Evaluating Diagnostic Algorithms for Heparin-Induced Thrombocytopenia using Two Combined Automated Rapid Immunoassays.

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder caused by antibodies against platelet factor 4 (PF4) and heparin complexes. Rapid immunoassays (IAs) for detection of these antibodies mark a milestone in HIT diagnosis, despite a higher false-positive rate compared with functional platelet-activation assays. However, combining different rapid IAs may help to improve their diagnostic specificity. Here, we compared the individual performance of the latex immunoturbidimetric assay (LIA; HemosIL HIT-Ab [PF4-H]; sensitivity 91.7%, specificity 68.4%) and chemiluminescence immunoassay (CLIA; HemosIL AcuStarHIT-Ab [PF4-H]; sensitivity 92.4%, specificity 85.8%) with their combined performance using two unique diagnostic algorithms in a single prospective cohort of suspected HIT patients. Using the simultaneous algorithm adapted from Warkentin et al, the combined LIA-CLIA had a sensitivity of 99.0% and specificity of 64.3%. The sequential algorithm adapted from Rittener-Ruff et al was applied in two theoretical scenarios to reflect real-world circumstances in diagnostic laboratories where access to clinical information is limited: (1) assuming all patients had an intermediate 4Ts score and (2) assuming all patients had a high 4Ts score. This algorithm correctly predicted HIT in 94.5% (high 4Ts) and 96.0% (intermediate 4Ts) and excluded HIT in 82.6% (high 4Ts) and 80.1% (intermediate 4Ts) of patients in either scenario, respectively. Although both combined algorithms improved diagnostic performance of individual IAs, the simultaneous algorithm showed fewer false predictions (7.9%) than the sequential algorithm (intermediate 4Ts: 37.6% and high 4Ts: 41.5%) and proved more practical as it does not rely on physician evaluations. Our findings highlight the importance of accounting for clinician and interlaboratory variability when evaluating diagnostic tests for HIT.

Investigation of anti-PF4 versus anti-PF4/heparin reactivity using fluid-phase enzyme immunoassay for 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia (HIT), autoimmune HIT, vaccine-induced immune thrombotic thrombocytopenia, and spontaneous HIT.

BACKGROUND: Four platelet-activating anti-platelet factor 4 (PF4) disorders have been recognized: classic heparin-induced thrombocytopenia (cHIT), autoimmune heparin-induced thrombocytopenia (aHIT), spontaneous heparin-induced thrombocytopenia (SpHIT), and vaccine-induced immune thrombotic thrombocytopenia (VITT). All test immunoglobulin G (IgG) positive using solid-phase enzyme immunoassay (solid-EIA) against PF4/heparin (PF4/H) and/or PF4 alone. Fluid-phase EIA (fluid-EIA) should better discriminate between anti-PF4 and anti-PF4/H antibodies since conformationally altered PF4 bound to solid phase is avoided. OBJECTIVES: To compare anti-PF4 vs anti-PF4/H antibody profiles for anti-PF4 disorders using solid- and fluid-EIA. METHODS: We developed a novel fluid-EIA to measure anti-PF4 vs anti-PF4/H antibodies. RESULTS: Using fluid-EIA, 27 of 27 (100%) cHIT sera tested IgG positive with PF4/H, but only 4 of 27 (14.8%) tested positive against PF4 alone; all 27 exhibited heparin-enhanced binding. In contrast, 17 of 17 (100%) VITT sera tested IgG positive against PF4 alone, with markedly reduced binding against PF4/H; this distinct VITT antibody profile was not evident using solid-EIA. All 15 aHIT sera and all 11 SpHIT sera tested IgG positive against PF4 alone, with variable reactivity in PF4/H-EIA (heparin-enhanced binding in 14 of 15 and 10 of 11 aHIT and SpHIT sera, respectively). Remarkably, 1 SpHIT patient with a VITT-mimicking fluid-EIA profile (PF4 >> PF4/H) also clinically resembled patients with VITT (postviral cerebral vein/sinus thrombosis), with anti-PF4 reactivity correlating inversely with platelet count recovery; moreover, the single aHIT patient with a VITT-mimicking fluid-EIA profile also developed postviral cerebral vein/sinus thrombosis. CONCLUSION: cHIT and VITT sera showed opposite fluid-EIA profiles (cHIT: PF4/H >> PF4, with most testing negative against PF4 alone; VITT: PF4 >> PF4/H, with most testing negative against PF4/H). In contrast, all aHIT and SpHIT sera reacted against PF4 alone but with variable (usually enhanced) reactivity against PF4/H. VITT-mimicking clinical/serologic profiles occurred in only a minority of patients with SpHIT and aHIT.

The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5 to 30 days after adenoviral vector COVID-19 vaccination. Therefore, a practical evaluation of clinical assessments and laboratory testing for VITT is needed to prevent significant adverse outcomes as the global use of adenoviral vector vaccines continues. We received the clinical information and blood samples of 156 patients in Canada with a suspected diagnosis of VITT between April and July 2021. The performance characteristics of various diagnostic laboratory tests were evaluated against the platelet factor 4 (PF4)-14C-serotonin release assay (SRA) including a commercial anti-PF4/heparin immunoglobulin G (IgG)/IgA/IgM enzyme immunoassay (EIA, PF4 Enhanced; Immucor), in-house IgG-specific anti-PF4 and anti-PF4/heparin-EIAs, the standard SRA, and the PF4/heparin-SRA. Of those, 43 (27.6%) had serologically confirmed VITT-positive based on a positive PF4-SRA result and 113 (72.4%) were VITT-negative. The commercial anti-PF4/heparin EIA, the in-house anti-PF4-EIA, and anti-PF4/heparin-EIA were positive for all 43 VITT-confirmed samples (100% sensitivity) with a few false-positive results (mean specificity, 95.6%). These immunoassays had specificities of 95.6% (95% confidence interval [CI], 90.0-98.6), 96.5% (95% CI, 91.2-99.0), and 97.4% (95% CI, 92.4-99.5), respectively. Functional tests, including the standard SRA and PF4/heparin-SRA, had high specificities (100%), but poor sensitivities for VITT (16.7% [95% CI, 7.0-31.4]; and 46.2% [95% CI, 26.6-66.6], respectively). These findings suggest EIA assays that can directly detect antibodies to PF4 or PF4/heparin have excellent performance characteristics and may be useful as a diagnostic test if the F4-SRA is unavailable.

Post-cardiac surgery thrombotic thrombocytopenic purpura: presence of anti-ADAMTS13 autoantibodies at preoperative baseline.

Thrombotic thrombocytopenic purpura (TTP) rarely complicates acute inflammatory conditions such as surgery, including post-cardiac surgery. Review of 32 previously-reported cases of post-cardiac surgery TTP indicates that this disorder often occurs as early as 2-3 days following surgery, which seems too soon to implicate new formation of anti-ADAMTS13 autoantibodies as a consequence of surgery itself. We diagnosed post-cardiac surgery TTP in a 60-year-old female that began approximately 3 days post-coronary artery bypass surgery in which anti-ADAMTS13 autoantibodies were implicated. We therefore investigated whether anti-ADAMTS13 autoantibodies were also present in a preoperative blood sample. Inhibitory (neutralizing) anti-ADAMTS13 autoantibodies were detectable in the preoperative blood sample, suggesting that the role of surgery in precipitating TTP might be due to effects such as abrupt increase in postoperative von Willebrand factor levels and associated proinflammatory factors, rather than effects of surgery itself leading to the formation of de novo anti-ADAMTS13 autoantibodies.

Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects.

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia.

BACKGROUND: Thrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID-19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID-19 patients may be suspected of heparin-induced thrombocytopenia (HIT), given similar clinical features. OBJECTIVES: We investigated the presence of platelet-activating anti-platelet-factor 4 (PF4)/heparin antibodies in critically ill COVID-19 patients suspected of HIT. PATIENTS/METHODS: We tested 10 critically ill COVID-19 patients suspected of HIT for anti-PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti-human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS-CoV-2 antibodies were measured using an in-house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) activity and the presence of anti-ADAMTS13 antibodies. RESULTS: Heparin-induced thrombocytopenia was excluded in all samples based on anti-PF4/heparin antibody and SRA results. Notably, six COVID-19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)-mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID-19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy. CONCLUSIONS: Our study identifies platelet-activating ICs as a novel mechanism that contributes to critically ill COVID-19.

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.

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

BACKGROUND: 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]). OBJECTIVES: 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. PATIENTS/METHODS: 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. RESULTS: 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%). CONCLUSION: 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.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

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

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 ß-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.

Producing megakaryocytes from a human peripheral blood source.

BACKGROUND: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of µ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.

Pitfalls in the diagnosis of heparin-Induced thrombocytopenia: A 6-year experience from a reference laboratory.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic.

Congenital thrombotic thrombocytopenic purpura (cTTP) with two novel mutations.

Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare disorder of childhood that has clinical and laboratory similarities to other, more common conditions. Prompt recognition is required as delays in therapy are associated with significant morbidity and failure to treat may lead to death. While the principles of treatment have not changed, enormous progress in the genetic and molecular understanding has taken place. Emerging treatment options may offer some hope of improved quality of life in future. We describe a Chinese patient with cTTP which resulted from two previously undescribed mutations in the ADAMTS13 gene.

Studies of the immune response in heparin-induced thrombocytopenia.

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). Patients with HIT produced higher levels of IgG antibodies, but similar IgA and IgM levels, compared with seropositive non-HIT controls. Among all 48 seroconverting patients, the first day of a positive antibody test (median, day 6) did not differ among the immunoglobulin classes. Thus, the HIT immune response does not exhibit the classic paradigm of IgM class precedence/immunoglobulin class switching; rather, relatively rapid formation of IgG antibodies is observed, sometimes with concomitant IgA and IgM formation. Compared with seropositive non-HIT controls, HIT patients develop significantly higher anti-PF4/heparin IgG levels that are detectable before the onset of thrombocytopenia.

Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: a survey of North American specialized coagulation laboratories.

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. As HIT is considered a clinico-pathologic entity, laboratory practices have an important role in diagnosing or excluding HIT. It was the objective of this study to assess the current status of laboratory testing for HIT in North America. An online survey consisting of 67 questions related to laboratory testing for HIT was developed by the North American Specialized Coagulation Laboratory Association (NASCOLA), and distributed to its 59 members. The survey included queries about HIT test ordering practices, HIT immunoassay and activation assays performed, and reporting practices. Data was collected from the 44 NASCOLA laboratories who responded. Of these sites, 88% performed immunoassays for HIT, commonly using commercial assays. However, sites varied in practices related to use of controls, immunoglobulin class of antibody detected, and in result interpretation and reporting. Platelet activation assays for HIT were performed by 36% of sites, commonly using assays of serotonin release (50%) or heparin-induced platelet aggregation (43%). Sites varied in the use of washed platelets versus platelet-rich plasma, controls, and heparin concentrations. This survey is the first comprehensive assessment of patterns of practice in HIT testing among diagnostic coagulation laboratories in North America. We observed site-specific variability of testing methods encompassing all stages of testing, including pre-analytical handling, testing methodologies, and result interpretation and reporting. The variability in HIT platelet activation assay methods among institutions indicates a need for proficiency testing to assess assay performance, and for consensus guidelines on HIT laboratory testing.

The prevalence of antibodies to the platelet factor 4 -heparin complex and association with access thrombosis in patients on chronic hemodialysis.

INTRODUCTION: Heparin-induced thrombocytopenia is a serious complication that can lead to thrombocytopenia, venous and arterial thrombosis. Patients with this disorder develop antibodies to the platelet factor 4-heparin (PF4-H) complex. Hemodialysis patients are repeatedly exposed to heparin and are at risk for developing PF4-H antibodies. We sought to determine the prevalence of PF4-H antibodies in a large cohort of patients on chronic hemodialysis and to evaluate the relationship between PF4-H antibodies and hemodialysis vascular access thrombosis in a case-control study. MATERIAL AND METHODS: Pre-dialysis blood samples were drawn on 419 patients; 107 cases with access thrombosis and 312 controls that never had access thrombosis. All samples were screened for PF4-H antibodies using an ELISA assay (GTI PF4 Enhanced, GTI Diagnostics). All positive and indeterminate samples were then tested using an IgG-specific PF4-H ELISA assay and a platelet serotonin-release assay. RESULTS: Antibodies to PF4-H were positive in 54 (12.9%) patients using the screening ELISA assay. Nine (2.1%) patients had IgG-specific PF4-H antibodies. None of the patient's had a positive platelet serotonin-release assay. No relationship between hemodialysis access thrombosis and PF4-H antibodies was noted using the screening ELISA assay (unadjusted odds ratio 0.63; 95% CI 0.30-1.30; P = 0.21), the IgG-specific ELISA assay (unadjusted odds ratio 0.83; 95% CI 0.17-4.06; P = 0.82) or indeterminate platelet serotonin-release assay results (unadjusted odds ratio 0.97;95% CI 0.10-9.44;P = 0.98). CONCLUSIONS: Hemodialysis with repeated exposure to unfractionated heparin was associated with a moderately elevated prevalence of PF4-H antibodies. However, our results do not support a relationship between PF4-H antibodies and hemodialysis vascular access thrombosis.

A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors.

PURPOSE: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study. EXPERIMENTAL DESIGN: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2). RESULTS: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C(max) correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure. No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >or=6 months. CONCLUSION: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: how much class do we need?

Heparin-induced thrombocytopenia (HIT) is usually caused by platelet-activating antibodies of immunoglobulin G class that recognize platelet factor-4 (PF4) bound to heparin or certain other polyanions. Commercial enzyme immunoassays (EIAs) for PF4/polyanion-reactive antibodies detect two immunoglobulin classes (IgA and IgM) besides IgG. To investigate whether the additional detection of these antibody classes improves or worsens assay operating characteristics, we compared the sensitivity and specificity of EIAs that detect these 3 immunoglobulin classes individually with that of a commercial EIA (Genetic Testing Institute, GTI), as well as a platelet-activation assay, the serotonin-release assay (SRA). We compared the operating characteristics of these 5 assays by evaluating 448 patients, in 14 of whom clinical HIT developed, who received either unfractionated or low molecular weight heparin in prospective studies that included systematic platelet-count monitoring and serologic evaluation for anti-PF4/polyanion antibodies. We found that the SRA and IgG and commercial EIAs had similar high sensitivity for HIT; however, diagnostic specificity (for unfractionated and low molecular weight heparin, respectively) varied considerably, as follows: SRA (95.1%, 97.2%) > IgG EIA (89.0%, 93.7%) > GTI EIA (74.2%, 87.6%). Additional detection of IgA and IgM antibodies by the GTI EIA worsened test specificity by detecting numerous nonpathogenic antibodies. Moreover, the frequency and magnitude of IgA and IgM antibody formation in non-HIT immune responses did not differ from that exhibited by patients in whom clinical HIT developed. We conclude that an EIA that detects anti-PF4/polyanion antibodies of only the IgG class has greater diagnostic usefulness in revealing clinical HIT than does an assay that also detects IgA and IgM class antibodies.