombocytopenia is warranted. One approach that I suggest is to administer an indirect, antithrombin (AT)–dependent factor Xa inhibitor (danaparoid or fondaparinux) based upon the following rationale: (1) effectiveness in treating and preventing HIT-associated thrombosis; (2) effectiveness in treating and preventing thrombosis in diverse non-HIT situations; (3) both prophylactic- and therapeutic-dose protocols exist, permitting dosing appropriate for the clinical situation; (4) body weight–adjusted dosing protocols and availability of specific anti-factor Xa monitoring reduce risk of under- or overdosing (as can occur with partial thromboplastin time [PTT]–adjusted DTI therapy); (5) their long half-lives reduce risk of rebound hypercoagulability; (6) easy coumarin overlap; and (7) relatively low cost.

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