Combination of two complementary automated rapid assays for diagnosis of heparin-induced thrombocytopenia (HIT).

HIT diagnosis typically uses complementary diagnostic assays (eg, a PF4-dependent enzyme-immunoassay [EIA] and a platelet activation assay such as the serotonin-release assay [SRA]). To determine whether the combination of two automated assays-a latex immunoturbidimetric assay (LIA) that evaluates competitive inhibition of a HIT-like monoclonal antibody and a chemiluminescence immunoassay (CLIA) for detecting anti-PF4/heparin IgG-optimizes diagnostic sensitivity while also yielding good specificity, particularly at high assay reactivities. We determined operating characteristics using combined LIA/CLIA results from a HIT observational trial (n = 430; derivation cohort) and 147 consecutive patients with HIT (n = 147; supplementary derivation cohort). We also evaluated 678 consecutive samples referred for HIT testing (replication cohort). LIA/CLIA reactivities were scored individually as "negative" (<1.00 U/mL, 0 points), "weak" (1.00-4.99 U/mL, 1 point), "moderate" (5.00-15.99 U/mL, 2 points) and "strong" (≥16.00 U/mL, 3 points), thus contributing up to 6 points (maximum) when LIA/CLIA results were combined. We also examined whether higher LIA/CLIA scores predicted presence of platelet-activating antibodies by conventional and modified (PF4- or PF4/heparin-enhanced) SRA. Combined LIA/CLIA testing yielded high diagnostic sensitivity (~99%) similar to EIA. Interpretation of LIA/CLIA results using the 6-point scale indicated progressively greater likelihood for the presence of platelet-activating antibodies with increasing scores (semi-quantitative reactivity). A LIA/CLIA score ≥ 4 points predicted the presence of platelet-activating antibodies by SRA or PF4-enhanced SRA with high probability (~98%). Combined LIA/CLIA testing optimizes diagnostic sensitivity, with progressively greater probability of detecting platelet-activating antibodies with higher assay reactivity that reaches 98% when both automated assays yield moderate or strong results.

Warkentin TE ，Sheppard JI ，Smith JW ，Li N ，Moore JC ，Arnold DM ，Nazy I ... -  《-》

Laboratory diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a clinical-pathological disorder; thus, laboratory testing for the pathogenic platelet-activating antiplatelet factor 4 (PF4)/heparin antibodies is central for diagnosis. The "iceberg" model summarizes the inter-relationship between platelet activation assays and PF4-dependent immunoassays, with platelet-activating antibodies comprising a subset of anti-PF4/heparin antibodies. The platelet serotonin-release assay (SRA), performed by reference laboratories, has high sensitivity and specificity for HIT (~95% each), and is especially suited for detecting highly pathogenic HIT sera containing both heparin-dependent and heparin-independent platelet-activating antibodies; this latter subgroup of antibodies explains "autoimmune HIT" disorders (delayed-onset, persisting, spontaneous, heparin "flush," fondaparinux-associated). Recently, SRA-negative HIT has become recognized, in which serum from some HIT patients contains subthreshold levels of platelet-activating antibodies (by SRA) that become detectable using a PF4-enhanced platelet activation assay. Unusual immunologic features of HIT include early antibody detectability (at onset of platelet count fall) and antibody transience (seroreversion). Widely available PF4-dependent enzyme immunoassays (EIAs) have high sensitivity but poor specificity for HIT, although specificity is enhanced with IgG-specific EIAs and strong positive results; unfortunately, EIA results are usually not available in real time. Automated rapid immunoassays, such as the chemiluminescence immunoassay (CLIA) and latex immunoturbidimetric assay (LIA), facilitate real-time laboratory diagnosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR-) test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian analysis, into real-time posttest probabilities that are dramatically increased (test positive) or decreased (test negative). Moreover, (semi-)quantitative CLIA- and LIA-positive results (weak, moderate, strong positive) can further refine the posttest probability of HIT.

Warkentin TE 《-》

Performance characteristics of an automated latex immunoturbidimetric assay [HemosIL(®) HIT-Ab((PF4-H))] for the diagnosis of immune heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-PF4/heparin antibodies. Given time-sensitive treatment considerations, a rapid and accurate laboratory test for HIT antibodies is needed. To determine operating characteristics for the HemosIL® HIT-Ab(PF4/H), a rapid, on-demand, fully-automated, latex immunoturbidimetric assay (LIA), for diagnosis of HIT. We evaluated LIA sensitivity, specificity, negative (NPV) and positive predictive value (PPV), negative (LR-) and positive likelihood ratio (LR+), using citrated-plasma from 429 patients (prospective cohort study of 4Ts scoring; HIT, n=31), and from consecutive HIT patients (n=125), using reference standard serotonin-release assay (SRA). Comparators included two PF4-dependent enzyme-immunoassays (EIAs). We used stratum-specific likelihood ratios (SSLRs) to determine how differing magnitudes of LIA-positivity influenced post-test probability of HIT. LIA operating characteristics were: sensitivity=97.4% (152/156); specificity=94.0% (374/398); PPV=55.6% (30/54); and NPV=99.7% (374/375). At manufacturers' cutoffs, LIA specificity and PPV were superior to the EIAs. Although a negative LIA pointed strongly against HIT (LR-, 0.034), the post-test probability was ~2% with high 4Ts score. The LIA's LR+ was high (16.0), with SSLRs rising substantially with greater LIA-positivity: 5.7 (1.0-4.9U/mL), 31 (5.0-15.9U/mL), and 128 (≥16U/mL). A LIA-positive result (at 1.0 cutoff) indicated at least 24% HIT probability (low 4Ts score), rising to 90% with high 4Ts score. Although approximately 1 in 40 SRA-positive patients tested LIA-negative, the LIA's high NPV and PPV indicate that this rapid assay is useful for the diagnostic evaluation of HIT, including in low pre-test situations.

Warkentin TE ，Sheppard JI ，Linkins LA ，Arnold DM ，Nazy I ... -  《-》

Timeline of heparin-induced thrombocytopenia seroconversion in serial plasma samples tested using an automated latex immunoturbidimetric assay.

HIT is caused by platelet-activating IgG that recognize multimolecular PF4/heparin complexes. HIT antibodies are generally detectable by PF4-dependent enzyme immunoassay (EIA) and by platelet serotonin-release assay (SRA) at the beginning of the HIT-related platelet count fall. We determined whether an automated immunoassay for HIT, the latex immunoturbidimetric assay (LIA), also detects antibodies early during the course of HIT. The LIA was also used to evaluate a patient with putative SRA-negative HIT. We evaluated the timing and magnitude of LIA reactivity in serial plasma samples obtained from 19 SRA-positive patients (17 with abnormal platelet count changes indicating HIT; two with subclinical seroconversion) and one putative SRA-negative HIT patient, all obtained from patients who participated in a clinical trial of heparin thromboprophylaxis. We determined LIA status at the onset of the HIT-related platelet count fall. The LIA was positive in all 19 SRA-positive patients (median value, 7.3 U/mL [range, 1.2-35.5]; cutoff, 1.0 U/mL); for all 13 evaluable patients for whom an informative plasma sample was available at (or shortly before) the onset of the HIT-related platelet count fall, LIA reactivity was positive. Heterogeneity in seroconversion using the LIA was observed; some patients exhibited gradual increases in reactivity, whereas other patients showed rapid increase in reactivity over a few days. The single clinical trial patient who met clinical-pathological criteria for "SRA-negative HIT" tested LIA-positive. The LIA detects HIT antibodies at the beginning of the HIT-associated platelet count fall. The LIA was also positive in a patient with SRA-negative HIT.

Warkentin TE ，Sheppard JI ，Smith JW ，Arnold DM ，Nazy I ... -  《-》

Serotonin-release assay-positive but platelet factor 4-dependent enzyme-immunoassay negative: HIT or not HIT?

IgG-specific and polyspecific PF4-dependent enzyme-immunoassays (EIAs) have exceptionally high sensitivity (≥99%) for diagnosis of heparin-induced thrombocytopenia (HIT), a drug reaction caused by platelet-activating antibodies detectable by serotonin-release assay (SRA). The IgG-specific EIAs are recommended for screening, as their high sensitivity is accompanied by relatively high specificity vis-à-vis polyspecific EIAs. We investigated the frequency of SRA-positive/EIA-negative (SRA+/EIA-) HIT, prompted by referral to our reference HIT laboratory of serial blood samples from a patient ("index case") with false-negative IgG-specific EIAs. Despite initial clinical suspicion for HIT, repeat negative IgG-specific EIAs prompted heparin resumption, which triggered recurrent thrombocytopenia and near-fatal cardiac arrest, indicating likely post-heparin HIT-associated anaphylactoid reaction. Further investigations revealed a strong-positive SRA, whether performed with heparin alone, PF4 alone, or PF4/heparin, with inhibition by Fc receptor-blocking monoclonal antibody (indicating IgG-mediated platelet activation); however, five different IgG-specific immunoassays yielded primarily negative (or weak-positive) results. To investigate the frequency of SRA+/EIA- HIT, we reviewed the laboratory and clinical features of patients with this serological profile during a 6-year period in which our reference laboratory investigated for HIT using both SRA and IgG-specific EIA. Although ~0.2% of 8546 patients had an SRA+/EIA- profile, further review of 15 such cases indicated clerical/laboratory misclassification or false-positive SRA in all, with no SRA+/EIA- HIT case identified. We conclude that while SRA+/EIA- HIT is possible-as shown by our index case-this clinical picture is exceptionally uncommon. Moreover, the requirement for a positive EIA is a useful quality control maneuver that reduces risk of reporting a false-positive SRA result.

Warkentin TE ，Smythe MA ，Ali MA ，Aslam N ，Sheppard JI ，Smith JW ，Moore JC ，Arnold DM ，Nazy I ... -  《-》