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The aim. Mechanisms underlying the development of neonatal alloimmune thrombocytopenia (NAIT) in in Russia have been studied. Materials and methods. Genetic polymorphisms of human platelet alloantigens (HPA) -1, -2, -3, -4, -5, and -15 were evaluated in 27 families having the newborns with NAIT. NAIT was diagnosed according to the following criteria: (1) newborn with thrombocytopenia; (2) mother with no thrombocytopenia and no increase of platelet associated IgG, (3) presence of antibodies reacting with paternal platelets in maternal plasma / serum. HPA genotyping revealed incompatibilities in 23 out of 27 tested families. In these 23 families HPA-1 conflicts were detected in 16 ones (70%). In 8 cases mothers were homozygous carriers of rare HPA-1b allele and in another 8 cases - of HPA-1a allele which cased incompatibilities with fetal HPA-1a and HPA-1b respectively. In 5 out of 23 families (22%) there were incompatibilities with fetal HPA-15 (HPA-15a, n=2 and HPA-15b, n=3), in 1 family - with HPA-5b (4%), and in 1 family - with HPA-3b (4%) alloantigens. In conclusion the main causes of NAIT in Russia were HPA-1a and -1b conflicts and HPA-15 conflicts were the second frequent ones.

We report the case of a 14-year-old boy affected by ulcerative colitis (UC) and acute thrombocytopenic purpura (ITP) with simultaneous onset. UC diagnosis was based on symptoms, endoscopy and histology findings. ITP diagnosis was based on the normal bone marrow megakaryocyte count, the presence of platelet associated IgG and the absence of splenomegaly. Medical treatments including high doses of steroids, intravenous immunoglobulins were ineffective on ITP course, while UC course was mild for several months after the onset. When colonic inflammation became untractable not responsive at steroids and immunosuppressive agents, colectomy resolved both pathologies.

Thrombocytopenia is common in patients with myelodysplastic syndromes (MDS) and immune destruction of platelets could be an important factor for its occurrence. We prospectively analyzed platelet-associated IgG (PAIgG) through platelet immunofluorescence test (PIFT), mean platelet volume (MPV), platelet size deviation width (PDW) and glycocalicin index (GCI) of 54 patients with MDS, classified according to the International Prognostic Scoring System (IPSS). Thrombocytopenia (platelet count

4 children with idiopathic thrombocytopenic purpura were treated with high dose i.v. IgG. Platelet associated immunoproteins were determined with an Elisa technique. Platelet counts increased in all patients, while simultaneously platelet associated IgG, IgA, IgM, C3 and C4 decreased. Serum antiplatelet antibody increased during treatment suggesting that administration of high doses of IgG interferes with the binding of antiplatelet antibody.

In a prospective study, the hypothesis of whether the quantitative determination of platelet-associated IgG (PAIgG) in patients with chronic autoimmune thrombocytopenic purpura (ATP) can predict the efficacy of splenectomy, was investigated. PAIgG levels were repeatedly determined in 16 patients with definite ATP pre- and postsplenectomy, and related to platelet counts and platelet mean life span. It was found that patients with an immediate remission after splenectomy tended to have lower PAIgG levels (less than 6%) than failures, but this difference was not statistically significant. We conclude that PAIgG is of limited value for the prediction of the efficacy of splenectomy in ATP.

An immunoradiometric assay was developed for determining platelet-associated IgG (PAIgG) both on intact and solubilized platelets. In 20 healthy subjects PAIgG was 0.28 +/- 0.20 ng/10(6) platelets and 2.2 +/- 1.1 ng/10(6) platelets on intact and on solubilized platelets, respectively. 13 children with acute ITP all had increased concentrations of PAIgG, but no correlation was found between the severity of thrombocytopenia and PAIgG concentrations, either on intact or on solubilized platelets. Neither was there any correlation of the increase in PAIgG between intact and solubilized preparations. 3 out of 4 children who received high-dose i.v. gammaglobulin showed a concomitant normalization of the platelet count and of the PAIgG concentration both on intact and lyzed platelets. The remaining child did not respond to gammaglobulin and continued to have abnormal PAIgG.

12 patients with severe chronic idiopathic thrombocytopenic purpura (ITP) were treated with i.v. polyvalent intact immunoglobulin (0.14-0.4 g/kg body weight for 5 d) because of various bleeding episodes or prior to surgery. In 9 patients a significant rise in platelet counts was noted, starting on d 2 and reaching its maximum between d 4 and 11. Thereafter, platelet counts decreased slowly, approaching pre-treatment values after 20 d in most cases. Response to immunoglobulin was reproducible, when infusions were repeated. Response to high-dose immunoglobulin was independent of age of patients and duration of disease. Platelet-associated IgG decreased in all patients tested. A pepsin-treated immunoglobulin preparation was ineffective in 3 patients.

Platelet-associated IgG (PAIgG) was studied by a quantitative platelet radioactive anti-IgG test (PRAT) in 298 patients. At the time of investigation, 171 patients were thrombocytopenic (platelet count100 X 10(9)/l), 127 had normal platelet counts. Patients fell into the following disease categories: Idiopathic thrombocytopenic purpura (ITP) (N = 81), possible ITP (19), acute ITP (9), systemic lupus erythematosus (22), autoimmune haemolytic anaemia of warm-type (18), systemic blood disease (65), liver diseases (35), other (49). A significant elevation of PAIgG was found in all disease categories. There was a significant correlation between PAIgG and the reciprocal values of platelet counts for most disease groups. No relationship was discernible between PAIgG and hypergammaglobulinaemic states (serum IgG1.8 g/l). Platelet survival studies (N = 30) revealed that normal and increased values of PAIgG were associated with normal or shortened platelet mean life span. It is concluded that an elevated PAIgG is only one of several factors involved in the development of immunologically medicated thrombocytopenia.

Blood was collected from nine normal volunteers into each of four different anticoagulants and in order to simulate transport conditions, was stored at room temperature for 96 h. The platelet associated IgG (PAIgG) was determined from aliquots thereof over this period. Blood in EDTA gave slightly higher initial values (day 0) than in other anticoagulants. PAIgG levels increased at different rates in all anticoagulants thereafter. In contrast to recent reports which suggested that falsely elevated levels were likely to be seen after storage in EDTA, we found little difference in these values in blood in different anticoagulants at 72 h and all PAIgG measurements remained within our quoted normal range at this time. After 96 h storage however, one of nine (11%) in CPD-A, two of nine (22%) in EDTA, two of nine (22%) in Na-citrate and six of nine (66%) in EDTA paraformaldehyde gave falsely elevated results. The possible mechanisms of these changes are discussed. The assay system employed in this study measures 'total' platelet IgG in a platelet extract in contrast to some other assays which quantify surface platelet IgG alone and it is possible that this difference in technique is responsible for the relatively smaller percentage changes after storage than reported by others. All anticoagulants tested in this study proved satisfactory both for handling and for the measurement of PAIgG at 48 h, with the proviso that normal ranges should be established for each.

Summary A double antibody radioimmunoassay has been developed for the detection of platelet associated IgG (PAIgG). The assay employs conventional radioassay technology and, as such, readily may be adopted by laboratories familiar with radioassay techniques. The assay is sensitive to 0.6 ng IgG and can be undertaken on 106-107 platelets, or fewer in the case of thrombocytopenia with raised PAIgG. The assay can be completed within 2 working days. Normal PAIgG levels in EDTA blood were 0.8 to 5.7 μg IgG/109 platelets. However, normal levels depended upon the anticoagulant used for blood collection and were on average five-fold higher for blood taken into EDTA than for blood taken into ACD; normal range 0.1–1.2 μg/IgG 109 platelets. PAIgG was assayed on blood taken simultaneously into ACD and EDTA from 15 patients with chronic idiopathic thrombocytopenia (ITP). PAIgG was raised in 14 out of 15 EDTA samples, but in only 11 of 15 ACD samples. EDTA was therefore regarded as the anticoagulant of choice. A total of 20 of 23 (87%) patients with chronic ITP had PAIgG values above the normal range. The PAIgG in chronic ITP was weakly related to platelet count, weakly and inversely related to platelet volume, but was independent of serum IgG concentration.

Summary A modification to the enzymoimmunoassay for measuring plateletassociated IgG (PAIgG) reported by Nel & Stevens (1980) is described. This modification makes use of microtitration plates which greatly facilitate the practical aspects of the procedure.

A report is presented of a patient with neonatal erythema infectiosum who developed petechiae, transient thrombocytopenia and transient cardiac failure due to transplacental transmission of human parvovirus B19 (HPV B19) infection. It is suggested that the thrombocytopenia was caused by platelet-associated IgG produced by the patient, and that the cardiac failure may have been caused by direct entry of HPV B19 into the cardiac tissue.

Backgrounds/Aims: Relationships of mean platelet volume (MPV) and thrombopoietin (TPO) with platelet turnover assessed by glycocalicin measurement were evaluated in thrombocytopenic patients. Methods: MPV, glycocalicin and platelet-associated IgG (PA-IgG) were measured in 107 patients with idiopathic thrombocytopenic purpura (ITP) and 19 patients with hypoproductive thrombocytopenia (HPT; aplastic anemia or leukemia), and TPO was measured in 53 ITP and 12 HPT patients. All the included ITP patients had PA-IgG ≥300% and glycocalicin ≥50% of control values, and HPT patients had PA-IgG Results and Conclusions: MPV was higher in ITP than in HPT patients: 9.56 ± 1.69 and 7.59 ± 0.90 fl (p < 0.001). In the ITP group a direct correlation was detected between MPV and glycocalicin (r = 0.344, p < 0.001). This interaction was essentially expressed in patients with normal/increased glycocalicin (≥100% of control; r = 0.470, p < 0.001, n = 64). TPO was greatly enhanced in HPT in comparison with ITP patients (958 ± 659 and 11 ± 27 pg/ml, p < 0.001). In the ITP group a reverse correlation was detected between TPO and glycocalicin (r = -0.373, p = 0.006). © 2014 S. Karger AG, Basel

Hematologic abnormalities, including thrombocytopenia, are seen in HIV infection. We have previously reported elevated platelet-associated IgG (PAIgG) in thrombocytopenia in children associated with human immunodeficiency virus (HIV). In this study we prospectively monitored 40 HIV-infected infants and children to determine the significance of elevated PAIgG levels as they relate to thrombocytopenia. We also examined platelet eluates for the presence of HIV antibody and antigen. Of 16 patients with thrombocytopenia, 15 (93.7%) had elevated PAIgG. Of 24 patients with normal platelet counts, 21 (87.5%) had elevated PAIgG. On follow-up, none of the children with normal platelet counts and elevated PAIgG levels developed thrombocytopenia. Examination of the platelet eluates was negative for HIV antibody or P24 antigen. Although the sensitivity of an elevated PAIgG level in predicting thrombocytopenia is 93%, its specificity is only 13%. Elevated PAIgG levels are therefore not causally related to the development of thrombocytopenia in children.

Tranilast (Rizaben®)-induced thrombocytopenia occurring in a 17-year-old man was reported. After withdrawal of the drug, he recovered within a week with oral prednisolone administration. Serological examination revealed no anti-platelet antibody, but platelet-associated IgG (PAIgG) was found. After incubation of peripheral blood of the patient with the drug in vitro, the level of PAIgG was significantly increased. These findings suggest the presence of a drug-dependent anti-platelet IgG in the patient's serum. This is the first report of immune thrombocytopenia caused by Tranilast. Our method for detecting drug-dependent platelet antibody in vitro is safe and useful for diagnosing drug-induced thrombocytopenia.

A 24-year-old Japanese male having ulcerative colitis and idiopathic thrombocytopenic purpura is described. When the symptoms of ulcerative colitis were manifest during his clinical course, the platelet life-span was shortened and the platelet associated IgG level was increased. Thus, causal association between the two diseases may be suggested.

A modified, sensitive, solid-phase assay for platelet-associated IgG is reported. Direct comparisons were made between a 125I monoclonal radioimmunoassay (RIA) and the polyclonal antibody latex particle assay. In 209 simultaneous comparisons with the RIA, the sensitivity of the latex test was 91 percent, specificity was 100 percent, and overall efficiency was 97 percent. Commencing with platelet-rich plasma, the direct latex particle test for platelet-bound IgG requires 45 minutes; 90 minutes are required to crossmatch one patient with 12 donors. The advantages of the latex assay are absence of radioactivity, stability of reagents, economy, speed, specificity, and sensitivity.

A number of methods have been developed to measure platelet-associated IgG (PAIgG). The antiglobulin consumption assay directly quantitates IgG on the platelet and is sensitive and specific. A fluorescent anti- IgG assay has recently been described and has the advantage of simplicity. We compared the results of these two PAIgG assays in immune and nonimmune thrombocytopenia and nonthrombocytopenic controls. The antiglobulin consumption assay was negative in 61 of 62 and the fluorescent negative in 54 of 62 assays in nonthrombocytopenic controls, and they were negative in 11 of 13 and 8 of 13 assays, respectively, in nonimmune thrombocytopenic patients. The antiglobulin consumption assay was positive in 54 of 58 and the fluorescent positive in 24 of 58 assays of patients with immune thrombocytopenia (ITP and SLE). The overall sensitivity and specificity of the antiglobulin consumption assay was 94% and 95% and of the fluorescent assay was 44% and 82%.

Elevated levels of platelet-associated lgG (PAIgG) found by direct assay in patients with immune thrombocytopenia, may in part reflect passive absorption of lgG to megathrombocytes present in increased proportions in thrombocytopenia caused by increased platelet destruction. Using a direct assay for PAIgG based on the inhibition of lysis of IgG-coated sheep red blood cells, we correlated these levels with two platelet volume parameters, median platelet volume and percentage megathrombocytes, using a Coulter Channelyzer. The mean PAIgG was 4.7 fg lgG/platelet in 30 controls, 6.6 fg lgG/platelet in 15 patients with nonimmune thrombocytopenia, and 41.6 fg lgG/platelet in 32 patients with immune thrombocytopenia. Only in the Immune thrombocytopenlc patients was there a correlation between platelet count and PAIgG (r = 0.66, p< 0.001); however, there was no correlation between the PAIgG and either of the two parameters of platelet volume. Furthermore, all 4 patients with nonlmmune thrombocytopenia whohad an Increase percentage megathrombocytes (20%-36%) had normal levels of PAIgG. These results indicate that the elevated levels of PAIgG in patients with immune thrombocytopenia is specific for this disorder and not due to passive absorption of lgG to megathrombocytes, whIch are present in increased proportions in these conditions.

Summary. Most assays that measure platelet associated IgG (PAIgG) relate the IgG associated with the test platelets to the platelet count. This could lead to a systematic error if platelet fragments were present in the washed platelet sample but not counted. To address this issue, we studied platelets from patients with idiopathic thrombocytopenic purpura (ITP), thrombocytopenia complicating cardiopulmonary bypass, and laboratory synthesized platelet fragments (freezethawed) using electron microscopy. Scanning electron microscopic (SEM) examination of the platelet samples demonstrated appreciable numbers of fragments only in the freeze-thawed specimens. Yet, ‘fragments’ could be seen in all specimens using transmission electron microscopy (TEM). Most of these ‘fragments’ proved to be artefacts: we found that the ratio of ‘fragments’ to intact platelets observed in the TEM specimens was similar to the estimated ratio of ‘fragments’ to platelets that would have been generated had the specimens been sectioned at 90° to the plane of the actual section. Platelets labelled with the membrane label 125I-iodosulfanilic acid were fragmented by repetitive freeze-thawing. Measurement of pellet radioactivity following washing indicated that the fragments were lost during the washing procedure. These studies indicate that elevated levels of platelet associated IgG in ITP do not represent artefacts due to contamination of the test platelets by platelet fragments.

We present measurements of total platelet-associated immunoglobulin G following platelet lysis (PAIgG tot) and surface-restricted IgG (PAIgG ext) on intact, gel-filtered platelets from 36 normal human donors and 9 patients with human immunodeficiency virus (HIV) infection (CDC stages IIb-IV). For this purpose, an indirect micro ELI-SA technique was developed involving competition between PAIgG and solid-phase absorbed IgG for fixation of conjugated anti-human IgG antibody. In normal donors, the mean values of PAIgG tot was 8.3 +/- 7.4 (mean +/- 2 SD) and of PAIgG ext 4.2 +/- 4.4 fg/platelet. In HIV-infected subjects, PAIgG tot was 37.2 +/- 62.8 and PAIgG ext 17.1 +/- 23 fg/platelet. In healthy subjects the comparison of individual levels of PAIgG tot and PAIgG ext revealed a significant correlation (r:0.763; p: 0.003). These results are compared to those which have been reported in the literature. In addition, the major immunopathological mechanisms considered responsible for immune-mediated idiopathic thrombocytopenic purpura are discussed.

Anti-platelet antibody has been generally recognized as an important etiologic factor in idiopathic thrombocytopenic purpura (ITP) and the detection of platelet associated IgG (PAIgG) is essential to establish the diagnosis and also useful to predict the clinical course. Several methods to detect PAIgG have been proposed, most of which, however, require complicated manipulation and are not practical for clinical use. This paper describes a simple and rapid method to detect PAIgG.This method was devised, utilizing the ability of staphylococcal protein A (SpA) to bind specifically to human IgG. An adhesive percentage of sample platelets to sheep red blood cells coated with SpA was calculated by enumeration of sample platelet before and after treatment. It takes only several hours to complete the assay from the drawing of blood. In a total of 37 cases including 15 cases of ITP, PAIgG was studied, using this method and Fab, anti-Fab assay of Luiken. Binding Index (an adhesive percentage) was 9.3 ± 3.8% in mormal subjects and 26.5 ± 10% in ITP patients, which differnce was statistically significant (pThese results suggest that this new method is reliable to detect PAIgG in addition to its simplicity and rapidity and practical in clinical use.

An ELISA test for platelet associated immunoglobulins was performed at disease onset in 36 consecutive patients with acute idiopathic thrombocytopenic purpura. All case histories were later reviewed. Platelet associated IgM was elevated in 91% of the patients, correlating with platelet associated IgG. It was found that neither platelet associated IgG nor IgM levels could predict duration of thrombocytopenia or the transition to chronic disease observed in three patients. High-dose intravenous IgG given to six patients had a significantly better antithrombocytopenic effect than oral prednisolone in eleven patients. Platelet associated immunoglobulin levels were reduced after IgG treatment, suggesting a possible interference with the binding of antibodies or immune complexes to platelets. IgG, IgM, platelets, prednisolone, thrombocytopenia

Evidence suggests that as platelets age in the circulation they become progressively smaller and less dense through the loss of protein. The smallest, least dense platelets have a significantly shortened survival, but the mechanism of clearance of these platelets is not known. To evaluate whether the binding of IgG could play a role in the clearance of senescent platelets, we measured platelet size, total protein, and platelet-associated IgG on subpopulations of platelets isolated from 6 healthy individuals using a discontinuous iso-osmotic arabinogalactan (stractan) gradient. There was a close correlation between density, size, and total protein content (r greater than 0.9) for all platelet fractions. There was also a relationship between the amount of platelet-associated IgG (PAIgG), total protein, and platelet size (r greater than 0.9) for the first 3 progressively less dense platelet factions. However, the fourth platelet fraction containing the smallest, least dense, and on current evidence, oldest platelets had very elevated amounts of IgG. This amount was approximately 10 times higher than the mean platelet IgG for the same individual and was similar to the amount of PAIgG found on platelets from patients with immune thrombocytopenia. A progressive increase in the ratio of PAIgG measured after platelet solubilization to PAIgG measured on intact platelets was also noted for the first three populations, indirectly suggesting that platelets clear IgG from their surface during aging. Increased binding of IgG to senescent platelets may mediate their destruction.

The measurement of platelet-associated IgG (PAIgG) is a potentially useful diagnostic test for idiopathic thrombocytopenia purpura (ITP). However, the restricted application of PAIgG measurements to thrombocytopenic populations primarily comprised of ITP patients will artificially enhance its diagnosis specificity. For this reason, we performed a prospective study in which the results of a sensitive technique for quantitating PAIgG were related to the cause of the thrombocytopenia. Over a 1-yr period, clinicians were invited to submit patient blood samples encompassing as wide a spectrum of thrombocytopenic disorders as possible for PAIgG measurements. The physician was then contacted and requested to indicate the likeliest cause for the thrombocytopenia. The PAIgG was elevated in only 24 of 254 samples obtained from nonthrombocytopenic patients. In contrast, 134 (79%) of the 169 thrombocytopenic patients had elevated PAIgG results, and the increased levels were apparent in all diagnostic categories. The sensitivity of the PAIgG test for clinically diagnosed idiopathic thrombocytopenic purpura was 91% and the specificity was 27%. The positive predictive value for a raised PAIgG as a diagnostic test for ITP in a thrombocytopenic patient was only 46%, while the negative predictive value was 82%. This study indicates that the presence of increased PAIgG provides little additional information in the diagnosis of ITP. This study also suggests that immune mechanisms may mediate many more thrombocytopenic disorders than have been previously thought likely.

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

A quantitative immunofluorescence platelet-associated immunoglobulins (PA-IgG) assay was used to detect alloimmunity to platelets in 8/12 multitransfused patients and to perform platelet crossmatching in the 8 alloimmunized patients. The correct separation of multitransfused patients into alloimmune and nonalloimmune groups was substantiated with chromium-51-labeled platelet survival studies. For 5 alloimmunized patients, compatible and incompatible donor platelets were demonstrated by PA-IgG crossmatching and were confirmed by platelet survival studies. With the other 3 alloimmunized patients, only studies with 5 of these incompatible donor platelets showed markedly reduced survival times on 4 occasions. PA-Igg compatible donor platelets survived 3.5- 8.7 days, while PA-IgG incompatible platelets showed survival times of 0.1–2.4 days. Overall, PA-IgG testing correctly indicated survival results on 15/17 occasions (88%), whereas platelet aggregation, serotonin release, and lymphocytotoxicity testing showed correct predictions for only 41%-59% of the survival studies. PA-IgG testing predicted which times, thus indication patients with platelet-specific alloantibodies. the PA-IgG assay provides a sensitive method to detect platelet alloantibodies and to perform platelet crossmatching, which can complement HLA typing in the selection of donor platelets for alloimmunized patients.

A method for the measurement of immunoglobulin G associated with gel- filtered platelets is described and finding in 70 control subjects and 37 patients with immune thrombocytopenic purpura (ITP) are reported. Control platelet-associated IgG (PAIgG) levels (nanograms IgG per 10(9) platelets) averaged (+/-SD) 1231+/-424; samples studied after 24 and 48 hr remained within the control range. PAIgG values of 19 adult and 12 childhood patients with chronic ITP averaged 4711+/-3025 and 4923+/- 3955, respectively, and differed significantly from controls (p less than 0.001). There was an inverse correlation between PAIgG values and the chronic ITP patient's platelet count. Six patients with childhood acute ITP had PAIgG levels ranging from 5588 to 56,250 and appeared to represent a different statistical population from those with chronic ITP. In chronic ITP patients responding to splenectomy, there was an immediate normalization of PAIgG levels; however, a certain percentage of patients studied several months after splenectomy evidenced elevated PAIgG levels in association with normal platelet counts. These data showed that the direct measurement of platelet associated antibody is a useful technique in the diagnosis and follow-up of patients with chronic ITP. Preliminary studies in patients with acute ITP have suggested that this method may be useful in differentiating acute and chronic childhood ITP.

An electroimmunoassay was applied to the quantitation of platelet- associated IgG (PAIgG). Protein solubilized by Triton X100 from washed platelets was electrophoresed at pH 5.0 in agarose gels containing carbamoylated rabbit anti-human IgG (pI approximately equal to 5.0). Because the rabbit antibody is immobilized under these conditions, while PAIgG is freely mobile, rocket precipitates were produced, the heights of which were directly proportional to the amount of antigen (PAIgG) present. By this method, PAIgG for normal individuals was found to be 4.3 +/- 1.7 fg/platelet (mean +/- 2 SD; n = 35). Increased PAIgG levels (direct assay) were found in 27 of 29 patients with a diagnosis of clinically active idiopathic thrombocytopenic purpura (ITP), ranging from 10.5 to 101.5 fg/platelet. Moderately elevated PAIgG was found in 8 of 10 patients in an early stage of recovery from ITP (range 7.5–9.5 fg/platelet) and in 3 of 6 patients with apparent nonimmune thrombocytopenia (range 14.5–24.0 fg/platelet). Electroimmunoassay for PAIgG can be performed on patients with platelet counts as low as 2000/microliters, yields results in less than 24 hr, is highly reproducible, and appears to provide a useful tool for the evaluation of patients with immunologically mediated thrombocytopenia.

The mechanism of thrombocytopenia, a frequent complication of septicemia, is obscure, but indirect evidence suggests that the immune system may be involved. To investigate this possibility, we quantitated platelet-associated IgG on platelets obtained from 44 patients during 46 episodes of septicemia. Thrombocytopenia occurred in 21 of 46 episodes (46 per cent). Platelet-associated IgG was elevated in eight of 11 episodes of gram-negative septicemia and thrombocytopenia (47.3 +/- 11.7 fg of IgG per platelet [mean +/- S.E.]) and in one of 20 patients with gram-negative septicemia and normal platelet counts (5.9 +/- 1.1) (P less than 0.001). Elevated levels occurred in eight of 10 patients with gram-positive septicemia and thrombocytopenia (55.3 +/- 14.7 fg of IgG per platelet) and in none of 11 patients with gram-positive septicemia and normal platelet counts (5.6 +/- 1.7) (P less than 0.001). Serial testing during the thrombocytopenia and recovery showed an inverse relation between the platelet count and platelet-associated IgG. Thrombocytopenia in some patients with septicemia may be related to the binding of IgG to platelets.

Levels of platelet-associated IgG (PA-IgG) were studied in 72 patients with Hodgkin–s (HD) and non-Hodgkin–s lymphoma (NHL). Thirty-nine percent of patients with HD and 20% of patients with NHL had elevated PA-IgG levels. There was a positive correlation between disease activity and the presence of PA-IgG in HD and NHL. In patients with HD, PA-IgG strongly correlated with extent of disease and may serve as a marker of disease activity. PA-IgG may have facilitated platelet destruction in 5 of 11 thrombocytopenic patients with HD and increased PA-IgG and in 2 patients with HD and increased PA-IgG who developed severe thrombocytopenia when treated with chemotherapy.

For an analysis of relationship between platelet sialic acid and platelet associated IgG (PA-IgG), PA-IgG and sialic acid level in both platelet and plasma were measured. PA-IgG was measured by immunfluorescent assay. This immunofluorescent assay is based on the treatment of platelets with neuraminidase. Sialic acid levels of platelets and plasma were measured by the thiobarbituric acid method.In patients with thrombocytopenia (ITP, acute leukemic patients who received platelet concentrates from random donors), the PA-IgG positive rates (% PA-IgG) in neuraminidase-treated and non-treated platelets were 37.0±4.18% and 7.9±2.33%, respectively. The results indicated that % PA-IgG was significantly elevated by neuraminidase treatment of platelets.Platelets from normal controls had a mean sialic acid of 0.72+0.30mg/108 platelets. Platelets from the patients had a mean sialic acid of 0.86+0.44mg/108 platelets. Sialic acid levels of platelets were significantly elevated in the patients. Sialic acid levels of plasma were elevated in the patients, but there was no correlation between platelet and plasma sialic acid in the patients.These findings suggest that the higher quality of sialic acid on the patient platelets might inhibit the detection of the PA-IgG.

Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In- labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.

We investigated the effects of gravitational force and three different plastic formulations of the storage bags on the quality of stored platelets by measuring the changes in platelet-associated immunoglobulin G (IgG) and two antigenic markers of the third component of human complement (C3), C3d and C3c. Pooled platelets were stored in parallel at microgravity (MG) and on the ground (1 g) using three different plastic polymers: (1) polyvinylchloride (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP); (2) PVC plasticized with trioctyl trimellitate (TOTM), and (3) unplasticized polyolefin (PO). The IgG and C3 were quantified by an automated antiglobulin consumption test in freeze/thaw disrupted platelets (total IgG or C3). The baseline values for platelet associated IgG and C3, measured after 2.5 days of storage at 1 g just prior to the launch, fell within the normal range. Following an additional 6.5 day storage, platelets stored at MG had accumulated significantly less C3d than those stored at 1 g, suggesting that MG storage was beneficial. Specific plastic formulations also exerted a significant effect on the accumulation of these immunoproteins, the effect being particularly pronounced at MG. The smallest increases of IgG and C3 were seen in platelets stored in TOTM and the largest in those stored in DEHP. It is possible that further studies at MG would permit a clear characterization of the effects of other independent storage variables.

Platelet-associated IgG (PAIgG) levels were measured in 60 children with ITP (46-chronic, 14-acute) using Fab-anti Fab radioimmunoassay method described by McMillan et al. In some patients platelet binding IgG in serum (PBIgG) was also determined at the same time. Patients with ITP had significantly greater PAIgG levels than 30 normal subjects and 13 non-immune thrombocytopenic controls. Elevated PAIgG values did not correlate with parameters of platelet size (mean platelet volume; MPV and percentage of large platelet; PLP) and so these data indicated that high levels of PAIgG in ITP were not due to nonspecific adhesion of serum IgG to megathrombocytes usually increased in this disorder, but due to specific immunological reaction. PBIgG IgG values were also elevated in patients with pretreated chronic ITP, but high levels remained even after successful splenectomy. Furthermore, serial determination of PAIgG values were obtained in some patients with chronic ITP who underwent splenectomy and with acute ITP who achieved spontaneous remission. PAIgG returned to normal levels when thrombocytopenia disappeared. PAIgG seems to be the most reproducible indicator which reflects transition of the clinical picture in this disorder.

A quantitative ELISA assay for the measurement of in vivo bound platelet-associated IgG (PAIgG) using intact patient platelets is presented. The assay requires quantitation and standardization of the number of platelets bound to microtiter plate wells and an absorbance curve using quantitated IgG standards. Platelet-bound IgG was measured using an F(ab')2 peroxidase labeled anti-human IgG and o-phenylenediamine dihydrochloride (OPD) as the substrate. Using this assay, PAIgG for normal individuals was 2.8 +/- 1.6 fg/platelet (mean +/- 1 SD; n = 30). Increased levels were found in 28 of 30 patients with clinical autoimmune thrombocytopenia (ATP) with a range of 7.0-80 fg/platelet. Normal PAIgG levels were found in 26 of 30 patients with nonimmune thrombocytopenia. In the sample population studied, the PAIgG assay showed a sensitivity of 93%, specificity of 90%, a positive predictive value of 0.90, and a negative predictive value of 0.93. The procedure is highly reproducible (CV = 6.8%) and useful in evaluating patients with suspected immune mediated thrombocytopenia.

A variety of assays are available for measuring platelet-associated IgG (PAIgG) but the complexity of these assays increases the potential for technical errors. These errors are difficult to detect and, if possible, known positive and negative control platelets should be included with each run. However, patient platelets with elevated levels of IgG are seldom available. This report describes a method for producing positive control platelets that can be labeled with differing amounts of IgG. Normal serum IgG (Cohn fraction II) was incubated with washed 2 percent formalin-fixed platelets for 60 minutes at 37 degrees C. The amount of IgG on the platelets was proportional to the concentration of soluble IgG and the number of incubations. Normal platelet IgG levels were 1.2 +/- 0.1 fg per platelet (mean +/- SEM, n = 34) and positive control platelets had 4.6 +/- 0.2 (n = 12) or 8.4 +/- 0.4 (n = 7). There was no change in the level of PAIgG when stored at 4 degrees C for 1 week, although there was a 28 percent loss in recoverable platelets. When mixed 1:1 with 60 percent glycerol and stored at -70 degrees C, the level of PAIgG was stable for 3 months, with less than 12 percent platelet loss on recovery (n = 12). These positive control platelets have proved useful for monitoring assay performance.

Platelet-associated IgG (PAIgG) was measured by a simple radial immunodiffusion technique using washed solubilized platelets and commercially available immunoplates. Subjects with normal platelet counts had PAIgG levels of 1.5--7.0 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels ranging from 5.7 to 70.5 fg/platelet. All patients with recurrent ITP and 85% of patients with acute ITP had elevated PAIgg. Elevated PAIgG was also found in 17% of patients with recovered ITP, 40% of patients with SLE and thrombocytopenia, 57% of patients with thrombocytopenia occurring during the course of septicemia, and 100% of patients with IgG myeloma in whom the serum IgG level was clearly elevated, regardless of the platelet count. The results are similar to reports that used more complex techniques.

Twenty-two patients with idiopathic thrombocytopenic purpura were treated with high-dose intravenous immunoglobulin infusions using an intact intravenous immunoglobulin preparation and a product designed for intramuscular administration. Seventeen patients (77%) responded favourably (rise in platelet count to 100 × 109/1 or above). Children responded better than the adults, and the acute cases better than the chronic. Splenectomy and the presence of detectable platelet antibody had no effect on the response rate. Both products induced identical responses.

Summary. An enzyme linked immunoassay incorporating antihuman globulin coupled with alkaline phosphatase has been developed to measure platelet associated IgG (PAIgG). Using a method in which platelet IgG is extracted into the fluid phase after appropriate procedures, we were able to bind the ‘solubilized’ PAIgG to commercially obtained antihuman IgG (AHG) which had previously been coated onto polystyrene. The amount of PAIgG thus bound was subsequently measured by the addition of the enzyme reagent using p-nitro phenyl phosphate as substrate. With this technique platelets from normal donors were found to have 2·6–17·4 ng/106 platelets (mean ± 2 SD). These values are higher than those obtained when assay systems using intact platelets are employed. Platelets from patients with immune thrombocytopenia had PAIgG values of 8·2–98·0 ng/106 platelets. In a few patients with disorders other than autoimmune thrombocytopenia (AITP) increased levels of PAIgG were also demonstrated. The assumption that increased PAIgG always represents platelet autoantibody may not be valid. The relevance of PAIgG as a parameter in the diagnosis and clinical management of patients with AITP is discussed.

High-dose gammaglobulin therapy for patients with idiopathic thrombocytopenic purpura (ITP), introduced by Imbach et al., was applied to 5 adults with chronic refractory ITP to investigate the mechanism of the increase in platelet counts. In 4 of the 5 cases, transient increase in platelet count was observed. Platelet-associated IgG was decreased in 3 cases, increased in 1 case and unchanged in 1 case after treatment. In 4 cases having a variety of autoantibodies, the antibody titers decreased after treatment. No significant changes in antiplatelet antibody titers in serum, or in circulating immune complexes were observed during or after these treatments. No side effects were noted in any of the cases. These results indicate that the treatment is suitable for the treatment of patients prior to surgery and of patients at high risk of intense bleeding and death. It can be reasonably assumed that the increased platelet count is mainly due to a defective removal of antibody-coated platelets.