Intravenous immunoglobulin as an adjunct therapy in persisting heparin-induced thrombocytopenia.

Heparin induced thrombocytopenia (HIT) is a serious adverse drug reaction caused by transient antibodies against platelet factor 4 (PF4)/heparin complexes, resulting in platelet activation and potentially fatal arterial and/or venous thrombosis. Most cases of HIT respond to cessation of heparin and administration of an alternative non-heparin anticoagulant, but there are cases of persisting HIT, defined as thrombocytopenia due to platelet activation/consumption for greater than seven days despite standard therapy. These patients remain at high risk for thrombotic events, which may result in limb-loss and mortality. Intravenous immunoglobulin (IVIg) has been proposed as an adjunct therapy for these refractory cases based on its ability to saturate FcγRIIa receptors on platelets, thus preventing HIT antibody binding and platelet activation. We describe 2 cases of persisting HIT (strongly positive antigen and functional assays, and persisting thrombocytopenia >7 days) with rapid clinical response to IVIg. We performed in-vitro experiments to support IVIg response. Healthy donor platelets (1 × 10e6) were treated with PF4 (3.75 µg/mL) for 20 min followed by 1-hour incubation with patients' sera. Platelet activation with and without addition of IVIg (levels equivalent to those reached in a patient after treatment with 2 gm/Kg) was evaluated in the PF4-dependent P-selectin expression assay (PEA). A significantly decreased platelet activation was demonstrated after the addition of IVIg to both patient samples, which correlated well with the rapid clinical response that each patient experienced. Thus, our study supports the use of IVIg as an adjunct therapy for persisting HIT.

Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management.

Drug-induced immune thrombocytopenia (DITP) can be triggered by a wide range of medications. Although many cases of DITP are mild, some are characterized by life-threatening bleeding symptoms. The pathogenesis of DITP is complex, in that at least six different mechanisms have been proposed by which drug-induced antibodies can promote platelet destruction. It is possible in many cases to identify antibodies that react with platelets in the presence of the sensitizing drug, but the required testing is technically demanding and not widely available. Therefore, a decision on whether to discontinue an implicated medication in a patient suspected of having DITP must be made on clinical grounds. An algorithm is available that can be helpful in assessing the likelihood that a particular drug caused thrombocytopenia, but the most important aspects of patient management are a high index of suspicion and a careful history of drug exposure in an individual who presents with acute, often severe thrombocytopenia of unknown etiology. How drugs induce platelet-reactive antibodies and how, once formed, the antibodies cause platelet destruction following exposure to the drug is poorly understood. Further studies to address these issues and characterize more completely the range of drugs and drug metabolites that can cause DITP are needed.

Heparin-induced thrombocytopenia and thrombosis.

Heparin, employed clinically for more than 50 years, is still a widely used anticoagulant. Unfortunately, some patients given this agent develop thrombocytopenia and thrombosis. Because this side effect can have catastrophic consequences, it is imperative that all clinicians caring for patients who receive heparin have at least a basic understanding of its pathogenesis, diagnosis, and management.

Prospective evaluation of a new platelet glycoprotein (GP)-specific assay (PakAuto) in the diagnosis of autoimmune thrombocytopenia (AITP).

Assays measuring platelet-associated immunoglobulin G (PAIgG), while highly sensitive, lack specificity in diagnosing autoimmune thrombocytopenia (AITP). We prospectively evaluated a new commercially available glycoprotein (GP)-specific assay, the PakAuto (GTI, Brookfield, WI), for its clinical usefulness in distinguishing immune from nonimmune thrombocytopenia (TP), in 216 patients with autoimmune TP (both primary "idiopathic" and "secondary") and 46 patients with TP due to other causes. This assay is designed to detect both platelet-associated (direct assay) and plasma (indirect assay) antiplatelet antibodies specific for GPs IIb/IIIa, Ib/IX, and Ia/IIa. The mean platelet counts of the immune (79 +/- 7 x 10(9)/L) and nonimmune groups (78 +/- 7 x 10(9)/L), were similar (P=0.95). The direct assay was positive in 114/216 patients with AITP (53%), and 13/46 with nonimmune TP (28%). Among the AITP group, the majority (61%) of patients with positive test results had autoantibodies reactive against all three GP targets. The sensitivity, specificity, positive, and negative predictive values for the direct PakAuto were 53%, 72%, 90%, and 24%, respectively, comparable to previously published experience of GP-specific assays. However, in some cases of TP due to nonimmune cause, the PakAuto was highly specific. Only 3 of 22 patients with gestational and 1 of 8 with familial/congenital TP had a positive direct assay, indicating that the test may be particularly useful for excluding an immune etiology for TP in certain patient subgroups.

Integrin clustering mechanisms explored with a soluble alphaIIbbeta3 ectodomain construct.

The purpose of this study was to test the hypothesis that residues critical for ligand- and temperature-induced clustering of integrin alphaIIbbeta3 are present on its extracellular domain. Sucrose density gradient sedimentation was used to examine the effects of ligand-mimetic peptides and physiological temperature on the oligomeric state of a soluble recombinant ectodomain variant of the alphaIIbbeta3 integrin, alphaIIbDelta962beta3Delta692, and its full-length counterpart. Both the ectodomain construct, isolated from High Five insect cell culture supernatants, and alphaIIbbeta3, isolated from human blood platelets, exhibited similar weight-average sedimentation coefficients at 23 degrees C, in the absence and presence of the ligand-mimetic peptide eptifibatide. These observations indicate that alphaIIbbeta3's ectodomain exhibits a similar extended conformation in both its free and ligand-bound states. Oligomerization was examined by incubation of both alphaIIbDelta962beta3Delta692 and full-length receptors at 37 degrees C, in the presence or absence of ligand-mimetic. Minimal oligomerization was observed with alphaIIbDelta962beta3Delta692. In contrast, full-length alphaIIbbeta3 exhibited substantial temperature-induced increases in its distribution of sedimenting species, indicative of thermal aggregation. These observations suggest that optimum oligomerization requires the participation of the integrin's transmembrane and cytoplasmic regions. In vivo, clustering of ligand-bound integrins may enhance signaling by increasing the local concentration of intracellular integrin-associated proteins.

Delayed thrombocytopenia after treatment with abciximab: a distinct clinical entity associated with the immune response to the drug.

BACKGROUND: Acute thrombocytopenia is a recognized side-effect of treatment with the fibrinogen receptor antagonist, abciximab, a chimeric (human/mouse) Fab fragment. The etiology of this complication is not fully understood. Generally, abciximab-induced thrombocytopenia occurs within a few hours of starting treatment with the drug. We have characterized a group of 13 patients who first developed thrombocytopenia 3-6 days after abciximab was discontinued. OBJECTIVE: To characterize clinical and serological aspects of this newly recognized clinical entity. PATIENTS AND METHODS: Clinical information was obtained from attending physicians and review of hospital records. Antibodies reactive with abciximab-coated platelets were characterized by flow cytometry. RESULTS: In each patient, IgG and/or IgM antibodies reactive with abciximab-coated platelets were identified. These antibodies could be distinguished from similar antibodies present in many normal persons by two criteria-they were relatively resistant to inhibition by normal Fab fragments, and they reacted preferentially with platelets coated with 7E3, the murine monoclonal antibody from which peptide sequences in abciximab are derived. Antibodies with these characteristics were not found in pretreatment serum from three of the thrombocytopenic patients or in patients given abciximab who did not develop thrombocytopenia. CONCLUSIONS: 'Delayed thrombocytopenia' after treatment with abciximab is caused by antibodies produced in response to the drug. These antibodies may be specific for murine peptide sequences in abciximab but could recognize other target epitopes on abciximab-coated platelets. Physicians administering abciximab should be aware of this potential complication of treatment, which usually occurs after discharge from hospital.

Severe neonatal alloimmune thrombocytopenia caused by antibodies to human platelet antigen 3a (Baka) detectable only in whole platelet assays.

BACKGROUND: Maternal antibodies that cause neonatal alloimmune thrombocytopenia are commonly identified by solid-phase assays that detect the causative antibodies on the basis of their reactions with specific PLT glycoproteins. Two cases of severe neonatal alloimmune thrombocytopenia caused by maternal antibodies specific for human PLT antigen 3a (HPA-3a [Baka]) that failed to give the expected reactions in some solid-phase assays were recently encountered. STUDY DESIGN AND METHODS: PLT-reactive antibodies were characterized by three different solid-phase assays and by flow cytometry. RESULTS: The two maternal antibodies gave negative reactions in the antigen capture ELISA, modified antigen capture ELISA, and MoAb immobilization of PLT antigens tests but reacted strongly in flow cytometry with intact PLTs that were HPA-3a+. Other sera samples specific for HPA-3a reacted equally well in all assays. CONCLUSIONS: The two antibodies appear to recognize an epitope on the HPA-3a+ form of glycoprotein IIb that is lost when PLTs are solubilized in detergent, as required for solid-phase assays. The diagnosis was made in these cases because no HLA antibodies were present, allowing an HPA-3a-specific reaction to be identified with intact PLTs as targets. Such antibodies are likely to be overlooked when HLA antibodies are also present.

TRALI due to granulocyte-agglutinating human neutrophil antigen-3a (5b) alloantibodies in donor plasma: a report of 2 fatalities.

BACKGROUND: TRALI is usually an immunologic reaction to WBC antibodies in infused plasma and ranks second only to ABO mismatch as a cause of transfusion-associated death. Implicated donors are usually multiparous women (>/=3 pregnancies). STUDY DESIGN AND METHODS: Two fatal cases of TRALI were evaluated by reviewing clinical and laboratory findings and characterizing alloantibodies present in donor plasma. Investigation for WBC antibodies was by lymphocytotoxicity (LCT), FlowPRA (FlowPRA, One Lambda, Inc.) and granulocyte immunofluorescence and agglutination assays. Patient 1 was a 62-year-old man with chronic T-cell lymphocytic leukemia, and Patient 2 was a 54-year-old woman undergoing a cadaveric kidney transplant. Both patients developed acute respiratory distress and hypotension during (Patient 1) and approximately 30 minutes after (Patient 2) transfusion. Fulminant pulmonary edema ensued in both cases necessitating mechanical ventilation and both patients died within 24 hours of the onset of respiratory complications. RESULTS: The donors of the implicated blood components were women with a history of two pregnancies but no blood transfusions. Weak apparently panreactive granulocyte antibodies were detected with flow cytometry. However, in the granulocyte agglutination test, strong antibodies specific for human neutrophil antigen (HNA)-3a (5b) were identified in both donors. CONCLUSION: It is concluded that female blood donors with only two previous pregnancies can form clinically important granulocyte-reactive alloantibodies leading to fatal TRALI reactions in recipients. The sometimes devastating consequences of TRALI should prompt the development of strategies to prevent or reduce its incidence. Further research is warranted to investigate recipient and donor factors responsible for TRALI, including whether 5b (HNA-3a) alloantibodies are especially prone to cause severe reactions, and to better characterize the HNA-3a (5b) antigen, particularly at the molecular level.

Serologically documented loracarbef (Lorabid)-induced immune thrombocytopenia.

We report here the first case of severe immune thrombocytopenia induced by a second-generation cephalosporin antibiotic, Loracarbef. A 56-year old white female developed acute severe thrombocytopenia associated with acute respiratory symptoms following administration of Loracarbef. She responded to Loracarbef withdrawal and systemic corticosteroid administration. Loracarbef-dependent platelet-reactive antibodies were demonstrable in her serum by flow cytometry.

Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/thrombosis.

Heparin-induced thrombocytopenia (HIT), with or without thrombosis, is a common and often serious complication of heparin therapy. Platelet-activating, heparin-induced antibodies characteristic of HIT are thought to be specific for complexes formed between platelet factor 4 (PF4) and heparin, and such complexes are routinely used for antibody detection. We studied the binding of HIT antibodies to PF4 complexed with heparin fractions of uniform molecular size or linear polyanions other than heparin and found that many compounds other than heparin form complexes with PF4 that are suitable for antibody detection, provided they carry strong negative charges spaced about 0.5 nm apart along the molecular backbone and are of sufficient length to span about 40% of the circumference of the PF4 tetramer. Polyvinyl phosphonate was among the compounds that were equivalent to heparin. Thus neither a polysaccharide chain nor sulfate side groups--the hallmarks of heparin structure--are required for HIT antibody detection. The findings support the view that antibodies associated with HIT are specific for conformational changes that take place in the positively charged PF4 molecule when it reacts with a suitable, linear polyanion.

Can drugs cause autoimmune thrombocytopenic purpura?

A wide range of medications can cause life-threatening immune thrombocytopenia (ITP), hemolytic anemia, or neutropenia in sensitive individuals. The antibodies associated with these conditions usually require soluble drug to be present in order to react with the cell membrane glycoproteins for which they are specific. However, some patients make drug-independent antibodies (autoantibodies) as well. Occasionally, only autoantibodies are produced following exposure to a drug. Although drugs and other small molecules can become conjugated to proteins in vivo, which may induce an immune response, only fragmentary information is available to explain how exogenous substances sometimes perturb the immune system in such a way that antibodies capable of causing immune cytopenia are produced. Platelets are affected by drug-induced antibodies more often than any other blood element. For many drug-induced thrombocytopenias, the targeted membrane glycoproteins are readily accessible for laboratory investigation and methods for detecting the responsible antibodies are well developed. Techniques for studying cellular aspects of the immune response induced by drugs through in vitro manipulation of T and B lymphocytes are also advancing rapidly. Studies of drug-induced ITP may provide clues to the general mechanisms whereby drugs and other xenobiotics induce immune diseases. Clinicians should consider the possibility of an exogenous trigger in patients who present with apparent autoimmune thrombocytopenia.

Blood group A and B antigens are strongly expressed on platelets of some individuals.

It is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A(1) and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II ("high expressers"). Serum A(1)- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A(1) and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A(1) Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A(1)-transferase gene of one Type II A(1) high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications. (Blood. 2000;96:1574-1581)

Immune hemolytic anemia caused by sensitivity to a metabolite of etodolac, a nonsteroidal anti-inflammatory drug.

BACKGROUND: Immune hemolytic anemia can be caused by sensitivity to many different drugs. In some instances, the sensitizing compound can be identified by in vitro testing, but results are often negative. One reason for this is that a drug metabolite formed in vivo can be the sensitizing agent, but the responsible metabolites have rarely been identified at a chemical level. This report describes a patient who developed severe, Coombs-positive hemolytic anemia on two occasions after taking the nonsteroidal anti-inflammatory drug etodolac. Studies were performed to characterize etodolac metabolites to which this patient was sensitive. CASE REPORT: Serum was tested for antibody in the presence and absence of drug using conventional methods and urine from individuals taking etodolac as a source of drug metabolites. Urinary metabolites of etodolac were identified by high-pressure liquid chromatography analysis. Glucuronide conjugates of etodolac and the 6-OH metabolite of etodolac were synthesized in a rat liver microsomal system to obtain reference standards. RESULTS: The patient's serum gave only trace (+/-) reactions with normal RBCs in the presence of etodolac but reacted strongly (4+) in the presence of urine from an individual taking this drug. The active urinary metabolites were identified as etodolac glucuronide and 6-OH etodolac glucuronide. CONCLUSION: This patient appears to have experienced acute, severe immune hemolytic anemia on two occasions because of sensitivity to the glucuronides of etodolac and 6-OH etodolac. In patients suspected of having drug-induced immune hemolytic anemia, RBC-reactive antibodies can sometimes be detected by using urine from an individual taking the implicated medication as the source of drug metabolites in in vitro reactions. For patients who present with acute immune hemolysis, a careful history of drug exposure should be taken, and, where indicated, confirmatory testing should be performed to identify the sensitizing drug and prevent inadvertent reinduction of hemolysis at a later time.

Minimal evidence of platelet and endothelial cell reactive antibodies in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microvascular thrombosis with thrombocytopenia and end-organ injury. Evidence suggests that platelet or endothelial cell injury may be initial pathological events in TTP. A number of factors in patient plasma, including immunoglobulins, have been proposed to mediate cellular injury in TTP. However, systematic analyses of TTP patient plasma for the presence of platelet or endothelial cell antibodies are lacking. We, therefore, analyzed 48 TTP patient plasma samples for the presence of platelet and endothelial cell antibodies by using enzyme-linked immunosorbent assay, flow cytometry, and microlymphocytotoxicity. Twelve of 48 TTP patient samples (25%) reacted against purified platelet glycoproteins. Nine (19%) also contained antibodies that bound to allogeneic target platelets in flow-cytometric assays. Nine of 48 samples (19%) contained antibodies to human umbilical vein endothelial cells in flow-cytometric assays, and seven of 48 patient samples (15%) bound to human dermal microvascular endothelial cells. Six of 48 (13%) patient plasma samples contained antibodies that bound to human umbilical vein endothelial cells activated with gamma-interferon and tumor necrosis factor-alpha. Of twenty samples that were reactive in one or more platelet or endothelial cell assay, eight contained human leukocyte antigen antibodies reactive in microlymphocytotoxicity. These studies demonstrate that antibodies reactive against platelet or endothelial cell antigens are not prevalent in TTP, and that more than a third of antibodies detected are human leukocyte antigen alloantibodies. Our findings suggest that autoantibodies against platelets or endothelial cells are not important in the pathogenesis of this syndrome.