Donald M. Arnold

Adjudicating bleeding events in a platelet dose study: impact on outcome results and challenges.

BACKGROUND: In the SToP platelet dose study, the World Health Organization (WHO) bleeding grade was assigned using adjudication. This study describes the challenges associated with adjudicating bleeding events and compares the adjudicated and bedside results for bleeding grade.

An algorithm for resolving ‘indeterminate’ test results in the platelet serotonin release assay for investigation of heparin‐induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin anticoagulation that results in thrombocytopenia and increased risk of thrombosis. It is caused by IgG antibodies that recognize complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions, and that result in Fcc receptor (FccR)-mediated platelet activation [1–3]. HIT is a clinicopathological syndrome as the diagnosis relies on a compatible clinical picture of otherwise unexplained thrombocytopenia temporally related to heparin (typically, beginning 5– 10 days after exposure), and the detection of PF4/heparinreactive antibodies [1,4]. The 4T s scoring system has been used to predict the pretest probability of HIT using the criteria of thrombocytopenia, timing, thrombosis and the exclusion of other causes [4]; however, the diagnosis is most accurate if platelet-activating antibodies are detected [5–7]. Assays addressing both the function and the target antigen of these HIT antibodies are available [5–9]. The C-serotonin-release assay (SRA) is a functional (platelet activation) assay that has high sensitivity and specificity for detecting the antibodies that cause HIT [4,6–11]. The SRA has similar sensitivity, but superior specificity, compared with the anti-PF4-heparin enzyme-immunoassay (EIA), and is used as our gold standard assay. However, on occasion, SRA results are difficult to interpret because of heparin-independent platelet activation or other non-specific reaction profiles, yielding an indeterminate result [6]. We developed a two-step algorithm for resolving indeterminate SRA profiles. First, the SRA was repeated with another aliquot of patient serum that was newly heat inactivated, using pooled target platelets from two different platelet donors. Second, if the results were still indeterminate, then an in-house anti-PF4-heparin-IgGEIAwas used to determine the diagnosis of HIT [2,7,9]. The purpose of this study was to evaluate prospectively the ability of this algorithm to resolve indeterminate SRA reactions, with the ultimate aim of improving the diagnostic yield of HIT testing. This was a descriptive study of consecutive samples referred to the McMaster University Platelet Immunology Research Laboratory for HIT testing from patients suspected of having HIT between 2005 and 2006 (n = 2091 samples). All procedures were approved by the institutional research ethics board. The SRA was performed as previously described [8,10] using pooled platelets obtained from two pre-screened donors whose platelets were known to react well with HIT antibodies. Test serum was heat inactivated, and tested with buffer, pharmacologic (0.1 and 0.3 U mL) and high (100 U mL) concentrations of UFH (Leo Pharmaceuticals, Ballerup, Denmark), and a pharmacologic concentration (0.1 U mL) of the low molecular weight heparin (LMWH), enoxaparin (Lovenox, Sanofi-Aventis, NJ, USA). Platelets pre-incubated with IV.3 were also added to samples with 0.1 U mL UFH. The SRA result was considered positive if the sample caused ‡ 20% serotonin release with pharmacologic UFH and LMWH concentrations, and < 20% serotonin release (inhibition) with high concentrations (100 U mL) of UFH and with the FccR-blocking monoclonal antibody, IV.3. A negative SRA was defined as < 20% serotonin release at all heparin concentrations [8]. Any serum sample that induced ‡ 20% release in any of the reaction wells (including the buffer control, i.e. with no heparin added), but otherwise did not fit the HIT profile (i.e. inhibition was not observed at 100 U mL heparin or in the presence of FccR-blocking monoclonal antibody) was designated as indeterminate . An in-house PF4-heparin, IgG-specific EIA was performed in parallel with the SRA [6,9]. The EIA was used to assess for the presence (OD405 ‡0.45; EIA positive ) or absence (OD405 <0.45; EIA negative ) of anti-PF4/heparin antibodies in SRA-indeterminate samples. Samples from patients with clinical HIT tend to yield OD405 values > 1.0 [7,12]. Correspondence: Jane Moore, HSC 3H42, McMaster University, 1200 Main St W, Hamilton L8N3Z5, ON, Canada. Tel.: +1 905 5259140 ext. 22414; fax: +1 905 5296359. E-mail: moorej@mcmaster.ca

The effect of rituximab on anti-platelet autoantibody levels in patients with immune thrombocytopenia

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case‐control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet‐bound anti‐glycoprotein (GP) IIbIIIa and anti‐GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti‐GPIIbIIIa levels (P = 0·02) but not anti‐GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.

Development of a high-yield expression and purification system for platelet factor 4

Abstract Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.

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

BACKGROUNDnHeparin-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.nnnAIMSnTo determine operating characteristics for the HemosIL® HIT-Ab(PF4/H), a rapid, on-demand, fully-automated, latex immunoturbidimetric assay (LIA), for diagnosis of HIT.nnnMETHODSnWe 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.nnnRESULTSnLIA 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 LIAs 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.nnnCONCLUSIONSnAlthough approximately 1 in 40 SRA-positive patients tested LIA-negative, the LIAs high NPV and PPV indicate that this rapid assay is useful for the diagnostic evaluation of HIT, including in low pre-test situations.

High sensitivity and specificity of an automated IgG-specific chemiluminescence immunoassay for diagnosis of HIT

TO THE EDITOR:nnHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder caused by platelet-activating antibodies that recognize PF4/heparin complexes.[1][1][⇓][2]-[3][3] Although PF4-dependent enzyme-immunoassays (EIAs) have high diagnostic sensitivity for HIT (∼97% to 99%),[4][4],[5