[Immuno-allergic cytopenias]. / Cytopénies sanguines immuno-allergiques.

Drug-induced cytopenias are sometimes related to the so-called immuno-allergic mechanism which involves an unusual and unpredictable (i.e.: allergic) immune mediated reaction against the drug, leading to the cell-lysis of either peripheral blood granulocytes, thrombocytes; or erythrocytes. Agranulocytosis is typically induced by amidopyrine, whereas thrombocytopenia and hemolytic anemia are observed with drugs like quinine-quinidine, betalactams antibiotics, sulfonamides, rifampicin etc. In vitro methods for identification of the offending drug are cumbersome and poorly suitable for routine use. Therefore, definite diagnosis in these cases is first based on clinical and hematological grounds. These accidents recover spontaneously. However, potentially severe complications (infection, bleeding) deserve to prevent the risk of recurrence by a lifetime prohibition of the identified or suspected drug.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a target glycoprotein in drug-induced thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a serious complication of drug treatment. Previous studies demonstrated that most drug-dependent antibodies (DDAbs) react with the platelet membrane glycoprotein (GP) complexes IIb/IIIa and Ib/IX/V. We analyzed the sera from 5 patients who presented with DITP after intake of carbimazole. Notably, thrombocytopenia induced by carbimazole was relatively mild in comparison to patients with DITP induced by quinidine. The sera reacted with platelets in an immunoassay on addition of the drug. In immunoprecipitation experiments with biotin-labeled platelets and endothelial cells, reactivity with the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) could be demonstrated, whereas neither GPIIb/IIIa nor GPIb/IX was precipitated in the presence of the drug. These results could be confirmed by GP-specific immunoassay (MAIPA) using monoclonal antibodies (mabs) against PECAM-1. In addition, the binding of DDAbs could be abolished by preincubation with soluble recombinant PECAM-1. Carbimazole-dependent antibodies showed similar reactivity with platelets carrying the Leu(125) and Val(125) PECAM-1 isoforms, indicating that this polymorphic structure, which is located in the first extracellular domain, is not responsible for the epitope formation. Binding studies with biotin-labeled mutants of PECAM-1 and analysis of sera with mabs against different epitopes on PECAM-1 in MAIPA assay suggested that carbimazole-dependent antibodies prominently bound to the second immunoglobulin homology domain of the molecule. Analysis of 20 sera from patients with quinidine-induced thrombocytopenia by MAIPA assay revealed evidence that DDAbs against PECAM-1 are involved in addition to anti-GPIb/IX and anti-GPIIb/IIIa. We conclude that PECAM-1 is an important target GP in DITP. (Blood. 2000;96:1409-1414)

Stereospecificity of antibody: quinine, the optical isomer of quinidine and anti-malarial drug chloroquine do not cross-react with quinidine immunoassays.

Quinine is an optical isomer of quinidine. Both quinine and chloroquine (an aminoquinoline derivative) are used in treating malaria. The authors studied cross-reactivity of quinine and chloroquine with the quinidine immunoassays using the TDx and AxSYM analyzers (Abbott Laboratories, Abbott Park, IL). The authors observed no cross-reactivity of chloroquine with quinidine immunoassays (TDx and AXSYM) even when drug-free serum was supplemented with 1000 microg/mL chloriquine. The authors observed no cross-reactivity of quinine up to a concentration of 250 microg/mL. At higher concentrations, the authors observed a small cross-reactivity. The cross-reactivity of a substance should be studied in the presence of the primary analyte. When serum pools prepared from patients receiving quinidine were supplemented with various concentrations of quinine or chloroquine, the authors observed statistically significant declines in quinidine concentrations with higher concentrations of both quinine and chloroquine. The authors observed significant cross-reactivity of L-amphetamine with the amphetamine immunoassay also marketed by Abbott Laboratories and run on the AxSYM analyzer. The authors conclude that although the antibody used in the quinidine assay is stereospecific, the antibody used in the amphetamine assay by the same manufacturer is not stereospecific.

Three autacoids--endogenous digitalis-like factor, clonidine-displacing substance, and quinidine-like immunoreactivity.

The existence of endogenous ligands of opioid receptors prompted research of a potential endogenous digitalis-like factor (DLF) and of endogenous clonidine-displacing substance (CDS). Within eleven years of research, endogenous ouabaine was identified as DLF. It originates in the adrenal cortex. Its physiological role is not yet clear. Most probably, endogenous ouabaine is primarily active in regulation of natriuresis and of blood pressure. The CDS originates in the brain and is active in regulation of blood pressure as well. Its chemical formula was recently identified as agmatine. The potential presence of further autacoids like, e.g. endogenous quinidine-like substance remains to be clarified.

Endogenous quinidine-like immunoreactivity in the serum of rats with cardiac overload, stress or hyperthyroidism. Rapid communication.

Quinidine-like immunoreactivity (Abbott TDx Quinidine fluorescence polarization immunoassay) was measured in control, sham-operated, abdominal aorta stenotic and hyperthyroid (thyroxine feeding 0.1 mg/rat/day rats. The mean quinidine levels (mumol quinidine/1 serum) were 0.07, 0.14, 0.13 and 0.25, respectively. The elevation in experimental hyperthyroidism was statistically (analysis of variance, Duncan's test) significant. The possibility of the existence of an endogenous antiarrhythmic, immunologically cross-reacting in quinidine immunoassay, is discussed.

Quinidine-induced thrombocytopenic purpura: clinical presentation in relation to drug-dependent and drug-independent platelet antibodies.

We have studied the clinical course of quinidine-induced thrombocytopenia in relation to the presence of drug-dependent (dd-ab:s) and drug-independent antibodies in 14 patients. Thrombocytopenia was reversible in 9 d after discontinuation of quinidine treatment in 10 patients. In four it lasted more than 1 month. Drug-dependent antibodies of IgG class were detectable in seven patients: in six by an immunofluorescence test applying flow cytometry and in one patient by a monoclonal antibody-immobilized platelet protein assay (MAIPA) only. The dd-ab:s of this patient had glycoprotein (GP) IIb/IIIa specificity. Five of the six patients with dd-ab:s by immunofluorescence test had GPIb/IX-specific dd-ab:s by MAIPA. They recovered within 5 d after discontinuation of the drug. All four patients with prolonged thrombocytopenia had elevated platelet-associated IgG (PAIgG) in the acute phase as studied by a direct platelet immunofluorescence test. The remaining five patients displayed a relatively rapid clinical recovery but less uniform pattern of immunological findings. The results suggest that patients with GPIb/IX-specific dd-ab:s recover promptly despite an acute and profound thrombocytopenia. Another sub-group with prolonged thrombocytopenia had persistently elevated PAIgG during the convalescent phase.

Drug purpura due to surreptitious quinidine intake.

Three patients had recurrent episodes of thrombocytopenia that resembled drug purpura, but the drug history in each case did not support the diagnosis. Although the patients specifically denied taking quinidine, serologic testing with this drug was done because the patients had access to it, and it is the commonest cause of drug purpura. Highly specific quinidine-dependent antiplatelet antibodies were found in the sera of all three patients. After being informed of the laboratory findings, the patients have had no recurrences of purpura. Serologic tests for quinidine- or quinine-dependent antibodies can help elucidate some obscure cases of purpura that may be self-induced.

Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F(ab')2 fragments of goat antibody specific for the Fc portion of human IgG, while rosette formation between antibody-coated protein A-Sepharose and platelets was inhibited by F(ab')2 fragments directed against the F(ab')2 portion of the IgG molecule. Since binding of IgG to protein A is known to occur via the Fc region, these findings suggest that binding of drug-induced antibodies to platelets occurs at the Fab domains of the IgG molecule.

Identification of platelet proteins that bind alloantibodies and autoantibodies.

We have used the techniques of radioimmunoprecipitation (RIP) and Western blot to identify the membrane proteins that bind certain alloantibodies. Anti-PlA1 sera precipitated two bands, corresponding to platelet glycoproteins IIb and III, whether or not calcium was present during the procedure. By Western blot, this antibody bound only glycoprotein III. Anti-PlA1 serum does not precipitate proteins from the platelets of a patient with Glanzmann's thrombasthenia. Two monoclonal antibodies reacting with lymphocyte HLA antigens, as well as sera from highly allosensitized patients, precipitated bands of 38,500 and 13,500 daltons. These bands correspond to the molecular weights of the two subunits of the HLA antigen, as it has been described for other cell types. The patients' sera also precipitated a protein of 72,000 daltons from some platelets. The sera of two patients with quinidine-induced thrombocytopenia precipitated a 138,000-dalton band (glycoprotein Ib-alpha) in the presence of quinidine. The purified IgG antibody from one patient did not require other plasma factors to bind to platelets in the presence of quinidine, while purified antibody from a second patient required plasma factors other than, or in addition to von Willebrand factor. Although several sera from patients with idiopathic thrombocytopenic purpura (ITP) were tested, only one precipitated membrane proteins by the RIP method; this serum identified binding proteins corresponding to glycoproteins IIb and III.

Structural features of the quinidine and quinine molecules necessary for binding of drug-induced antibodies to human platelets.

Although most quinidine- and quinine-induced platelet antibodies react only in the presence of the drug that provoked sensitization, some are active with either quinidine or its stereoisomer, quinine; that is, they are "cross-reactive." This suggests that the activity of drug-dependent antibodies is dependent on different structural features of the quinidine or quinine molecules. To investigate this possibility, we studied reactions of 16 quinidine- and quinine-induced antibodies with quinidine, quinine, and analogues of these drugs modified at the quinoline ring (desmethoxy-derivative), the quinuclidine ring (dihydro-derivative), or the asymmetric C(9)-hydroxyl position. It was found that the antibodies could be classified into three groups on the basis of their reactions with platelets in the presence of these compounds. Eight antibodies (group 1) reacted only in the presence of the sensitizing drug or its dihydro- or desmethoxy-derivative. Three antibodies (group 2) differed from those in group 1 only in that their reactions were markedly weakened when the primary desmethoxy-derivative was used. Five antibodies (group 3) reacted in the presence of the sensitizing drug, its stereoisomer, and one or more of the analogues tested, including at least one of the C(9)-derivatives. Antibodies in this group gave stronger reactions with the sensitizing drug than with its stereoisomer. These results provide further evidence for heterogeneity among drug-induced platelet antibodies by demonstrating that noncross-reactive antibodies (groups 1 and 2) are dependent for their activity on a specific configuration at the optically active C(9)-hydroxyl position, and that some of these (group 2) also require the methoxy group for full reactivity. In contrast, cross-reactive antibodies (group 3) appear to be dependent on the quinoline ring common to all the analogues tested but also require a specific configuration at C(9) for full reactivity.

The human platelet receptor(s) for quinine/quinidine-dependent antibodies.

Substantial evidence now exists to associate platelet membrane glycoprotein Ib (GP Ib) with a receptor for quinine/quinidine-dependent platelet-specific antibodies. A direct relationship between GP Ib and this receptor activity has been difficult to establish for several reasons, including: the apparent existence of additional receptor activity not directly attributable to the presence of GP Ib; the variable reactivity of different sera observed by some investigators; the instability of receptor activity in semi-purified, soluble form; and differences in methods used by various laboratories to identify and quantitate either quinine/quinidine-dependent antibodies or platelet receptor activity. Moreover, little attention has been paid to the possibility that the Bernard-Soulier syndrome may represent a more heterogeneous collection of functional and molecular platelet abnormalities than hitherto supposed. As more patients are identified and studied, this possibility can also be addressed. A role for factor VIII-related antigen (VIIIR:Ag) in platelet destruction and/or clearance by drug-antibody complexes remains controversial. The observation that VIIIR:Ag is required for platelet activation in vitro (serotonin release, aggregation and increased platelet factor 3 availability) has been made, yet recent evidence indicates that VIIIR:Ag is not required for binding of antibody to platelets in the presence of drug or for complement-mediated lysis of platelets by antibody and drug. Evidence that VIIIR:Ag participates as part of the initial immunogenic complex is intriguing, yet still unconfirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-antibody-platelet interaction in quinine- and quinidine-induced thrombocytopenia.

Binding of quinine- and quinidine-dependent antibodies to platelets was studied using an electroimmunoassay to measure platelet-bound IgG. Antibodies from four patients with drug-induced thrombocytopenia differed significantly in their interaction with platelets: association constants for binding to platelets at high drug concentrations ranged from 0.29 to 2.6 x 10(7) M(-1), the maximum number of antibody molecules bound ranged from 36,000 to 161,000/platelet, the amount of drug necessary to achieve half-maximum binding of antibodies to platelets ranged from 2 to 60 muM, and only one of the antibodies cross-reacted with the stereoisomer of the drug to which the patient was sensitized. Binding of the antibodies to platelets was enhanced at the highest achievable molar ratio of drug:antibody, 10,000:1, rather than being inhibited, as would be expected in a conventional, hapten-dependent reaction. The drug-antibody-platelet reaction was unaffected by Factor VIII/von Willebrand protein, nonspecifically aggregated IgG, or heat-labile complement components. After pretreatment with tritiated quinine, platelets retained several hundred thousand molecules of drug each, but failed to bind detectable amounts of antibody. However, platelets treated simultaneously with quinine-dependent antibody and tritiated quinine retained significantly more drug after repeated washes than platelets treated with drug and normal serum. These findings support the proposition that in quinine- and quinidine-induced thrombocytopenia, drug and antibody combine first in the soluble phase to form a complex, which then binds with high affinity to a receptor on the platelet surface (innocent bystander reaction), and demonstrate that these antibodies are heterogeneous in respect to the amount of drug required to promote their binding to platelets, the number of platelet receptors they recognize, and their binding affinities.

Studies on quinine- and quinidine-dependent antibodies against platelets and their reaction with platelets in the Bernard-Soulier syndrome.

The sera of 14 patients with quinine/quinidine-dependent thrombocytopenia were studied in the platelet suspension immunofluorescence test (PSIFT), the 51Cr-lysis assay and the complement fixation test (CFT). When anti-Ig or anti-IgG reagents were used, the PSIFT proved to be a little more sensitive than the 51Cr-lysis assay and far more sensitive than the CFT. With the PSIFT, it was possible to determine the immunoglobulin class of the antibodies, which in all sera was IgG of the subclass IgG1. In two sera there were also IgG3 antibodies, and in the sera of three patients additional IgM drug-dependent platelet antibodies were detected. With the anti-C3 reagent, however, lower titres were observed in the PSIFT than in the 51Cr-lysis assay, in contrast to the results with other complement-binding IgG platelet antibodies. The F(ab')2 fragments of the antibodies did not react in the PSIFT. This indicates that the Fc part of the antibody is essential for the fixation on the platelet membrane. These findings support the theory that in this syndrome drug-antibody complexes area formed in he absence of platelets that selectively adhere to the Fc receptor on platelets. The platelets of patients with the Bernard-Soulier syndrome (BSS) reacted normally with the quinine/quinidine-dependent antibodies in some of the sera, but not with those in other sera in the PSIFT. However, lysis of BSS platelets was never observed. The results of absorption studies showed a reflection of the results in the PSIFT, indicating that the receptor for the quinine/quinidine-dependent antibodies is not absent in this disease but altered. Whether a reaction with BSS platelets will take place depends on characteristics of the antibodies.

Experience with immunological tests in drug-induced hepatitis.

Nine patients are reported who developed hepatic injury following the administration of therapeutic agents. The drugs incriminated were prajmalium bitartrate (4 patients), quinidine (3 patients), procainamide and nifedipine (one patient each). In all patients the clinical features indicated an allergic process, which in three cases was substantiated by positive response to reexposure. The detection of immunologic processes in our patients by various in vitro methods supports the assumption that immune mechanisms may be involved in the production of drug-induced hepatic disease and confirm allergy.

Further studies of the human platelet receptor for quinine- and quinine-dependent antibodies.

Previous studies have shown that the receptor for quinine- and quinidine-dependent antibodies is not expressed on the surface of platelets from patients with the Bernard-Soulier (B-S) syndrome. We now report data to suggest that these platelets lack the receptor for these antibodies. Since B-S platelets are also missing 2 related components of the GP I complex, GP lb and glycocalicin, our findings suggest that the receptor for quinine- and quinidine-dependent antibodies may be associated with this complex on normal platelets. An antibody previously shown to be directed against a surface antigen that migrated in the GP I position on SDS-polyacrylamide gel electrophoresis specifically blocked the reaction of the receptor with quinine- or quinidine-dependent antibodies. Purified glycocalicin, however, lacked detectable receptor activity. In contrast, a mixture of GP lb and a putative structural analog of this glycoprotein (Mr 210,000) eluted from wheat germ affinity columns after chromatography of Triton X soluble preparations of platelets or membranes was shown to contain at least 80% of the total receptor activity. Our results strongly suggest that the receptor is associated with GP lb and/or its high m.w. structural analog(s).

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.

Autoimmune hemolytic anemia.

In autoimmune hemolytic anemia, individuals produce antibodies directed against one of their own erythrocyte membrane antigens. The hemolysis in autoimmune hemolytic anemia is most commonly extravascular rather than intravascular, and the liver and spleen play a major role in the clearance of the antibody-coated cells. The importance of complement in the destruction of IgM-coated cells has also been recognized.

Detection of quinidine-specific antibodies with platelet 125I-labeled staphylococcal protein A test.

Remarkably higher titers (80--160) of quinidine-specific antibodies were detected in the serum of 5 patients with suspected quinidine purpura, when 125I-labeled staphylococcal protein A was used as the marker of IgG bound onto platelets. During a follow-up of 3 of the patients, the antibody titers declined. The antibodies could be detected even when the final drug concentration was reduced from 0.3 to 0.003 mmol/l (i.e. therapeutic levels). The reaction between serum and platelets was independent of the type of quinidine salt used. No cross-reactivity occurred with quinine. Heat inactivation of the serum did not decrease the quantity of specific antibodies detected on the platelets. The sedimentation behavior of antiplatelet activity in density gradient centrifugation resembled that of 7 S antibodies. When sedimentation was, however, effected on the drug-serum mixture, the reactivity of 7 S decreased.