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• W2000745319 abstract "SummaryChronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-induced platelet destruction. To better define the role of antigen-specific assays in adult chronic ITP, we prospectively measured platelet-associated autoantibody against either glycoprotein (GP) IIb/IIIa or GPIb/IX in 282 patients with chronic ITP and 289 patients with thrombocytopenia of other causes. We divided chronic ITP into four subgroups: presplenectomy, mild (platelet count >30 000 µL−1 requiring no therapy), presplenectomy, severe (platelet count <30 000 µL−1 requiring therapy but not splenectomy), postsplenectomy, remission (postsplenectomy partial or complete remission without further therapy) and postsplenectomy refractory (required therapy after splenectomy failure). Positive results: total ITP group, 55.4%; presplenectomy, mild, 31.1%; presplenectomy, severe, 42.6%; postsplenectomy, remission, 50.0%; and postsplenectomy, refractory, 87.8%. In addition, the degree of positivity increased with the severity of the patient's disease. The assay had a minimum specificity of 84.4% if clinical factors, consistent with immune thrombocytopenia, were not considered in patients with thrombocytopenia associated with other diseases. However, if clinical factors consistent with immune thrombocytopenia were considered and only patients with questionable immune thrombocytopenia and patients ‘lost to follow-up’ were included in the false-positive group the specificity was 93.1%. We conclude that the presence of immune thrombocytopenia is highly probable if the immunobead assay is positive and that antigen-specific assays are diagnostically useful in adult chronic ITP. Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-induced platelet destruction. To better define the role of antigen-specific assays in adult chronic ITP, we prospectively measured platelet-associated autoantibody against either glycoprotein (GP) IIb/IIIa or GPIb/IX in 282 patients with chronic ITP and 289 patients with thrombocytopenia of other causes. We divided chronic ITP into four subgroups: presplenectomy, mild (platelet count >30 000 µL−1 requiring no therapy), presplenectomy, severe (platelet count <30 000 µL−1 requiring therapy but not splenectomy), postsplenectomy, remission (postsplenectomy partial or complete remission without further therapy) and postsplenectomy refractory (required therapy after splenectomy failure). Positive results: total ITP group, 55.4%; presplenectomy, mild, 31.1%; presplenectomy, severe, 42.6%; postsplenectomy, remission, 50.0%; and postsplenectomy, refractory, 87.8%. In addition, the degree of positivity increased with the severity of the patient's disease. The assay had a minimum specificity of 84.4% if clinical factors, consistent with immune thrombocytopenia, were not considered in patients with thrombocytopenia associated with other diseases. However, if clinical factors consistent with immune thrombocytopenia were considered and only patients with questionable immune thrombocytopenia and patients ‘lost to follow-up’ were included in the false-positive group the specificity was 93.1%. We conclude that the presence of immune thrombocytopenia is highly probable if the immunobead assay is positive and that antigen-specific assays are diagnostically useful in adult chronic ITP. Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by destructive thrombocytopenia due to antiplatelet autoantibodies. Based upon the American Society of Hematology (ASH) Practice Guidelines, the diagnosis of ITP is one of exclusion [1George J.N. Woolf S.H. Raskob G.E. Wasser J.S. Aledort L.M. Ballem P.J. Blanchette V.S. Bussel J.B. Cines D.B. Kelton J.G. Lichtin A.E. McMillan R. Okerbloom J.A. Regan D.H. Warrior I. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology.Blood. 1996; 88: 3-40Crossref PubMed Google Scholar]. Assuming that the patient's history, physical examination, complete blood count and peripheral blood smear are compatible with this diagnosis, no further diagnostic tests are considered necessary. As noted by Chong and Keng [2Chong B.H. Keng T.B. Advances in the diagnosis of idiopathic thrombocytopenic purpura.Semin Hematol. 2000; 37: 249-60Crossref PubMed Scopus (38) Google Scholar] and, as will be discussed later, there are potential problems with a diagnosis of exclusion. The availability of a dependable assay for antiplatelet autoantibodies would be helpful in supporting or confirming the diagnosis of immune thrombocytopenia. Following the classic studies of Harrington et al. [3Harrington W.J. Sprague C.C. Minnich V. Moore C.V. Aulvin R.C. Dubach R. Immunologic mechanisms in idiopathic and neonatal thrombocytopenic purpura.Ann Intern Med. 1953; 38: 433-69Crossref PubMed Scopus (195) Google Scholar] and Shulman et al. [4Shulman N.R. Marder V.J. Weinrach R.S. Similarities between known antiplatelet antibodies and the factor responsible for thrombocytopenia in idiopathic purpura. Physiologic, serologic, and isotopic studies.Ann NY Acad Sci. 1965; 124: 499-542Crossref PubMed Scopus (239) Google Scholar, 5Shulman N.R. Weinrach R.S. Libre E.P. Andrews H.L. Shannon J.A. The role of the reticuloendothelial system in the pathogenesis of idiopathic thrombocytopenic purpura.Trans Assoc Am Physicians. 1965; 78: 374-90PubMed Google Scholar] which showed that infusion of blood or plasma from ITP patients into normal recipients resulted in thrombocytopenia, multiple attempts have been made to develop a useful assay for antiplatelet antibodies. In the 1970s, direct platelet-associated IgG assays were noted to be positive in about 90% of patients with chronic ITP. However, the assays were also positive in a large percentage of patients with thrombocytopenia of non-immune origin and were deemed too non-specific for diagnostic purposes [6Kelton J. Powers P.J. Carter C.J. A prospective study of the usefulness of the measurement of platelet-associated IgG for the diagnosis of idiopathic thrombocytopenic purpura.Blood. 1982; 60: 1050-3Crossref PubMed Google Scholar, 7Mueller-Eckhardt C. Kayser W. Mersch-Baumert K. Mueller-Eckhardt G. Breidenbach M. Kugel H.G. Graubner M. The clinical significance of platelet-associated IgG: a study on 298 patients with various disorders.Br J Haematol. 1980; 46: 123-31Crossref PubMed Scopus (144) Google Scholar]. In 1982, van Leeuwen et al. [8Van Leeuwen E.F. Van Der Ven J.T.H. Von Engelfriet C.P. Dem Borne A.E.G. Specificity of autoantibodies in autoimmune thrombocytopenia.Blood. 1982; 59: 23-6Crossref PubMed Google Scholar] demonstrated that each of 42 antibody eluates from chronic ITP patients would bind to normal platelets but only 10 (23.8%) would bind to thrombasthenic platelets, suggesting that many of these ITP patients had antibodies specific for platelet glycoprotein IIb/IIIa. Subsequently, using a variety of antigen-specific assays, several laboratories demonstrated the presence of autoantibodies in chronic ITP which bound to several platelet surface glycoproteins, particularly the GPIIb/IIIa and GPIb/IX complexes [9Woods V.L. Oh E.H. Mason D. McMillan R. Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP.Blood. 1984; 63: 368-75Crossref PubMed Google Scholar, 10Woods V.L. Kurata Y. Montgomery R.R. Tani P. Mason D. Oh E.H. McMillan R. Autoantibodies against platelet glycoprotein Ib in patients with chronic idiopathic thrombocytopenic purpura.Blood. 1984; 64: 156-60Crossref PubMed Google Scholar, 11Beardsley D.S. Spiegel J.E. Jacobs M.M. Handin R.I. Lux S.E. Platelet membrane glycoprotein IIIa contains target antigens that bind antiplatelet antibodies in immune thrombocytopenias.J Clin Invest. 1984; 74: 1701-7Crossref PubMed Scopus (143) Google Scholar]. In 1989, two antigen-specific assays were reported which are clinically useful: the immunobead assay and the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay [12McMillan R. Tani P. Millard F. Berchtold P. Renshaw L. Woods V.L. Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP.Blood. 1987; 70: 1040-5Crossref PubMed Google Scholar, 13Kiefel V. Santoso S. Weisheit M. Mueller-Eckhardt C. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA). A new tool for the identification of platelet-reactive antibodies.Blood. 1987; 70: 1722-6Crossref PubMed Google Scholar]. These assays can measure both platelet-associated and plasma autoantibodies. Comparative studies show that the results obtained with these assays are similar [14Warner M.N. Moore J.C. Warkentin T.E. Santos A.V. Kelton J.G. A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.Br J Haematol. 1999; 104: 442-7Crossref PubMed Scopus (152) Google Scholar]. Two prospective studies have been reported using antigen-specific assays. Brighton et al. [15Brighton T.A. Evans S. Castaldi P.A. Chesterman C.N. Chong B.H. Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias.Blood. 1996; 88: 194-201Crossref PubMed Google Scholar] using the direct MAIPA assay to study 94 patients with immune thrombocytopenia, noted a sensitivity of 49% and a specificity of 78%. Their results excluded 13 patients with severe ITP who could not be tested due to their low platelet count and, as acknowledged by the authors, their relatively high percentage of ‘false positives’ may have been influenced by the inclusion of patients with diseases which can be associated with immune thrombocytopenia (e.g. lymphoproliferative disorders). Warner et al.[14Warner M.N. Moore J.C. Warkentin T.E. Santos A.V. Kelton J.G. A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.Br J Haematol. 1999; 104: 442-7Crossref PubMed Scopus (152) Google Scholar] studied 81 patients and reported a sensitivity of 66% and a specificity of 92%. They obtained identical results using either the MAIPA or an antigen capture assay. In the present study, we report our results using the immunobead assay for evaluation of a large number of patients with the diagnosis of chronic ITP, as well as, patients with thrombocytopenia associated with other disorders. We prospectively studied 282 patients with adult chronic ITP (≥18 years old) who had platelet-associated autoantibodies measured in our laboratory between 3 January 1986 and 1 April 1998. All chronic ITP patients fulfilled the criteria advised by the ASH Practice Guidelines [1George J.N. Woolf S.H. Raskob G.E. Wasser J.S. Aledort L.M. Ballem P.J. Blanchette V.S. Bussel J.B. Cines D.B. Kelton J.G. Lichtin A.E. McMillan R. Okerbloom J.A. Regan D.H. Warrior I. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology.Blood. 1996; 88: 3-40Crossref PubMed Google Scholar]: each patient's history, physical examination, complete blood count and peripheral blood smear were compatible with ITP and other causes of thrombocytopenia were excluded as much as possible. This study was approved by the Institutional Review Board of our institution. We subdivided the ITP patients into four subgroups (Table 1):Table 1Platelet-associated autoantibody results in patients with ITPPatient group*ITP- pre-mild, platelet count >30 000 µL−1 requiring no therapy; ITP-pre-severe, did not require splenectomy either due to spontaneous remission, safe counts on therapy, refusal or non-candidacy for splenectomy; ITP-CR/PR, splenectomy induced a complete or partial remission; ITP-ref, failed to maintain a platelet count of >30 000 µL−1 after splenectomy and required additional therapy.Total patientsAge (years) Mean ± SD (median)Follow-up (years) Mean ± SD (median)Positive (%)Negative (%)Total ITP group282––159 (56.4)128 (43.6)ITP-pre-mild7756.2 ± 18.7 (55)5.8 ± 4.4 (5.0)24 (31.1)53 (68.9)ITP-pre-severe6147.4 ± 19.4 (46)6.5 ± 4.5 (6.7)26 (42.6)35 (57.4)ITP-CR/PR46†All studied prior to splenectomy.43.6 ± 18.8 (37)7.2 ± 4.1 (6.2)23 (50.0)23 (50.0)ITP-ref9846.6 ± 17.7 (42)11.2 ± 9.3 (8.8)86 (87.8)16 (12.2)* ITP- pre-mild, platelet count >30 000 µL−1 requiring no therapy; ITP-pre-severe, did not require splenectomy either due to spontaneous remission, safe counts on therapy, refusal or non-candidacy for splenectomy; ITP-CR/PR, splenectomy induced a complete or partial remission; ITP-ref, failed to maintain a platelet count of >30 000 µL−1 after splenectomy and required additional therapy.† All studied prior to splenectomy. Open table in a new tab ITP-pre-splenectomy, mild (ITP-pre-mild), 77 patients This patient group had a mean platelet count of 83 000 µL−1 (range 40–130 000 µL−1) and required no treatment. The mean age for this group was 56.2 ± 18.7 years with a median age of 55. The mean follow-up time [65 patients, 12 lost to follow-up (LTF)] was 5.8 ± 4.4 years (median 5.0 years). ITP-pre-splenectomy, severe (ITP-pre-severe), 61 patients This group had platelet counts <30 000 µL−1 but did not undergo splenectomy for a variety of reasons including: (i) patients who remitted spontaneously either with or without treatment (usually corticosteroids, IV gammaglobulin or platelet transfusion); (ii) patients whose platelet count was maintained at safe levels with some form of mild therapy (usually low-dose corticosteroids); or (iii) patients who refused or were not candidates for splenectomy. The mean age for this group was 47.4 ± 19.4 years with a median age of 46. The mean follow-up time (45 patients, 17 LTF) was 5.8 ± 4.4 years (median 6.7 years). ITP post-splenectomy, remission (ITP-CR/PR), 52 patients Patients who had a complete remission (CR, normal platelet count) or a partial remission (PR, platelet count >30 000 µL−1) following splenectomy and required no additional therapy. Of these, 46 patients were studied prior to splenectomy and six patients from 8 months to 14 years postsplenectomy. The mean age for this group was 43.6 ± 18.8 years with a median age of 37. The mean follow-up time (39 of the 46 patients studied presplenectomy, 7 LTF) was 7.2 ± 4.1 years (median 6.2 years). ITP post-splenectomy, failure (ITP-ref), 98 patients Patients refractory to splenectomy, with a postsplenectomy platelet count <30 000 µL−1, who required additional therapy. The mean age for this group was 46.6 ± 17.7 years with a median age of 42. The mean follow-up time (83 patients, 19 LTF) was 11.2 ± 9.3 years (median 8.8 years). Of this group, nine patients died of causes related to their ITP, either CNS bleeding (seven patients) or sepsis associated with prolonged immunosuppression (two patients). In addition to the chronic ITP patients, we studied 289 patients with thrombocytopenia associated with other illnesses (Table 2) including: aplastic anemia (four patients); carcinoma (11 patients); hereditary disorders (10 patients: Gaucher's disease, six patients; Bernard–Soulier, one patient; familial thrombocytopenia, two patients; storage pool disease, one patient); congestive splenomegaly, 15 patients; pseudothrombocytopenia, two patients; qualitative platelet disorders, nine patients; gestational thrombocytopenia, 14 patients; chronic lymphocytic leukemia, 82 patients; non-Hodgkin's lymphoma, 57 patients; hairy cell leukemia, 35 patients; other leukemias (12 patients: acute granulocytic leukemia, five patients; acute lymphocytic leukemia, two patients; chronic granulocytic leukemia, two patients; prolymphocytic leukemia, three patients); myelodysplastic syndrome, 28 patients; myeloproliferative disorders (10 patients: myelofibrosis, five patients; chronic myelomonocytic leukemia, two patients; essential thrombocytosis, one patient; polycythemia rubra vera, one patient; unclassified, one patient).Table 2Platelet-associated autoantibody results in patients with other diagnoses*ITP, immune thrombocytopenic purpura; ?ITP, data not sufficient to rule in or out immune thrombocytopenic purpura; false positive, data do not support the presence of immune thrombocytopenia.DisorderPatient numberNegativePositiveITP?ITPFalse positiveLost to follow-upAplastic anemia440––––Carcinoma11110––––Hereditary10100––––Congestive splenomegaly15150––––Pseudothrombocytopenia220––––Qualitative disorders990––––Gestational thrombocytopenia141310010Chronic lymphocytic leukemia82631912313Non-Hodgkin's lymphoma574985021Hairy cell leukemia353140400Other leukemias121111000Myelodysplastic syndrome282262211Myeloproliferative disorder10465001Total group2892444525956* ITP, immune thrombocytopenic purpura; ?ITP, data not sufficient to rule in or out immune thrombocytopenic purpura; false positive, data do not support the presence of immune thrombocytopenia. Open table in a new tab When possible (86.7%), the medical history of patients with thrombocytopenia associated with other illnesses who had a positive test, was reviewed by one of us (RMc) in an attempt to determine if the thrombocytopenia was of immune origin or due to another cause. To be designated as immune thrombocytopenia, a clear response to ‘immune therapy’ must have occurred. We used a modification of the response criteria described by Cooper et al. [16Cooper N. Woloski B.M.R. Fodero E.M. Novoa M.V. Leber M. Beer J.H. Bussel J.B. Does treatment with intermittent infusions of intravenous anti-D allow a proportion of adults with recently diagnosed immune thrombocytopenic purpura to avoid splenectomy?.Blood. 2002; 99: 1922-7Crossref PubMed Scopus (77) Google Scholar], i.e. an increase in the platelet count of >20 000 µL−1 to a minimum platelet count of >30 000 µL−1 following therapy with either intravenous gammaglobulin, corticosteroids, splenectomy or danazol. In some cases, where multiple potential causes for thrombocytopenia were present (e.g. marrow infiltration, chemotherapy effect, etc.) or where therapy for thrombocytopenia was not required because the platelet count was at ‘safe’ levels, it was not possible to determine if immune thrombocytopenia was present. Patients with thrombocytopenia associated with another disorder and a therapeutic response compatible with an immune cause are designated as having ‘ITP’. If an immune component could not be either ruled out or confirmed, they are designated as having ‘?ITP’. All others were considered to be ‘false positives’. In six patients, we could not obtain medical information and these were termed ‘lost to follow-up’. Immunobead assay of platelet-associated autoantibody Assay details have been previously reported [17McMillan R. Antigen-specific assays in immune thrombocytopenia.Trans Med Rev. 1990; 4: 136-43Crossref PubMed Scopus (47) Google Scholar]. Briefly, patient platelets were isolated by differential centrifugation and washed six times with 0.05 mol L−1 citrate isotonic buffer, pH 6.2. Polystyrene beads (6.3 mm, one per assay) were coated for 2 h with monoclonal antibody [10 µg per bead of anti-GPIIb (2A9, provided by Dr Virgil Woods, UCSD) or 5 µg per bead of anti-GPIb (P3, provided by Dr Zaverio Ruggeri, Scripps Clinic) in 0.1 mol L−1 NaHCO3 buffer], blocked with 2% bovine serum antigen in 0.05% Tween in PBS (PBS-Tween) and washed four times with PBS-Tween. Platelet samples (108 platelets) were solubilized with 1% Triton X-100 and centrifuged for 5 min at 1200 × g. To 900 µL of each lysate was added one monoclonal antibody-coated bead of each type. After 2 h incubation and four washes with PBS-Tween, 1 mL of radiolabeled (about 400 000 cpm in PBS-Tween) monoclonal antihuman IgG (HB-43, American Type Culture Collection) was added followed by a final 60 min incubation. After four washes, bead-bound radioactivity was determined. Three normal control samples and positive controls were run with each assay. Results are expressed as a ratio of cpm patient sample devided by mean cpm of three control samples. A ratio of >3.0 was considered positive. In our experience, it is common for a sample, which is quite positive against one complex, to be mildly positive against the other complex. It is our bias that this is a technical problem during platelet solubilization, where a small amount of the nonantigenic complex is non-specifically bound to the antigenic complex and is ‘carried over’ when the autoantibody-antigen complex is bound to the monoclonal antibody. For this reason, we have expressed our antibody target specificities as follows: GPIIb/IIIa: autoantibody positive for only that complex; GPIIb/IIIa-predominant: autoantibody binds to both complexes but binding to GPIb/IX is <20% of GPIIb/IIIa; GPIb/IX: autoantibody positive for only that complex; GPIb/IX-predominant: autoantibody binds to both complexes but binding to GPIIb/IIIa is <20% of GPIb/IX; both: binding to both complexes. Assay of platelet-associated autoantibody IgG subtypes IgG subtype distribution of platelet-associated autoantibody was determined on a small group of selected patients: one patient (not included in the ITP group since he had a normal platelet count but severe bleeding due to anti-GPIIb/IIIa autoantibody directed to the ligand binding site) [18McMillan R. Bowditch R. Tani P. Anderson H. Goodnight S. A non-thrombocytopenic bleeding disorder due to an IgG4-kappa anti-GPIIb/IIIa autoantibody.Br J Haematol. 1996; 95: 747-9Crossref PubMed Scopus (31) Google Scholar], one patient with mild ITP and a platelet count of 105 000 µL−1 on no therapy but with high levels of anti-GPIIb/IIIa autoantibody and four patients with severe, refractory ITP with platelet counts <10 000 µL−1 who required therapy. Platelet-associated anti-GPIIb/IIIa autoantibodies were obtained by acid elution as previously described [19Fujisawa K. McMillan R. Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation-dependent antigens.Blood. 1993; 81: 1284-9Crossref PubMed Google Scholar]. Microtiter wells were coated with 100 µL of monoclonal anti-GPIIb (2A9, 5 µg mL−1) and, after blocking for 1 h with 200 µL of 2% BSA, 100 µL of normal platelet lysate (108 platelets mL−1 solubilized in 1% Triton X-100) were added as a source of GPIIb/IIIa and incubated for one hour; 2% BSA served as a control. After washing, 100 µL of autoantibody eluate was added and, after a one hour incubation and washing, IgG subtype autoantibody binding was detected with biotinylated monoclonal antibody to IgG1,2,3 or 4 (Pierce, Rockford, IL, USA) followed by avidin–biotin complexes and substrate (ABC System, Vector Laboratories, Burlingame, CA, USA). The percentage IgG subtype was calculated by dividing the individual IgG subtype OD by the sum of all IgG subtype ODs × 100. As shown in Fig. 1 and Table 1, positive assays were noted in 159 of 282 ITP patients (56.4%). In the ITP subgroups, the following positive assays were noted: ITP-pre-mild, 24 of 77 patients (31.1%); ITP-pre-severe, 26 of 61 patients (42.6%); ITP-CR/PR, 23 of 46 patients studied presplenectomy (50.0%) and one of six patients studied postsplenectomy (16.6%); and ITP-ref, 86 of 98 patients (87.8%). There was no correlation between the degree of assay positivity and the patient's platelet count in the group as a whole (r = −0.057) or within the individual ITP subgroups (ITP-pre-mild, r = 0.148; ITP-pre-severe, r = −0.037; ITP-CR/PR, r = −0.162; ITP-ref, r = 0.001). The degree of positivity was significantly higher between the ITP-Ref group and each of the other three ITP groups (Student's t-test: ITP-mild, P <0.0001; ITP-pre, P = 0.0001; ITP-CR/PR, P = 0.0003) while there were no significant differences when the other three groups were compared with each other. Figure 2 demonstrates the percentage of patients in each group with ratios of <3.0, 3.0–10.0, 10.0–20.0 and >20.0. Of the eight patients with ITP-related deaths, all were positive and had ratios >16.5.Figure 2The degree of positivity of the immunobead assay in ITP patients. The percentage of each ITP group (ITP-pre-mild, ITP-pre-severe, ITP-CR/PR or ITP-ref) with ratios (left to right) of: <3.0 (negative), 3–10, 10–20 or >20.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Table 3, most antibodies were directed to the GPIIb/IIIa complex either alone (51.6%) or predominantly (67.9%) while fewer bound to GPIb alone (11.9%) or predominantly (17.6%) or to both complexes (14.5%).Table 3Autoantibody specificity in chronic ITP*GPIIb/IIIa, autoantibody positive for only that complex; GPIIb/IIIa predominant, autoantibody binds to both complexes but binding to GPIb/IX is <20% of GPIIb/IIIa; GPIb/IX, autoantibody positive for only that complex; GPIb/IX predominant, autoantibody binds to both complexes but binding to GPIIb/IIIa is <20% of GPIb/IX; both, binding to both complexes.ITP subtypeGPIIb/IIIaGPIIb/IIIa predominantGPIb/IXGPIb/IX predominantBothTotalITP-pre-mild14540124ITP-pre-severe16132426ITP CR/PR11620423ITP-ref41141071486Total82 (51.6%)26 (16.3%)19 (11.9%)9 (5.7%)23 (14.5%)159* GPIIb/IIIa, autoantibody positive for only that complex; GPIIb/IIIa predominant, autoantibody binds to both complexes but binding to GPIb/IX is <20% of GPIIb/IIIa; GPIb/IX, autoantibody positive for only that complex; GPIb/IX predominant, autoantibody binds to both complexes but binding to GPIIb/IIIa is <20% of GPIb/IX; both, binding to both complexes. Open table in a new tab Of the 289 patients with thrombocytopenia associated with other disorders (Table 2), 244 (84.4%) had negative results. In patients, with other disorders, giving positive assay results (45 patients), 25 had evidence of immune thrombocyopenia based on their clinical response to ‘immune therapy’ as defined in the Methods section. The following platelet increases occurred following ‘immune treatment’ with either intravenous gammaglobulin, corticosteroids, splenectomy or danazol: an increase in the platelet count of >20 000 µL−1 to a minimum platelet count of >30 000 µL−1 (two patients); an increase in the platelet count of >50 000 µL−1 (nine patients) or attainment of a normal platelet count (14 patients). In nine additional patients, immune thrombocytopenia was considered a possibility but this could not be determined one way or the other because the patients had ‘safe’ platelet counts and required no therapy. Five patients gave false positive results and 6 patients were lost to follow-up. If all 20 patients without definite immune thrombocytopenia, as defined by therapeutic response, including those who were lost to follow-up or who had questionable immune thrombocytopenia, are assumed to have false-positive results, the specificity of the assay would be 93.1%. Only one of 65 patients, who had diagnoses where immune thrombocytopenia would be highly unlikely (patients in the following groups: carcinoma, hereditary, congestive splenomegaly, pseudothrombcytopenia, qualitative platelet disorders, pregnancy), had a positive assay. One possible explanation for either a ‘false-positive’ assay or an extremely high autoantibody result in a patient with mild thrombocytopenia could be the presence of a high percentage of an autoantibody IgG subtype, such as IgG4, which is not recognized by the reticuloendothelial system. We have studied the IgG subtypes of eluates from two such patients: one patient with a normal platelet count but severe bleeding due to anti-GPIIb/IIIa autoantibody directed to the ligand binding site (not included in the ITP group since he did not satisfy ASH criteria) and one patient with a platelet count of 105 000 µL−1 on no therapy but high levels of anti-GPIIb/IIIa autoantibody. As shown in Table 4, the patient with a normal platelet count (ITP-1) but bleeding due to blocking of the ligand binding site, had 100% IgG4 autoantibody and the other patient, with high antibody levels but a platelet count of 105 000 µL−1 (ITP-2), had 75% IgG4 autoantibody. The IgG subtype distribution of four other ITP patients, all with platelet counts <10 000 µL−1 prior to therapy (ITP 3–6) and high autoantibody levels, had IgG4 levels of <10%. How common this occurs is unknown since additional patient eluates were not available for study since the platelet samples were either used up to perform the immunobead assay or had been discarded before the potential importance of IgG subtypes was considered. Obviously, additional studies will be required to prove this hypothesis.Table 4IgG subtypes of platelet-associated anti-GPIIb/IIIa autoantibodiesPatientAutoantibody eluate IgG subtype binding (% total binding)*Microtiter wells were coated with monoclonal anti-GPIIb (2A9) and, after blocking with BSA, platelet lysate was added as a source of GPIIb/IIIa. After washing, autoantibody eluate was added. IgG subtype autoantibody binding was detected with biotinylated monoclonal antibody to IgG1,2,3 or 4 followed by avidin–biotin complexes and substrate. % binding was calculated by dividing the individual IgG subtype OD by the sum of all ODs × 100.IgG1IgG2IgG3IgG4ITP-1000100.0ITP-21021375ITP-387.34.93.64.2ITP-491.408.60ITP-586.17.80.55.6ITP-683.63.14.88.5* Microtiter wells were coated with monoclonal anti-GPIIb (2A9) and, after blocking with BSA, platelet lysate was added as a source of GPIIb/IIIa. After washing, autoantibody eluate was added. IgG subtype autoantibody binding was detected with biotinylated monoclonal antibody to IgG1,2,3 or 4 followed by avidin–biotin complexes and substrate. % binding was calculated by dividing the individual IgG subtype OD by the sum of all ODs × 100. Open table in a new tab Immune thrombocytopenia, associated with chronic ITP or other disorders (collagen vascular and lymphoproliferative disorders; viral infections, including HIV, etc.) is characterized by platelet destruction due to autoantibodies, often directed to the platelet glycoprotein complexes GPIIb/IIIa and/or GPIb/IX. The importance of these autoantibodies to disease pathogenesis is supported by the following: (i) infusion of plasma or IgG-rich plasma fractions, from ITP patients, into normal recipients results in thrombocytopenai [3Harrington W.J. Sprague C.C. Minnich V. Moore C.V. Aulvin R.C. Dubach R. Immunologic mechanisms in idiopathic and neonatal thrombocytopenic purpura.Ann Intern Med. 1953; 38: 433-69Crossref PubMed Scopus (195) Google Scholar, 4Shulman N.R. Marder V.J. Weinrach R.S. Similarities between known antiplatelet antibodies and the factor responsible for thrombocytopenia in idiopathic purpura. Physiologic, serologic, and isotopic studies.Ann NY Acad Sci. 1965; 124: 499-542Crossref PubMed Scopus (239) Google Scholar, 5Shulman N.R. Weinrach R.S. Libre E.P. Andrews H.L. Shannon J.A. The role of the reticuloendothelial system in the pathogenesis of idiopathic thrombocytopenic purpura.Trans Assoc Am Physicians. 1965; 78: 374-90PubMed Google Scholar]; (ii) the presence of platelet-associated and plasma autoantibodies in patients with immune thrombocytopenia and their absence in control subjects and non-immune thrombocytopenias, as demonstrated in this study; (iii) variation of autoantibody levels with the patient's response to therapy (decrease or disappearance with treatments which affect antibody production such as splenectomy, cyclophosphamide, combination chemotherapy but no change with treatments which influence phagocytosis such as danazol, vincristine) [20Fujisawa K. Tani P. Piro L. McMillan R. The effect of therapy on platelet-associated autoantibody in chronic immune thrombocytopenic purpura.Blood. 1993; 81: 2872-7Crossref PubMed Google Scholar]; and (iv) their ability, in some cases, to activate complement and induce platelet phagocytosis in vitro and presumably in vivo[21McMillan R. Martin M. Fixation of the third component of complement (C3) to platelets in vitro by antiplatelet antibody for patients with ITP.Br J Haematol. 1981; 47: 251-6Crossref PubMed Scopus (28) Google Scholar, 22Tsubakio T. Tani P. Curd J.G. McMillan R. Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura.Br J Haematol. 1986; 63: 293-300Crossref PubMed Scopus (60) Google Scholar, 23McMillan R. Longmire R.L. Tavassoli M. Armstrong S. Yelenosky R. In vitro platelet phagocytosis by ITP splenic leukocytes in idiopathic thrombocytopenic purpura.N Engl J Med. 1974; 290: 249-51Crossref PubMed Scopus (95) Google Scholar]. The present study is the largest study to date which prospectively evaluates the use of an antigen-specific autoantibody assay. The study was designed to, not only evaluate the findings in patients with chronic ITP but also to determine its usefulness in other illnesses where immune thrombocytopenia may be a complication. These results show that the immunobead assay has a high degree of specificity (>90%) in the diagnosis of chronic ITP. However, the assay has a sensitivity of only 55.4%. These results are similar to those of two previously published prospective studies which showed specificities of 78 and 92% and sensitivities of 49 and 66% [14Warner M.N. Moore J.C. Warkentin T.E. Santos A.V. Kelton J.G. A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.Br J Haematol. 1999; 104: 442-7Crossref PubMed Scopus (152) Google Scholar, 15Brighton T.A. Evans S. Castaldi P.A. Chesterman C.N. Chong B.H. Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias.Blood. 1996; 88: 194-201Crossref PubMed Google Scholar]. The results of these three prospective studies show that antigen-specific assays, when positive, have a high degree of diagnostic accuracy both in classic ITP and, as shown here, in patients with immune thrombocytopenia associated with other diagnoses. On the other hand, we must ask why only 49–66% of the patients satisfying the ITP diagnositic requirements of the ASH Practice Guidelines are positive using these antigen-specific assays. Perhaps it is because the Practice Guideline recommendations are not rigorous enough to make an accurate diagnosis of ITP, since they are based on the exclusion of other causes, or because the assays are deficient. Probably both possibilities are true to some degree. The following should be considered: Some patients who satisfy the Practice Guidelines' criteria may not have ITP This possibility is suggested by the rather large number of negative assays in the ‘ITP-pre-mild’ patient subgroup. Until the advent of automatic cell counters, most patients in this group would not have been diagnosed since they are asymptomatic from their mild thrombocytopenia, and require no therapy. Since only one-third of this subgroup have positive assays, do they all have ITP or some other cause of mild thrombocytopenia such as unrecognized drug reactions (many are older and taking multiple drugs), early myelodysplasia or other, as yet, unidentified causes? Similarly, in the ‘ITP-pre-severe’ subgroup, several patients had a subsequent spontaneous recovery either with or without therapy over a period of weeks to months and some had a history of a preceding viral illness prior to their thrombocytopenia. Do they have ITP, a post-viral syndrome similar to childhood acute ITP, or some other diagnosis? Other platelet surface antigens or Ig subtypes are involved Since laboratories using the immunobead or MAIPA assays routinely assay only for autoantibody against GPIIb/IIIa and GPIb/IX (and in some cases GPIa/IIa and GPIV), perhaps there are autoantibodies against other important antigens which are being missed. Some patients may also have only IgM or IgA autoantibodies. ITP therapy has suppressed autoantibody production prior to assay It has been well shown that autoantibody levels may become normal upon treatment with corticosteroids and other immunosuppressive agents [20Fujisawa K. Tani P. Piro L. McMillan R. The effect of therapy on platelet-associated autoantibody in chronic immune thrombocytopenic purpura.Blood. 1993; 81: 2872-7Crossref PubMed Google Scholar, 24Berchtold P. Wenger M. Autoantibodies against platelet glycoproteins in autoimmune thrombocytopenic purpura: their clinical significance and response to treatment.Blood. 1993; 81: 1246-50Crossref PubMed Google Scholar]. If assays are performed after therapy is introduced (which is often the case), false negative results may occur. There may be technical or sensitivity problems with the assays It is possible that low-levels of autoantibody, which are undetected by the present assays, may be sufficient to result in platelet destruction. It is also possible that the autoantibodies compete for the same antigenic site as the monoclonal antibodies used in the assays (steric hindrance). This latter possibility seems unlikely since we have tested several negative ITP samples, in the past, using multiple monoclonal anti-GPIIb/IIIa antibodies with different epitope specificities and obtained the same results. Obviously, there are still many unanswered questions. Antigen-specific assays have a definite place in the diagnosis of chronic ITP. A positive autoantibody assay is a strong diagnostic indicator for the presence of immune thrombocytopenia with a high degree of specificity and anti-GPIIb/IIIa or anti-GPIb/IX autoantibodies are present in a high percentage of patients with severe disease. A negative assay does not rule out the presence of ITP. It is possible that the ASH Practice Guidelines are too permissive in the diagnosis of chronic ITP and other criteria are needed to aid in diagnostic accuracy." @default.
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• W2000745319 title "Prospective evaluation of the immunobead assay for the diagnosis of adult chronic immune thrombocytopenic purpura (ITP)" @default.
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