Jane C. Moore

Quantitative interpretation of optical density measurements using PF4‐dependent enzyme‐immunoassays

Summary.  Background: Many laboratories test for heparin‐induced 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 (‘HIT antibodies’). 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 strong‐positive 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 strong‐positive 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.

Aortic stenosis and bleeding gastrointestinal angiodysplasia: is acquired von Willebrand's disease the link?

An association between aortic stenosis and haemorrhage from gastrointestinal angiodysplasia has been recognised for many years, but no explanation for this link has been found. Remarkably, aortic valve replacement, rather than bowel resection, corrects the bleeding. Aortic stenosis can be complicated by acquired von Willebrands disease type IIA (vWD-IIA), which is corrected after valve replacement, and gastrointestinal angiodysplasia is a common site of bleeding in older patients with acquired or congenital vWD. Could the stenotic aortic valve lead to an acquired, reversible deficiency of the largest multimers of plasma von Willebrand factor (equivalent to vWD-IIA) and thus explain the association with gastrointestinal haemorrhage?

A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet‐associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein‐ specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti‐GP Ib/IX autoantibodies in 76 patient samples. The protein‐specific assays were able to differentiate immune from non‐immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.

Detection of a Platelet-Agglutinating Factor in Thrombotic Thrombocytopenic Purpura

A sensitive and specific test was used to identify a platelet-agglutinating factor in sera from patients with thrombotic thrombocytopenic purpura. Serum from patients plus a preparation rich in large multimers of factor VIII: von Willebrand factor were added to target platelets, and agglutination occurred in 41 of 48 samples. Edetic acid, heparin, or heating, but not aspirin, monomeric IgG, or dansylarginine N-(3-ethyl-1,5-pentanediyl)amide inhibited the platelet-agglutinating factor. In-vitro agglutination requires the presence of a platelet-agglutinating factor and large multimers of von Willebrand factor. High concentrations of either component lowers the amount of the other required for platelet agglutination. Some patients may be more susceptible to the agglutinating factor because of a congenital or acquired abnormality in processing unusually large multimers of von Willebrand factor or because of infections or inflammatory disorders that lead to increased synthesis of large multimers of von Willebrand factor.

Calpain proteolysis of von Willebrand factor enhances its binding to platelet membrane glycoprotein IIb/IIIa: an explanation for platelet aggregation in thrombotic thrombocytopenic purpura

We have previously observed calpain activity (calcium‐dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain‐treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 μm) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mm) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain‐proteolysed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain‐proteolysed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolysed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity.

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 Ca2+ activity as well as Ca2+‐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.

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 IgG subclasses of platelet‐associated autoantibodies directed against platelet glycoproteins IIb/IIIa in patients with idiopathic thrombocytopenic purpura

Summary. The majority of patients with idiopathic thrombocytopenic purpura (ITP) have antiplatelet autoantibodies that are most frequently directed against platelet glycoproteins IIb/IIIa or Ib/IX/V. However, there is some debate whether the immune response is oligoclonal or polyclonal in nature. We investigated the subclass distribution of anti‐IIb/IIIa IgG autoantibodies in 59 prospectively studied patients with ITP. We also tested patients with a variety of thrombocytopenic disorders (n = 31) and healthy controls (n = 30). Platelet lysates were tested for IgG anti‐IIb/IIIa autoantibodies, and the specific IgG subclass distribution was measured using antigen capture assays. All testing was done blinded to diagnosis and other assay results. After unblinding, we found that 43 of the 59 ITP patients had anti‐IIb/IIIa autoantibodies (sensitivity = 73%). Anti‐IIb/IIIa autoantibodies were also detected in five of the 31 non‐ITP patients, but in none of the 30 healthy controls (specificity = 91%). The IgG subclass assay was positive in 39 of the 43 ITP patients with anti‐IIb/IIIa antibodies (sensitivity = 92%) and in 12 samples that had no detectable anti‐IIb/IIIa antibodies including two ITP patients (specificity = 83%). The most common subclass in the ITP patient samples was IgG1 (77%), either alone (n = 14) or with other IgG subclass antibodies (n = 19). However, there were also patients with only IgG2 (n = 2), IgG3 (n = 3) or IgG4 (n = 3) antibodies. Our results are consistent with the hypothesis that ITP is a heterogeneous disorder and that some patients have evidence of oligoclonality, whereas other patients have polyclonal autoantibodies.

The use of well‐characterized sera for the assessment of new diagnostic enzyme‐immunoassays for the diagnosis of heparin‐induced thrombocytopenia

Enzyme immunoassays (EIAs) are frequently used to detect PF4-dependent antibodies implicated in heparin-induced thrombocytopenia (HIT) [1]. One assay, the ‘PF4 Enhanced®’ (GTI Diagnostics, Waukesha, WI, USA), hereafter designated as ‘GTI-IgGAM’, utilizes PF4 bound to polyvinylsulfonate, and has

Relationship between platelet aggregating factor and von Willebrand factor in thrombotic thrombocytopenic purpura

The pathophysiology of the platelet thrombotic disorder, thrombotic thrombocytopenic purpura (TTP), is not well understood. Two apparently unrelated laboratory abnormalities have recently been described in patients with TTP: a platelet aggregating factor and abnormalities in von Willebrand factor (vWF). Although an interaction between these two abnormalities has been postulated to participate in the disease, this has not been proved. In this report we describe studies on a patient with relapsing TTP. These studies demonstrate that a consistent relationship exists between the platelet aggregating factor present in the patients serum and vWF. The patient had chronic low‐grade thrombocytopenic and schistocytic haemolytic anaemia that could be temporarily cured by infusions of plasma and certain other blood products. During acute exacerbations of the illness, a platelet aggregating factor was detectable in the patients serum and this was associated with the loss of the larger multimers of vWF. During remissions of the illness, abnormally large multimers of vWF were present. The results of this study support the concept that a platelet aggregating factor plus large multimers of vWF participate in the acute platelet thrombi that characterize TTP.