To cite this article: Warkentin TE, Sheppard JI, Moore JC, Sigouin CS, Kelton JG. Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays. J Thromb Haemost 2008; 6: 1304-12.Summary. Background: Many laboratories test for heparininduced thrombocytopenia (HIT) using a PF4-dependent enzyme-immunoassay (EIA). An advantage of the EIA is its simplicity; a disadvantage is that it only indirectly detects heparin-dependent, platelet-activating antibodies (ÔHIT antibodiesÕ). Objectives: To determine whether the magnitude of a positive EIA result, expressed in optical density (OD) units, predicts risk of HIT antibodies, defined as a strong-positive platelet serotonin-release assay (SRA) result ( ‡50% serotonin release). Patients/methods: We determined the risk of a strongpositive SRA result for five categories of OD reactivity (<0.40, 0.40-<1.00, 1.00-<1.40, 1.40-<2.00, and ‡2.00 OD units) using two EIAs (commercial anti-PF4/polyanion IgG/A/M and in-house anti-PF4/heparin-IgG). Results: For patient sera investigated for HIT antibodies, a weak-positive result (0.40-<1.00 OD units) in either EIA indicated a low probability (£5%) of a strong-positive SRA; the risk increased to 90% with an OD ‡ 2.00 units. Quantifying the EIA-SRA relationship for 1553 referred patient sera, we found that for every increase of 0.50 OD units in the EIA-IgG, the risk of a strongpositive SRA result increased by OR = 6.39 [95% confidence interval (CI), 5.13, 7.95; P < 0.0001]. For every increase of 1.00 OD units in the EIA-IgG, the risk increased by OR = 40.81 (95% CI, 26.35, 63.20; P < 0.0001). Conclusions: The probability of HIT antibodies (strong-positive SRA result) inferred by a positive PF4-dependent EIA varies considerably in relation to the magnitude of the EIA result, expressed as OD values. In our laboratory, the probability of HIT antibodies being present reached ‡50% only when the OD level was ‡1.40 units.

The frequency of immune heparin-induced thrombocytopenia (HIT) varies among prospective studies. It is unknown whether this is caused by differences in the heparin preparations, the patient populations, or the types of serologic assay used to confirm the diagnosis. Seven hundred forty-four patients were studied from 3 different clinical treatment settings, as follows: unfractionated heparin (UFH) during or after cardiac surgery (n = 100), UFH after orthopedic surgery (n = 205), and low-molecular-weight heparin (LMWH) after orthopedic surgery (n = 439). Both an activation assay and an antigen assay were used to detect heparin-dependent IgG (HIT-IgG) antibodies. By activation assay, the frequency of HIT-IgG formation ranged from a low of 3.2% in orthopedic patients receiving LMWH to a high of 20% in cardiac patients receiving UFH; by antigen assay, the corresponding frequencies ranged from 7.5% to 50%. Both UFH use (P = .002) and cardiac surgery (P = .01) were more likely to be associated with HIT-IgG formation. However, among patients in whom HIT-IgG formed and who were administered UFH, the probability for HIT was higher among orthopedic patients than among cardiac patients (by activation assay: 52.6% compared with 5%; odds ratio, 21.1 [95% CI, 2.2-962.8]; P = .001; by antigen assay: 34.5% compared with 2.0%; odds ratio, 25.8 [95% CI, 3.2-1141]; P < .001). It is concluded that there is an unexpected dissociation between the frequency of HIT-IgG formation and the risk for HIT that is dependent on the patient population. HIT-IgG antibodies are more likely to form in patients who undergo cardiac surgery than in orthopedic patients, but among patients in whom antibodies do form, orthopedic patients are more likely to develop HIT.

No abstract

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4) bound to heparin. Immunogenicity of heparins differs in that unfractionated heparin (UFH) induces more anti-PF4/heparin antibodies than low-molecular-weight heparin (LMWH) and UFH also causes more HIT. Fondaparinux, a synthetic anticoagulant modeled after the antithrombin-binding pentasaccharide, is believed to be nonimmunogenic. We tested 2726 patients for anti-PF4/heparin antibodies after they were randomized to receive antithrombotic prophylaxis with fondaparinux or LMWH (enoxaparin) following hip or knee surgery. We also evaluated in vitro cross-reactivity of the IgG antibodies generated against PF4 in the presence of UFH, LMWH, danaparoid, or fondaparinux. We found that anti-PF4/heparin antibodies were generated at similar frequencies in patients treated with fondaparinux or enoxaparin. Although antibodies reacted equally well in vitro against PF4/UFH and PF4/ LMWH, and sometimes weakly against PF4/danaparoid, none reacted against PF4/fondaparinux, including even those sera obtained from patients who formed antibodies during fondaparinux treatment. At high concentrations, however, fondaparinux inhibited binding of HIT antibodies to PF4/polysaccharide, indicating that PF4/ fondaparinux interactions occur. No patient developed HIT. We conclude that despite similar immunogenicity of fondaparinux and LMWH, PF4/fondaparinux, but not PF4/ LMWH, is recognized poorly by the antibodies generated, suggesting that the risk of HIT with fondaparinux likely is very low.

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT)is caused by antibodies against a "self" protein-platelet factor 4-bound to heparin. We observed an overrepresentation of the female gender in 290 patients who developed HIT after cardiac or orthopedic surgery compared with the representation found in national databases (study 1). Therefore, we investigated gender imbalance in HIT by logistic regression analysis of a randomized controlled trial of unfractionated heparin (UFH) and lowmolecular-weight heparin (LMWH) (study 2), and we analyzed individual patient data from 7 prospective studies comparing HIT frequency between UFH and LMWH, evaluating effects of gender, heparin (UFH vs LMWH), and patient type (surgical vs medical) (study 3). All 3 studies showed female overrepresentation, which for study 3 was a common odds ratio (OR) of 2.37 (95% confidence interval [95% CI], 1.37-4.09; P ‫؍‬ .0015). Study 3 also showed an interaction between gender, heparin, and patient type. Although UFH was more likely than LMWH to cause HIT (P < .0001), this effect was predominantly seen in women compared with men (common OR, 9.22 vs 1.83; P ‫؍‬ .020) and in surgical patients compared with medical patients (common OR, 13.93 vs 1.75; P ‫؍‬ .005). We conclude that females are at greater risk for HIT and that using LMWH to prevent HIT may have greatest absolute benefit in females undergoing surgical thromboprophylaxis. IntroductionHeparin-induced thrombocytopenia (HIT) is a relatively common drug-induced immune reaction affecting platelets. 1 The immunologic basis of this disorder is the transient production 2 of plateletactivating antibodies of IgG class 3-5 that recognize multimolecular complexes of platelet factor 4 (PF4) and heparin. [6][7][8] An evolving concept is that HIT resembles an autoimmune disorder 9 because the pathogenic antibodies recognize one or more neoepitopes found on the "self" protein, PF4, rather than on heparin. 10,11 Indeed, in some patients, HIT strongly resembles an autoimmune process because heparin treatment initiates the anti-PF4/heparin antibody formation but then is no longer required for the development of thrombocytopenia. [12][13][14] To date, only a minor role for patientspecific risk factors for HIT has been reported. In particular, no association with human leukocyte antigen (HLA) genes is observed, 15 and the potential role of Fc receptor polymorphisms in HIT is debated. [16][17][18] We previously observed in large retrospective studies that females constituted 56.4% 2 and 58.9% 19 of HIT patients, suggesting a possible gender imbalance. Together with the emerging concept of HIT as a drug-induced transient autoimmune disorder, we hypothesized that there might be a gender imbalance in risk for this adverse drug reaction, with a greater frequency among females, as observed in certain chronic autoimmune disorders. [20][21][22] We also examined whether the striking difference in risk for HIT between unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) 23 is consistent for both genders and in ...

Heparin-induced thrombocytopenia (HIT)is caused by platelet-activating antibodies that recognize PF4/heparin complexes. Uncertainties remain regarding HIT immunobiology, including the temporal relation of antibody formation to onset of thrombocytopenia, and whether immunoglobulin class switching occurs. Using serial plasma samples from 2 heparin thromboprophylaxis trials, we determined the time of onset, antibody levels, and immunoglobulin class distributions (IgG, IgA, IgM) for 12 patients with HIT and 36 patients who formed anti-PF4/heparin antibodies, but did not develop HIT ("seropositive non-HIT controls"). In patients with HIT, anti-PF4/heparin antibodies became detectable 4 days (median) after starting heparin; antibody detection preceded the platelet count decline by 2 days (median) . IntroductionHeparin-induced thrombocytopenia (HIT) is an adverse drug reaction caused by platelet-activating antibodies that recognize complexes of platelet factor 4 (PF4) bound to heparin. 1,2 In most patients, IgG class antibodies [3][4][5] are formed that bind to PF4/ heparin complexes 6 on platelet surfaces, 7 cross-linking platelet Fc receptors, 8 with resulting platelet activation 9 and an associated procoagulant response. 10 HIT is characterized by an increased risk of venous and arterial thromboses. 11 Antibodies against PF4/heparin can be detected in virtually every patient with HIT, indicating that the disorder is antibody mediated. [1][2][3][4][5][6] However, HIT has several atypical features that differ from the classically described antigen-induced immune response. For example, patients often develop HIT as early as 5 days after their first exposure to heparin. 12,13 In addition, these rapidly forming antibodies include relatively high levels of IgG, which is more typical of a secondary immune response. Second, and unlike antibodies formed during exposure to common viral and vaccine antigens, 14 heparin-dependent antibodies remain detectable for only a few weeks or months after an episode of HIT. 12 Third, an anamnestic (immune memory) response generally does not occur in patients with a previous history of HIT, because these patients can receive heparin months or years later without either regenerating antibodies quickly or developing recurrence of the syndrome. 12,15,16 Fourth, a significant proportion of patients who form antibodies against PF4/heparin complexes do not develop a fall in the platelet count and remain well without any clinical features of HIT.We participated in a series of prospective trials of heparin thromboprophylaxis in which patients were documented to have serologically proven HIT. Many of these patients had serial blood samples collected during the evolution of their anti-PF4/heparin immune response, allowing us to address the following questions:1. When do anti-PF4/heparin antibodies form in relation to the development of HIT?2. When do the immunoglobulin classes IgG, IgA, and IgM form in patients with HIT, and is there evidence that IgM formation precedes the other immunoglobulin clas...

The frequency of immune heparin-induced thrombocytopenia (HIT) varies among prospective studies. It is unknown whether this is caused by differences in the heparin preparations, the patient populations, or the types of serologic assay used to confirm the diagnosis. Seven hundred forty-four patients were studied from 3 different clinical treatment settings, as follows: unfractionated heparin (UFH) during or after cardiac surgery (n = 100), UFH after orthopedic surgery (n = 205), and low-molecular-weight heparin (LMWH) after orthopedic surgery (n = 439). Both an activation assay and an antigen assay were used to detect heparin-dependent IgG (HIT-IgG) antibodies. By activation assay, the frequency of HIT-IgG formation ranged from a low of 3.2% in orthopedic patients receiving LMWH to a high of 20% in cardiac patients receiving UFH; by antigen assay, the corresponding frequencies ranged from 7.5% to 50%. Both UFH use (P = .002) and cardiac surgery (P = .01) were more likely to be associated with HIT-IgG formation. However, among patients in whom HIT-IgG formed and who were administered UFH, the probability for HIT was higher among orthopedic patients than among cardiac patients (by activation assay: 52.6% compared with 5%; odds ratio, 21.1 [95% CI, 2.2-962.8]; P = .001; by antigen assay: 34.5% compared with 2.0%; odds ratio, 25.8 [95% CI, 3.2-1141]; P &lt; .001). It is concluded that there is an unexpected dissociation between the frequency of HIT-IgG formation and the risk for HIT that is dependent on the patient population. HIT-IgG antibodies are more likely to form in patients who undergo cardiac surgery than in orthopedic patients, but among patients in whom antibodies do form, orthopedic patients are more likely to develop HIT.