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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. Am. J. Hematol. 90:629–633, 2015. © 2015 Wiley Periodicals, Inc.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies (HIT-Abs) that bind to a complex of heparin and platelet factor 4. We investigated the epitope specificity of the HIT-Abs, and found that the HIT-Abs recognized solid-phase immobilized complexes with an optimum ratio of four to eight molecules of PF4 per molecule of heparin. To try to define the epitopes within the PF4 molecule, intact and reduced (linearized) PF4 was tested against 29 different sera from patients with HIT. In addition, eight different peptides that spanned the PF4 molecule were studied for their ability to bind to the HIT-Abs either alone or in the presence of heparin. With the exception of a subpopulation of patient samples (5/29, 17%), we found that reduced PF4 and the peptides were uniformly non-reactive with the HIT-Abs in the presence of heparin. Reduced PF4 and PF4 carboxy-terminal peptides with a minimum size of 19 amino acids were recognized by a minority (5/29) of HIT-Abs samples but only when heparin was present. The specificity of this subgroup of samples from patients with HIT was highly restricted and the loss of one amino acid (peptide reduced in length from 19 to 18 amino acids) rendered the peptides non-reactive. The clinical characteristics of these patients were similar to the other HIT patients. These studies demonstrate that the majority of HIT-Abs recognize a noncontiguous conformational epitope on the PF4 molecule that is produced when four to eight PF4 molecules are bound together by heparin.

Human platelets are activated by IgG immune complexes and undergo platelet aggregation and the release of granular contents. Recently, the receptor for IgG on human platelets has been identified and characterised. In contrast, it is not known if rabbit platelets carry Fc receptors. In this report we describe investigations to detect the presence of an Fc receptor on rabbit platelets. The Fc receptor was investigated using functional assays (platelet aggregation and (14)C-serotonin release), a direct binding assay, and an affinity precipitation assay. All of these methods gave consistently positive results using human platelets and consistently negative results using rabbit platelets, indicating that rabbit platelets do not carry Fc receptors. This inconsistency in the interactions of immune complexes with rabbit and human platelets indicates that rabbits are not ideal models for studying a number of human diseases that could be caused by immune complexes, including heparin-induced thrombocytopenia and certain immunological disorders such as systemic lupus erythematosus.

A small fraction of patients who receive heparin develop heparin-induced thrombocytopenia (HIT) and, of these patients, a still smaller proportion develop associated thrombotic complications. Heparin-induced thrombocytopenia is caused by the formation of antibodies that bind to specific complexes of platelet factor 4 (PF4) and heparin. However, it remains uncertain why certain patients form these antibodies and develop HIT or why certain patients have thrombotic events. In this report we describe studies on individuals with and without HIT to determined if a potential PF4 polymorphism could explain differences in susceptibility to HIT. In the 10 control individuals and the 10 patients we studied, we did not find a difference in the PF4 sequences. Genetic difference in the PF4 antigenic target does not explain the occurrence of HIT in susceptible patients.

We have characterized monospecific sheep antisera to human IgG, IgA and IgM using Ouchterlony double diffusion, immunoelectrophoresis, quantitative precipitation and nephelometric activity. The antisera were conjugated at high and low F/P ratios, absorbed and subsequently evaluated in both direct and indirect immunofluorescence. Antisera which exhibited excellent reactivity in double diffusion, IEP and quantitative precipitation but gave a poor nephelometric response (Ab dilution >1/25 gave no measurable response in the Technicon Fluoronephelometer) showed a marked decreased effectiveness in both direct and indirect fluorescence. Antisera which exhibited a strong nephelometric response were invariably excellent in double diffusion, IEP and quantitative precipitation and gave strong positive fluorescence at reasonable dilutions in both direct and indirect fluorescence. We suggest nεphelometry prior to conjugation is the method of choice in screening antisera usable in immunofluorescence.

Label from [1-14C]-, [2-14C]-, [3-14C]-, and [1,3-14C2]phenylalanine is incorporated non-randomly into the Lythraceae alkaloids, decodine and decinine, in Decodonverticillatus. The incorporation pattern, determined by chemical degradation of the alkaloids, demonstrates incorporation of two intact C6—C3 units.

Summary The majority of platelet alloantigens are located on platelet glycoproteins IIb/IIIa. This report describes a codominant allelic system carried on the glycoprotein la/IIa complex, which we originally designated as Zava/Zavb but which is identical to the Bra/Brb system. Furthermore Zava was found to be identical to Hca. The alloantigens could not be detected using a direct binding enzyme immunoassay (EIA) with intact platelets, but were readily detected using a glycoprotein capture EIA and by radioimmunoprecipitation techniques. The two index cases (designated as homozygous Zava and Zavb) had alloantibodies against the corresponding antigen and did not react with their own platelets. Using these alloantibodies and a monoclonal antibody that reacts with the platelet glycoprotein la/IIa complex (12F1). We demonstrated that all Ia/IIa molecules carry either Zava or Zavb and we found that Zava and Zavb are on discrete populations of Ia/IIa. Following immunodepletion using either anti-Zava or anti-Zavb, all detectable Ia/IIa complexes from the respective homozygous platelets were removed. Immunodepletion of heterozygous Zava/Zavb with either anti-Zava or anti-Zavb did not reduce the amount of Ia/IIa complexes precipitable using the alternate alloantiserum. Population studies (n= 50) indicated the phenotypic frequency of Zava/Zava is less than 1%; Zava/Zavb is 18% and Zavb/Zavb is 82%. Four different alloantisera that had either anti-Zava or anti-Zavb reactivity also carried reactivity against the Baka or Bakb antigens which may suggest an association in the immune response to these alleles.

The amount of IgG on the surface of washed platelets from healthy individuals varies according to the assay used. An immunoradiometric assay (IRMA) for IgG was developed, validated and used to measure platelet associated IgG. The platelet-associated IgG (PAIgG) on washed platelets was allowed to react with excess 125I-labelled anti-IgG. The unbound 125I-anti-IgG was then quantitated by the addition of sepharose beads to which IgG had been covalently bound. The 125I-anti-IgG/IgG-beads were separated from the platelet suspension by passage across a density gradient. The mean amount of IgG present on washed platelets from healthy individuals was 0.8 fg IgG/platelet, or approximately 4,000 molecules of IgG per cell. Inverse Scatchard analysis confirmed the validity of the assay calibration and identical results were obtained when five different anti-IgG antibodies were used. The specific binding of 125I-anti-IgG to platelet-bound IgG was complete by 30 min, but non-specific binding of radioactivity continued thereafter. This non-specific binding occurred not only with anti-IgG but with all antibodies tested and could give elevated estimates of PAIgG in direct binding assays.

The biosynthesis of decodine (7) and decinine (9), Lythraceae alkaloids containing a trans-fused quinolizidine nucleus, was studied in intact plants of Decodonverticillatus (L.) Ell. Nonrandom incorporation of radioactivity from 14C-labelled samples of lysine, cadaverine, and Δ1-piperideine was demonstrated by partial degradation. Pelletierine did not serve as a precursor of the alkaloids. It was also shown, by means of 3H,14C-labelled tracers, that the alkaloids are derived from L-lysine whereas pipecolic acid, which is present in the plant, is derived from D-lysine.

The glycoprotein localization of the platelet binding site for the Fc IgG has been the subject of debate. We attempted to resolve this issue by relating the binding of radiolabeled IgG immune complexes composed of heat-aggregated IgG to platelets from healthy individuals; an individual with Bernard-Soulier syndrome lacking glycoproteins IIb and IX; and a patient with Glanzmann's thrombasthenia lacking glycoproteins IIb and IIIa. The binding of IgG complexes to platelets was determined by measuring the specific binding of radiolabeled heat-aggregated IgG to washed platelets in a plasma-free mileu. 125I aggregated IgG bound to normal platelets in a saturable and concentration-dependent fashion. Specific binding could be inhibited by a 50-fold excess of purified Fc, but not by F(ab')2. Identical binding curves were obtained by using platelets from a patient with Glanzmann's thrombasthenia and a patient with Bernard-Soulier syndrome, indicating that the platelet Fc receptor is not carried on glycoproteins Ib, IIb, IIIa, or IX. We then measured the binding of radiolabeled detergent-solubilized platelets to IgG fixed to a solid matrix. A 40-kD platelet fragment bound to the immobilized IgG following passage across a density gradient. Confirmation of the Fc specificity of the interaction was shown by inhibition of platelet glycoprotein binding by excess IgG or purified Fc but not F(ab')2. The electrophoretic mobility decreased slightly after reduction, which indicated the existence of at least one intrachain disulfide bond. Treatment with high salt solutions or urea did not solubilize the receptor, which indicated that it was an integral protein. Enzyme studies showed that the platelet Fc receptor was not digested by neuraminidase, but neuraminidase treatment altered mobility by about 3%. In addition, treatment of platelets with trypsin or pronase did not affect its function as measured by the binding of 125I-IgG aggregates to enzyme-treated platelets, but did prevent its detection when using radioimmunoprecipitation studies. The platelet Fc receptor is a 40-kD, integral protein without interchain disulfide bonds.

Linear pharmacokinetic models were obtained for fentanyl in anaesthetized rabbits. A composite model was used to predict the bolus dose and infusion rates necessary to achieve rapidly a steady-state concentration of fentanyl in plasma. When this was tested in a further six rabbits, steady-state concentrations were greater than predicted. The most likely reasons for this are imperfect mixing between plasma and the central compartment, and substantial uptake of fentanyl by the lung. These sources of error were allowed for in modifications to the model. On further testing, a 4-h infusion set to achieve a plasma fentanyl concentration of 10 ng ml-1 yielded actual concentrations ranging from a mean low of 8.7 ng ml-1 to a mean high of 18.0 ng ml-1. The second model described more precisely the disposition of fentanyl, although a tendency to overestimate dosing requirement remained. This tendency is probably a consequence of continuing lung uptake of fentanyl. The demonstrated ability to obtain rapidly and maintain for 4h "steady" blood fentanyl concentrations suggests that the methods merits further investigation with other drugs both in animals and man.