Pneumococcus-induced serotonin release from human platelets. Identification of the participating plasma/serum factor as immunoglobulin.

Pneumococcus-induced serotonin release from human platelets is greatly facilitated by a factor present in normal human plasma and serum. We have identified this factor as immunoglobulin by: (a) removing if from plasma and serum with solid phase antiFab antibody; (b) demonstrating its absence from the serum of an individual with severe immunoglobulin deficiency; and, (c) showing its presence in IgG preparations isolated from normal individuals. Evidence suggesting that the release reaction is triggered by pneumococcal antigen-antibody complexes rather than by nonimmune interaction of immunoglobulin with pneumococcus includes: (a) the failure of isolated IgG myeloma proteins to support release; (b) a lack of correlation between IgG concentration and "releasing factor activity" in normal human sera; (c) the identification of a normal serum that supports release by types II and III pneumococci but not type VII; and, (d) the ability of most normal sera to support release by pneumococca serotypes II and VII, though these types have not shown nonimmune reactivity with the Fc portion of the IgG molecule. The ability of antibodies present in normal serum to support pneumococcus-induced serotonin release suggests that the thrombocytopenia seen in pneumococcal infection may at least in part be caused by pneumococcal antigen-antibody complexes.     

Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is a common immune-mediated drug reaction that can be complicated by life-threatening arterial thrombosis. The diagnosis can be confirmed by demonstrating heparin-dependent release of radiolabeled serotonin from washed normal platelets in the presence of patient serum. However, certain serum samples from these patients produce 14C-serotonin release from some but not other normal donor platelets. We investigated this problem of donor platelet variability by studying the reactivities of 10 serum samples from patients with heparin-induced thrombocytopenia with platelets from 10 normal donors (100 serum and platelet combinations). We observed a marked variability in reactivity for patient serum and platelets from normal donors; this initially appeared random. However, closer examination indicated that the reactivities varied hierarchically. Because heparin-induced thrombocytopenia is caused by binding of heparin-dependent IgG to platelet Fc receptors, we examined whether platelet Fc receptor number or function explained the variability in platelet reactivity. We observed that platelet Fc receptor function, as measured by platelet release associated with heat-aggregated IgG, was highly correlated with platelet reactivity to heparin-induced thrombocytopenia serum samples. No significant correlation, however, was found between Fc receptor number and platelet response. Reaction of murine monoclonal antibodies that activate human platelets by means of the platelet Fc receptors was not predictive of platelet reactivity to heparin-induced thrombocytopenia serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.     

Study of the factors that cause specific transformation in cultures of lymphocytes from patients with quinine- and quinidine-induced immune thrombocytopenia.

Quinine- or quinidine-induced thrombocytopenic purpura is caused by synthesis of an immunoglobulin (Ig)G antibody, which caused platelet damage in the presence of the offending drug. The nature of the antigenic stimulus has been examined by measuring incorporation of [3H]thymidine into DNA during lymphocyte transformation to blast cells in the presence of the drug. Although patients' lymphocytes responded normally to the nonspecific mitogen, phytohemagglutinin P, they did not respond to either drug or platelets alone. However, significant transformation occurred when patients' lymphocytes were cultured for 7 d with homologous or autologous platelets in the presence of therapeutic concentrations of the drugs (0.39-39 microM). Platelet membranes were more active than intact platelets on the basis of protein content, whereas platelets from a patient with Bernard-Soulier syndrome were inactive. Washed platelets pretreated with the drugs were inactive when cultured with lymphocytes in the absence of the drugs, whereas platelets pretreated similarly in plasma caused transformation. Control lymphocytes from 20 normal patients and 6 patients with nondrug-induced thrombocytopenia were not transformed by drugs and platelets in the presence of normal serum or serum containing drug-dependent antibody, showing that the observed response was specific for presensitized lymphocytes. Thus lymphocytes of patients with drug-induced thrombocytopenia are transformed by an antigen that forms after interaction of plasma, specific platelet membrane components and the drug.     

An enzyme-linked immunosorbent assay for the evaluation of thrombocytopenia induced by heparin

Five patients with heparin-associated thrombocytopenia (HAT) were evaluated by platelet aggregation and quantitation of immunoglobulin binding to intact target platelets in both the presence and absence of heparin. These patients developed thrombocytopenia (12,000 to 70,000 platelets/microliter) 7 to 15 days and embolic and hemorrhagic complications 9 to 15 days after the initiation of heparin therapy. Platelet aggregation after the addition of heparin was demonstrated in two of four HAT serum samples, whereas normal serum samples showed no significant platelet aggregation. The five HAT serum samples showed normal to elevated baseline serum platelet-bindable immunoglobulin (SPBIg) with a range of 4.3 to 11.4 fg/platelet (normal less than or equal to 1.0 to 6.5 fg/platelet). When HAT sera were incubated with target platelets and heparin (5 U/ml), the SPBIg increased to 8.5 to 37.5 fg/platelet, a mean increase of 148% in the presence of heparin. Normal and control serum samples (from 10 normal laboratory volunteers, nine patients without thrombocytopenia receiving heparin, nine patients with autoimmune thrombocytopenic purpura, and nine patients with nonimmune thrombocytopenia not receiving heparin) showed only a slight increase in SPBIg of 0 to 2.8 fg/platelet above baseline, a mean increase of 15% after heparin incubation with the serum samples. The measurement of SPBIg of washed platelets incubated with test serum samples in the presence and absence of heparin is potentially a specific and sensitive in vitro test for the diagnosis of HAT and may prove more sensitive than platelet aggregation studies with heparin.     

In vitro releasability of histamine and serotonin: studies of atopic patients

Sixteen atopic patients with anaphylaxis to food, eczema, asthma and/or rhinitis were investigated for in vitro reactivity of their leukocytes and platelets to various stimuli. Leukocytes from two patients with anaphylaxis to foods had very high spontaneous release of histamine. Compared to non-atopic volunteers without respiratory disease, leukocytes from the other atopic patients showed increased histamine release after stimulation with methacholine in concentrations between 10(-2) and 10(-6) M. Histamine release induced by anti-IgE or anti-kappa chain serum was slightly decreased in atopics compared to controls, and was significantly decreased at low concentrations of anti-IgE (p less than 0.05). There was no significant difference of means for histamine release induced by the calcium ionophore A-23815. The uptake of 3H serotonin from platelet-rich plasma of atopic patients appeared to occur more slowly than in non-atopics. Serotonin release from washed platelets after stimulation with aggregated IgG was significantly lower in the atopic group (p less than 0.01). There was no significant difference in serotonin release induced by thrombin, epinephrine, ionophore or methacholine. Alterations in releasability of mediator containing cells to immunologic and non-immunologic stimuli may play a role in the expression of atopic disease.     

In vitro detection of heparin-induced humoral antiplatelet activity

We investigated the etiology of thrombocytopenia, with or without platelet thrombi, occurring while patients are receiving parenteral heparin. We used two in vitro methods to detect possible humoral factors in the sera of patients who became thrombocytopenic while receiving heparin. In the presence of heparin, four of four such patients' serum caused platelet aggregation. Only serum from the patient most severely affected clinically caused release of platelet factor 3 (PF3). All control sera gave negative results by both methods. We propose that platelet aggregation studies may be a sensitive and reliable method of confirming that thrombocytopenia occurring during heparin therapy is due to a humoral factor requiring the presence of heparin.     

Mechanism of complement-mediated activation of human blood platelets in vitro: comparison of normal and paroxysmal nocturnal hemoglobinuria platelets.

The paroxysmal nocturnal hemoglobinuria (PNH) platelet differs from the normal human platelet in its interaction with activated complement components: (a) when complement is activated by the alternative pathway, greater amounts of C3 are fixed to the PNH platelet than to the normal platelet; (b) the platelet-release reaction, as measured by serotonin release, occurs after C3 fixation to the PNH platelet. This reaction does not occur with normal platelets; (c) although serotonin release mediated by antibody alone was the same for normal and PNH platelets, antibody-initiated complement activation resulted in the fixation of greater amounts of C3 to PNH platelets and greater consequent serotonin release; and (d) nearly maximal serotonin release; and (d) nearly maximal serotonin release from PNH platelets occurs after the fixation of C3 (or perhaps C5) to the membrane without completion of the terminal sequence. In contrast, completion of the terminal complement sequence beyond C5 is required for maximal serotonin release from normal platelets. These abnormalities of interaction of complement components and PNH platelets may explain the occurrence of thromboses in this disease.     

Diclofenac-induced antibodies against RBCs and platelets: two case reports and a concise review

BACKGROUND: Diclofenac has frequently been implicated as the cause of immune hemolytic anemias and less frequently of immune thrombocytopenia. The presence of the causative antibodies has only been demonstrated in patients with immune hemolytic anemia, but not yet in patients with thrombocytopenia. The cases of two patients in whom diclofenac simultaneously induced antibodies against platelets and RBCs are reported. STUDY DESIGN AND METHODS: The investigation was carried out with standard serologic tests for detection of antibodies against platelets and RBCs. The patients' sera were tested in the presence and absence of diclofenac and its metabolites. RESULTS: One of the two patients developed severe hemolysis and significant thrombocytopenic purpura. The other patient developed significant thrombocytopenia but no hemolysis. Both patients had a positive DAT and drug- and/or metabolite-dependent antibodies against RBCs and platelets. CONCLUSION: Based on our findings and those of other investigators, we believe that diclofenac leads to the production of antibodies against RBCs and/or platelets.     

Human platelet activation by C3a and C3a des-arg

C3a liberated from C3 by treatment with C3 convertase (or by trypsin) induced aggregation of gel-filtered human platelets and stimulated serotonin release. At concentrations of 10(-10) M to 8 X 10(-12) M, C3a induced aggregation when added alone to platelets. However, at lower concentrations (2 X 10(-12) M) C3a did not aggregate platelets directly but exhibited highly significant synergism (two-way analysis of variance P less than 0.0001) with ADP in mediating platelet aggregation and release of serotonin. Removal of the C-terminus arginine from C3a abolished anaphylotoxin activity but did not affect the platelet- stimulating activity of the peptide. C3a and C3a des-arg were equally reactive in mediating platelet aggregation and release of serotonin. Further C3a and C3a des-arg exhibited synergism with ADP of equal significance in both aggregation and the release reaction. The concentrations of C3a required for the platelet-stimulating activity involve relatively small number of molecules per platelet (4,000-10,000 for the synergistic reaction with ADP). These data suggest the possibility of a C3a (C3a des-arg) receptor on human platelets. This premise is strengthened by the demonstration ultrastructurally of C3a on the platelet membrane subsequent to C3a stimulation.     

Serotonin uptake at 22 degrees C of stored platelets

In order to ascertain the possibility that platelet serotonin uptake may occur during storage of platelet concentrates (PC) at 22 degrees C with agitation, the high-performance liquid chromatographic procedure was applied to determine serotonin uptake by platelets. Studies at 22 degrees C showed that platelets stored for 4 days exhibited a significant serotonin uptake with a Vmax value of 2.4 X 10(-19) mole/platelet/min and a Km value of 0.62 X 10(-6) M. Incubation of PC with 5 X 10(-6) M serotonin for 1 day at 22 degrees C increased their serotonin contents from 2.2 to 4.2 X 10(-7) mole/10(11) platelets. Thrombin stimulation caused about 80% release of intracellular serotonin from fresh as well as stored platelets, which contained standard serotonin in the same amount as the original amount. These results suggest that a significant serotonin uptake of platelets might occur during in vitro storage at 22 degrees C and stored platelets have retained abilities to sequester extracellular serotonin into dense granules.     

Inhibitory effects of ZK 36374, a stable prostacyclin analogue, on adhesion of rabbit platelets to damaged aorta and serotonin release by adherent platelets

The effects of ZK 36374, a prostacyclin analogue, were studied on adhesion of rabbit platelets to damaged rabbit aorta and on activation of platelets (judged by release of serotonin and formation of thromboxane-B2) in response to the processes of adhesion to the vessel surface and aggregation in response to microfibrillar collagen in suspension. In the presence of ZK 36374 (10-100 nmol/l), platelet adhesion and thromboxane-B2 formation were progressively reduced. The extent of serotonin release from adherent platelets was similar to that found for platelets aggregated with collagen. However, higher concentrations of ZK 36374 were required to inhibit serotonin release from adherent platelets than from aggregated platelets. The results indicate that ZK 36374 acts similarly to native prostacyclin on adhesion and collagen-induced aggregation and unlike previously described analogues is equipotent. The mechanism of release of serotonin induced by adhesion of platelets is less sensitive to the action of ZK 36374 than that of release induced by aggregation in response to microfibrillar collagen in suspension.     

Detection of platelet isoantibodies by (3H)serotonin platelet release and its clinical application to the problem of platelet matching.

The detection of platelet isoantibodies by the release of (3H)serotonin from platelets has been evaluated. The conditions for optimal release of (3H)serotonin with platelet isoantibodies using a microtechnique have been defined. A group of cardiac surgery patients were followed pre- and post-transfusions, with 48percent developing a positive serotonin release assay. Of these patients, 16percent also had a platelet complement-fixing and/or lymphocytotoxic isoantibody. There was variation in the degree of correlation between (3H)serotonin release and lymphocytotoxicity using individual National Institutes of Health typing serum. The matching obtained between family members by both techniques showed a close correlation when each technique was evaluated separately using the same NIH typing serum. The detection of iso-antibodies in patients with hematological malignancies correlated with the unresponsiveness to unmatched platelet transfusions in 15 out of 17 cases. The use of the patient's isoantibody to matched platelets of family members by (3H)serotonin release correlated well with the clinical response to transfusion with these platelets. The data suggest that (a) platelet isoantibodies can be detected with increased frequency by (3H)serotonin release; (b) (3H)serotonin release is a specific reaction depending on the surface antigen of the platelet; and (c) the method can be used to match compatible family members for platelet transfusions.     

Assays for heparin-induced thrombocytopenia

Tests for the presence of heparin-dependent antibodies (heparin-Ig) have evolved in parallel with improved understanding of the pathophysiology of heparin-induced thrombocytopenia (HIT). The first group of tests relied upon platelet aggregation or activation. Among tests in this group, the serotonin release assay has been reported to demonstrate the best performance characteristics. However, this test has not been widely adopted outside a few specialized laboratories owing to its complexity and need for radioactive materials. As a result, the less sensitive and specific platelet aggregation test is more commonly used for the diagnosis of heparin-Ig. The literature suggests that test sensitivity can be improved by the use of the patient’s own platelets, platelets from selected donors known to be reactive in the assay, or washed platelets. Test specificity has been enhanced by the use of two point assays that include neutralization of the reaction by a high dose of heparin. A second group of assays have focused on detection of heparin-dependent binding of immunoglobulins to the platelet membrane. Most of these tests are hampered by the fact that platelets in patients with suspected HIT and in conditions that are in the differential diagnosis of HIT frequently express high levels of platelet-associated immunoglobulin. The most recent tests for heparin-Ig are based on the recognition that patient antibodies are directed against the heparin-PF4 complex. This has led to the development of the PF4/heparin EIA assay. Because whole platelets are not used in this assay, problems related to under-reactivity or nonspecific reactivity are avoided. In addition, the ability of the test to predict clinical complications may be improved because the test can distinguish IgM from IgG heparin-Ig. Currently the laboratory diagnosis of heparin-Ig remains inexact. The sensitivity and specificity of laboratory assays cannot be firmly established. Much like the diagnosis of the phospholipid syndrome — where use of both the cardiolipin EIA and the lupus anticoagulant test offer overlapping advantages — the combination of the heparin-PF4 EIA plus either a test of platelet activation or a heparin-dependent antibody binding assay may prove to be a more sensitive and specific approach to the diagnosis of heparin-Ig. Despite the progress that has been made in the area of laboratory diagnosis of heparin-Ig, further improvement is needed. Heparin-induced thrombocytopenia is not rare and may be associated with devastating morbidity as well as mortality. Low-molecular-weight heparins usually cross-react with heparin-Ig. Therapy with Org 10172 appears to be the most promising alternative for patients with HIT. Because the clinical diagnosis is uncertain in sick hospitalized patients, further improvements in laboratory assays for heparin-Ig allowing earlier and more accurate diagnosis of patients at risk for HIT will be welcome.     

Serotonin in blood: Assessment of its origin by concomitant determination of β-thromboglobulin (platelets) and chromogranin A (enterochromaffin cells)

Abstract

Background. Serotonin is produced in enterochromaffin (EC) cells, taken up and stored in platelets and released during platelet activation. Measurement of platelet-poor plasma serotonin is difficult, mainly due to platelet activation during blood sampling. We aimed to establish a method to assess the influence of platelet release upon platelet-poor plasma serotonin measurement by concomitant determination of serotonin, β-thromboglobulin (β-TG) and chromogranin A (CgA). Methods. Blood samples from patients with thrombocytosis, thrombocytopenia and small intestinal neuroendocrine (EC-cell) tumors (SI-NETs) as well as healthy volunteers were analyzed. We also measured serotonin in venous and arterial samples from patients undergoing coronary angiography to evaluate peripheral serotonin metabolism. Results. Serotonin and CgA were significantly higher in patients with SI-NETs compared to all other groups implying EC cell origin of serotonin in patients with SI-NETs. We found that the serotonin concentration was similar in patients with thrombocytosis and thrombocytopenia, whereas plasma β-TG was higher and lower respectively. A high EDTA concentration in the sampling tubes gave significantly lower serotonin concentrations. Serotonin concentrations did not differ between arterial and venous blood. Conclusions. Our methodology to measure platelet-poor plasma serotonin was appropriate. Blood platelet numbers did not affect the level of serotonin in contrast to β-TG.     

Identification of nonserotonergic [3H]ketanserin binding sites associated with nerve terminals in rat brain and with platelets; relation with release of biogenic amine metabolites induced by ketanserin- and tetrabenazine-like drugs

In mammalian striatal tissue and cat platelets, [3H]ketanserin labels besides serotonin-S2 receptors nonserotonergic saturable binding sites. The sites have been distinguished and characterized in [3H]ketanserin binding assays by selective inhibition with tetrabenazine (Ki = 4 nM), a monoamine depleting agent. In rats, the nonserotonergic ketanserin sites were enriched in the striatum (KD = 12.4 +/- 0.3 nM, maximal number of binding sites = 53.2 +/- 11.8 fmol/mg of tissue at pH 7.7, 37 degrees C) and nucleus accumbens. The sites were decreased by 65 to 78% after 6-hydroxydopamine lesions, suggesting an association with dopaminergic nerve terminals. In in vitro superfusion experiments using [3H]dopamine, [3H]norepinephrine and [3H]serotonin loaded rat brain tissue and [3H]serotonin loaded human platelets, 5 min superfusion with 10(-6) M ketanserin, tetrabenazine and reserpine caused instantaneously a marked increase in tritium efflux. The effect was attenuated by the monoamine oxidase inhibitor, pargyline, in brain slices but not in platelets. High-performance liquid chromatography analysis of endogenous catecholamines, serotonin and metabolites in superfusates from striatal slices revealed that stimulation with these drugs provoked mainly release of 3,4-dihydroxybenzeneacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. Potencies of a series of ketanserin derivatives, benzoquinolizine derivatives and a variety of drugs affecting neurotransmission were assessed in the in vitro release test using [3H]dopamine loaded striatal slices, and in [3H]ketanserin binding assays to nonserotonergic sites in the striatum and to serotonin-S2 receptors in brain tissue. Activities of drugs in the release test correlated strongly with their binding affinities for nonserotonergic ketanserin sites (rs = 0.83, n = 30, P less than .001). High potency in the latter two tests was confined to few close structural congeners of ketanserin and tetrabenazine. Distinct structural activity relationships for interaction with nonserotonergic ketanserin sites and serotonin-S2 receptors were found. It was concluded that nonserotonergic ketanserin sites mediate release of oxidated metabolites of biogenic amines from nerve endings and of serotonin from platelets. Hence release of biogenic amine metabolites or of cytoplasmic amines is probably not a mere diffusion process but involves specific membranous molecules. Unlike tetrabenazine, ketanserin caused no obvious depletion of central catecholamine and indoleamine stores. Implications of these findings for the mechanism of action of the drugs are discussed.     

The mobilization of arachidonic acid in platelets exposed to thrombin or ionophore A23187. Effects of adenosine triphosphate deprivation.

In studies conducted with human gel-filtered platelets, we have found: (a) that the release of serotonin and transfer of [3H]arachidonic acid from phosphatidylcholine and phosphatidylinositol to plasmalogen phosphatidylethanolamine which are associated with the activation of platelets by thrombin are both strongly dependent upon the presence of metabolic ATP; (b) that serotonin release and arachidonic acid mobilization in labeled phosphatides are promoted by the calcium ionophore A-23187 in media free of calcium ions; (c) that inhibitors of ATP synthesis, while leading to impairment of the release reaction induced by ionophore, do not inhibit ionophore-stimulated mobilization of arachidonic acid. We conclude that the activation of phospholipase A2 responsible for freeing arachidonic acid from platelet phosphatides is solely dependent upon the increased cytoplasmic levels of calcium ions promoted by either ionophore or, in an energy-dependent fashion by thrombin. Phospholipase activation is not a function of latent hydrolytic activity made available by the release reaction.     

Platelet antibody determination by platelet factor 3 assay

Summary Platelet antibody determination by the PF3 test was carried out in 96 thrombocytopenic patients with various disorders, 31 repeatedly transfused patients with or without thrombocytopenia and 24 patients with autoimmune disease (SLE andmyasthenia gravis) without thrombocytopenia. The frequency of a positive test was greatest in the patients with ITP (61%), SLE (50%) or a history of numerous blood transfusions (60%). The patients withmyasthenia gravis also showed a considerable frequency (20%) of platelet antibodies detectable by the PF3 test. The PF3 test is less sensitive than the serotonin release test in detecting autoantibodies, but it is more sensitive than aggregometry in detecting isoantibodies and drug-related antibodies.     

Comparison of platelet immunity in patients with SLE and with ITP

Idiopathic thrombocytopenic purpura (ITP) is characterized by the development of a specific anti-platelet autoantibody immune response mediating the development of thrombocytopenia. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autoantibodies. In 15-20% of SLE cases, patients develop thrombocytopenia which appears to be autoimmune in nature (SLE-TP). To better understand the pathogenesis of the thrombocytopenia associated with SLE, we investigated the overlapping platelet and cellular immune features between SLE and ITP. Thirty-one patients with SLE, eight with SLE-TP, and 17 with ITP, were studied and compared to 60 healthy controls. We evaluated platelet-associated IgG, platelet microparticles, reticulated platelets, platelet HLA-DR expression, in vivo cytokine levels, lymphocyte proliferation, and the T lymphocyte anti-platelet immune response in these patients. Patients with SLE-TP and those with ITP had increased platelet-associated IgG, an increased percentage of platelet microparticles, a higher percentage of reticulated platelets and larger platelets, suggesting antibody-mediated platelet destruction and increased platelet production. More than 50% of patients with ITP had increased HLA-DR on their platelet surface whereas subjects with SLE-TP did not. Analysis of serum cytokines demonstrated increased levels of IL-10, IL-15 and TNF-alpha in patients with SLE, but in those with ITP, only increased levels of IL-15 were seen, no increases in any of these cytokines were observed in patients with in SLE-TP. The ability of lymphocytes to proliferate in response to phorbol myristate acetate (PMA) stimulation was increased in SLE-TP, but was normal in both SLE and ITP. Lymphocytes from subjects with ITP displayed an increased ability to proliferate on exposure to platelets, in contrast, those with SLE-TP did not. While the number of subjects evaluated with SLE-TP was small, these data reveal a number of differences in the immunopathogenesis between SLE-TP and ITP.     

Prospective observational evaluation of the particle immunofiltration anti-platelet factor 4 rapid assay in MICU patients with thrombocytopenia

Introduction

Heparin-induced thrombocytopenia (HIT) results from antibodies to PF4/heparin complexes and clinical diagnosis is difficult. We evaluated the particle immunofiltration anti-platelet factor 4 (PIFA) rapid assay, in conjunction with a clinical risk score, in the diagnosis of HIT.

Methods

We performed a prospective observational study in all patients admitted to the medical intensive care unit (MICU) in a large academic medical center. Patients were screened daily for thrombocytopenia defined as either a platelet count that decreased by at least 33% or an absolute platelet count less than 150,000/μL. Patients with suspected HIT underwent PIFA and ELISA testing for anti-PF4/heparin antibodies. Available residual frozen sera were sent to a reference laboratory for serotonin release assay (SRA) testing.

Results

During the study period, 340 patients were admitted to the MICU, of which 143 patients met criteria for thrombocytopenia. Forty-three patients had no evidence of recent heparin exposure. PIFA and ELISA testing were performed on 100 patients, of which 92 had samples available for SRA analysis. PIFA results were negative in 62, positive in 28 and inconclusive in 2 patients. The 4Ts score showed low to intermediate risk in 57 of the PIFA negative patients. The ELISA results were negative in 86 and positive in 6 patients. SRA testing identified 3 patients with a positive SRA test and 89 patients with a negative result. All patients with a negative PIFA result also had a negative SRA result. In the one patient deemed to have clinical HIT, the pretest probability was high (4Ts score of 6) and the anti-PF4/heparin antibody testing revealed a positive SRA, inconclusive PIFA and a negative ELISA result.

Conclusions

While thrombocytopenia in our population is common, the prevalence of HIT is low. The combination of a low to intermediate pretest probability with a negative PIFA test can rapidly exclude the presence of platelet activating anti-PF4/heparin antibodies and, therefore, HIT as the cause of the thrombocytopenia. Since a positive PIFA result has a low positive predictive value, a positive PIFA is not diagnostic of HIT and additional evaluation is warranted.