Quantification of platelet-associated IgG with competitive solid-phase enzyme immunoassay

In order to measure platelet-associated IgG (PAIgG), we devised a solid-phase enzyme immunoassay employing a competitive binding of peroxidase-conjugated anti-IgG antiserum between platelets and polystyrene tubes coated with IgG. The amounts of peroxidase bound to the tubes were measured in a spectrophotometer by an enzymatic reaction. This method is highly sensitive, reproducible and can be carried out more simply. the PAIgG values of normal controls averaged 21.6 +/- 6.6 (SD) ng/10(7) platelets. 27 (93%) of 29 patients with idiopathic thrombocytopenic purpura (ITP), who had a platelet count of less than 15 X 10(4)/microliter, had PAIgG values greater than those of controls by 2 SD and averaged 205.5 +/- 323 ng. There was a significant inverse correlation between platelet count and PAIgG value of ITP patients. the PAIgG values of patients with aplastic anemia were within normal range.     

The use of intravenous immunoglobulin (endobulin) in patients with immune thrombocytopenia

Six adults were treated for chronic immune thrombocytopenia (ITP) with high dose intravenous immunoglobulin (ENDOBULIN). Five patients were evaluable. In four patients the platelet associated IgG (PAIgG) levels were increased. All four responded to i.v. Ig and two subsequently underwent splenectomy. One patient had normal PAIgG and had no response to i.v. Ig. Endobulin is useful in the treatment of chronic ITP and is followed in some patients by a prolonged response. High levels of PAIgG may predict a favourable outcome.     

A double antibody radioassay for the detection of platelet associated IgG

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.     

Direct quantitation of platelet-associated IgG by electroimmunoassay

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.     

Quantitation of platelet-associated IgG by radial immunodiffusion

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.     

Effect of paraformaldehyde on platelet size and on measurement of surface IgG

In many platelet assays, as in measurement of platelet-adherent IgG (PAIgG), platelets are fixed in paraformaldehyde (PFA). To better clarify the effect of PFA on platelet size and on PAIgG measurement we compared PAIgG levels in a series of 40 samples, with or without PFA fixing. We used an ELISA which was set up on unfixed platelets and gave excellent results in terms of linearity (r = 0.923), precision (mean CV = 5%) and correlation with a platelet suspension immunofluorescence test. We found PAIgG values in unfixed platelets were about 10-fold higher than in PFA-fixed (0.411 +/- 0.172 fg/platelet vs. 0.035 +/- 0.019 fg/platelet). This discrepancy could be a consequence of the smaller mean platelet volume (MPV) of washed platelets when fixed in PFA (8.0 +/- 0.8 fl as compared to 10.1 +/- 1.07). This effect of PFA could decrease the amount of binding sites for IgG exposed on the platelet membrane and hence explain the significantly lower PAIgG values observed in fixed platelets. The PAIgG measurements on unfixed platelets from 200 healthy subjects displayed a Gaussian distribution with a mean +/- SD of 0.32 +/- 0.13 fg/platelet, i.e., 1200 +/- 500 molecules/platelet.     

Comparison of the measurement of surface or total platelet-associated IgG in the diagnosis of immune thrombocytopenia

Platelet-associated IgG (PAIgG) can be measured on intact platelets or following platelet lysis. Measurement of PAIgG following platelet lysis may provide different or additional information compared to PAIgG measured on intact platelets. The PAIgG of lysed platelets represents "total" PAIgG, ie, IgG on the surface of platelets plus any IgG that was inside the platelet. To investigate the clinical relevance of the two types of PAIgG assay we performed a prospective study on washed platelets collected from 47 patients with idiopathic thrombocytopenic purpura (ITP). The PAIgG was measured on intact and lysed platelets using an immunoradiometric assay. Platelet-associated IgG was 2-3 times higher when measured on lysed platelets from healthy controls or patients with ITP compared to PAIgG measured on the same intact platelets. The higher level of PAIgG observed following platelet lysis was not due to the reactions not achieving equilibrium. Using lysed platelets, PAIgG was elevated on 29 of 47 samples from different ITP patients and elevated in 31 samples when measured on intact platelets. The PAIgG is invariably higher when measured following platelet lysis compared measurements made on intact platelets. Neither technique offers a diagnostic advantage over the other.     

A rapid quantitation of platelet-associated IgG by nephelometry

Platelet-associated IgG (PAIgG) was measured by a simple rapid nephelometric technique using washed solubilized platelets and commercially available, prestandardized reagents. Normal subjects with normal platelet counts had PAIgG levels of 2.1-6.7 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels of 7.2-43.3 fg/platelet. Ninety percent of ITP patients had values exceeding 2 SD units of the mean of normal subjects. Elevated values were also found in 17% of patients with recovered ITP, patients with SLE with and without thrombocytopenia, patients with thrombocytopenia occurring during septicemia, and patients with IGg myeloma. Results can be obtained within several hours of receipt of blood specimen, and are similar to the reports that used more complex techniques.     

The use of electron microscopy in the investigation of the ultrastructural morphology of immune thrombocytopenic purpura platelets

Ultrastructural studies have proven useful for the accurate identification and classification of certain lymphoid and hematopoietic disorders; however, the value of electron microscopy in the diagnosis and clinical management of platelet pathology is less well defined. Electron microscopy has been used to evaluate inherited platelet disorders. In these disorders, certain platelet structural defects can be characteristic. Recently, we investigated the ultrastructural morphology and immunogold localization of IgG in platelets from patients with idiopathic thrombocytopenic purpura (ITP). The accelerated platelet destruction of ITP is mediated by antiplatelet autoantibodies directed against platelet surface glycoproteins and by the functional capacity of the reticuloendothelial system. The basic dysregulation that occurs in these patients remains unexplained. Traditionally, laboratory investigation of ITP has focused on the development of serologic assays to measure the autoantibodies. As an alternative investigative approach, determination of the immunomorphologic characteristics of platelet-associated IgG (PAIgG) in ITP platelets may prove useful in the diagnosis or clinical management of patients with ITP. Our results showed that the ultrastructural morphology of ITP platelets is similar to that observed for normal platelets. No structural abnormalities are observed in ITP platelets. Immunogold labeling of IgG within alpha-granules of ITP platelets is significantly higher than that of normal platelets. Additionally, in some ITP platelets, immunogold labeling is also observed on the platelet surface and within channels of the open-cannalicular system. In comparison, immunogold labeling of these structures in normal platelets, is rare, or absent. In conclusion, electron microscopic studies should contribute to furthering our understanding of this common autoimmune disorder, and may provide possible biological explanations for the increased levels of PAIgG in platelets from patients with ITP.     

Platelet-associated IgG in childhood idiopathic thrombocytopenic purpura: measurements on intact and solubilized platelets and after gammaglobulin treatment

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.     

Usefulness of measurement of reticulated platelets for diagnosis of idiopathic thrombocytopenic purpura.

Reticulated platelets (RP) and large platelets (LP) were measured by an automated hematology analyzer (modified R-2000) in 287 healthy volunteers and 131 patients with thrombocytopenia or thrombocytosis. RP was significantly higher in patients with idiopathic thrombocytopenic purpura (ITP), especially in active phase, while RP was markedly lower in patients with essential thrombocytosis (ET) or chronic myelocytic leukemia (CML). LP was significantly higher in patients with ITP, especially in active phase, while LP was markedly lower in patients with aplastic anemia (AA), ET, or CML. In ITP, RP and LP were significantly higher in patients positive for anti-glycoprotein (Gp) IIb/IIIa antibody. RP and LP were poorly correlated with platelet-associated IgG (PAIgG). RP and LP were poorly correlated with plasma thrombopoietin levels, and negatively correlated with platelet count. These results show that RP reflects the pathology of thrombocytopenic disorders, and that measurement of RP is useful for the differential diagnosis and analysis of platelet kinetics.     

Serum leptin levels in patients with idiopathic thrombocytopenic purpura

OBJECTIVES: The purpose of this study was to evaluate serum leptin levels in idiopathic thrombocytopenic purpura (ITP), in order to determine the influence of leptin on the pathogenesis of ITP. SUBJECTS AND METHODS: Forty-six untreated patients with chronic ITP were compared with 40 healthy people of similar age, sex and body mass index (BMI). Serum leptin levels (ng/mL) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that the mean serum leptin levels in patients with ITP (22.11 +/- 15.84 ng/mL) were significantly (P < 0.001) higher than that in healthy control volunteers (5.44 +/- 4.84 ng/mL). Furthermore, serum leptin levels in patients with ITP were inversely related (r = -0.86, P < 0.001) to the platelet counts and positively related to the platelet-associated IgG (PAIgG) levels (r = 0.7, P < 0.001). The levels of PAIgG and platelet counts were significantly different between leptin-positive (level greater than mean +/- 2 SD control value) and leptin-negative patients. CONCLUSION: These findings suggest that leptin might play an important role in the pathogenesis of ITP.     

T lymphocyte subsets and platelet-associated IgG in idiopathic thrombocytopenic purpura: effect of splenectomy

The modifications of cell-mediated immunity in idiopathic thrombocytopenic purpura (ITP) were investigated using the technique of Moretta et al. [J. exp. Med. 151: 969-974, 1980] to study T lymphocyte subsets in 35 ITP subjects at various clinical stages. In all these patients, platelet-associated IgG (PAIgG) were measured by a complement lysis inhibition technique. Untreated ITP patients did not show significant modifications of T lymphocyte subsets. Patients with thrombocytopenia not responsive to corticoids or splenectomy and increased PAIgG levels showed significant reduction of Fc-gamma-bearing T lymphocytes (T-gamma). Patients 'cured' by splenectomy (12 out of 17) showed a normal PAIgG level, a marked increase of T-gamma cells and a reduction of T-mu cells; patients 'not cured' by splenectomy showed an increased PAIgG level and a reduction of T-gamma cells. An increase of PAIgG levels and a reduction of T-gamma cells were found in 2 splenectomized patients after relapse. An association between chronic nonresponsive ITP and decrease of T-gamma cells was established.     

The amount of platelet-bound albumin parallels the amount of IgG on washed platelets from patients with immune thrombocytopenia

The biologic relevance of the increased platelet-associated IgG (PAIgG) on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is unclear. Platelets from ITP patients are often larger than normal, and it is possible that the increased IgG is not specific but passively related to platelet size. The measurement of platelet-bound albumin could provide information concerning the specificity of the platelet-bound IgG, since albumin, like IgG, is a plasma protein, but unlike IgG, is not an active participant in immunologic reactions. Albumin is also a normal constituent of platelet membrane, and increased platelet albumin could indicate an increased platelet mass. Platelet-bound albumin, IgG, and total platelet protein were measured on both intact and disrupted platelets from healthy individuals (n = 25) and patients with ITP (n = 21). Platelet IgG and albumin were measured in an immunoradiometric assay using intact antisera and F(ab')2 fragments prepared from the same antisera. There was no relationship between platelet-bound IgG or albumin, and platelet size measured by either platelet protein or platelet volume, (r less than 0.3 for all interactions). In contrast, there was a significant correlation between platelet-bound albumin and platelet-bound IgG (r = 0.7, n = 21, p less than 0.001). Those patients with elevated platelet PAIgG also had elevated platelet albumin, and this relationship was irrespective of the total platelet protein content or mean platelet volume. It is possible that the increased platelet-bound IgG in ITP reflects an increase in platelet surface area or contaminating platelet fragments that are not manifested as an increase in platelet volume or total platelet protein. Alternatively, a platelet membrane abnormality may occur in ITP that results in the uptake of significant amounts of plasma proteins. Either possibility implies that not all of the IgG on platelets from patients with ITP is pathologic IgG.     

Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) for the measurement of circulating platelet antibody and platelet-associated IgG (PAIgG) is described. The test is done in microtiter plates and rapidly provides quantitative and highly reproducible results. Alloantibodies from 28 of 30 multiple transfused patients and isoantibodies from 3 of 4 patients with immune thrombocytopenic purpura (ITP) were detected. PAIgG was elevated in all 4 patients with ITP, and HLA and platelet-specific antigens were reliably detected using HLA typing sera and anti-PIA1 antibody, respectively. Platelets preserved wither by dessication in the wells of the microtiter plates or in liquid suspension in saline at 4 degrees C gave results comparable to values using fresh platelets. Storage periods ranged from 30 days for dessicated platelets to more than 1 yr for platelets stored in suspension. The ability to utilize preserved platelets may allow relatively convenient screening of large numbers of potential platelet donors for alloimmunized patients.     

Effect of paraformaldehyde on platelet size and on measurement of surface IgG

In many platelet assays, as in measurement of platelet-adherent IgG (PAIgG), platelets are fixed in paraformaldehyde (PFA). To better clarify the effect of PFA on platelet size and on PAIgG measurement we compared PAIgG levels in a series of 40 samples, with or without PFA fixing. We used an ELISA which was set up on unfixed platelets and gave excellent results in terms of linearity ( r = 0.923), precision (mean CV = 5%) and correlation with a platelet suspension immunofluorescence test. We found PAIgG values in unfixed platelets were about 10-fold higer than in PFA-fixed (0.411 - 0.172 fg/platelet vs. 0.035 - 0.019 fg/platelet). This discrepancy could be a consequence of the smaller mean platelet volume (MPV) of washed platelets when fixed in PFA (8.0 - 0.8 fl as compared to 10.1 - 1.07). This effect of PFA could decrease the amount of binding sites for IgG exposed on the platelet membrane and hence explain the significantly lower PAIgG values observed in fixed platelets. The PAIgG measurements on unfixed platelets from 200 healthy subjects displayed a Gaussian distribution with a mean - SD of 0.32 - 0.13 fg/platelet, i.e., 1200 - 500 molecules/platelet.     

Platelet-Associated IgG in Acute and Chronic Hepatic Diseases

In order to investigate the role of an immune mechanism in the pathogenesis of thrombocytopenia in hepatic patients, we measured platelet associated IgG (PAIgG) in 84 patients with various hepatic diseases. Increased PAIgG levels were found in 84% of patients with chronic active hepatitis (mean 21.32 ± 8.45 fg/platelet) and in 75% of those with cirrhosis with hepatitis (mean 17.3 ± 11.2 fg/platelet). In these patients there was no significant correlation between PAIgG and platelet count. Increased PAIgG amounts were also observed in some patients with inactive cirrhosis (18%). Normal PAIgG values were found in all patients with acute viral hepatitis and chronic persistent hepatitis. An immunologic mechanism may play a role in the pathogenesis of thrombocytopenia in hepatic patients. Furthermore, measurement of PAIgG may have a great usefulness in the differential diagnosis of chronic hepatic diseases.     

Flow cytometry detection of platelets autoantibodies in children with idiopathic thrombocytopenic purpura

Abstract  

Immune thrombocytopenic purpura is an acquired disorder, in which accelerated platelet consumption is due to platelet autoantibodies. The aim of this study was to investigate the clinical value of platelet autoantibodies assay in children with ITP and to evaluate flow cytometry in the detection of platelet autoantibodies in comparison with monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay. We measured platelet autoantibodies by flow cytometry and MAIPA in 18 children with ITP (6 acute, 7 chronic and 5 in remission), in addition to 5 healthy children with matched age and sex as a control group. Significant elevation of platelet-associated immunoglobulin G (PAIgG), PAIgM and PAIgA was demonstrated in children with acute ITP compared to controls and children with chronic ITP (P < 0.05). There was significant elevation of PAIgG and PAIgM in children with acute ITP compared to children with ITP in remission (P < 0.05). There was significant negative correlation between platelet count and PAIgG levels in ITP children (r = −0.717; P = 0.001). Flow cytometry found PAIgG in 94.4% of ITP children. MAIPA has detected platelet specific IgG autoantibodies in 83.3% of ITP children. ROC analysis revealed sensitivity of 94%, specificity of 57% with overall accuracy of 83% for detection of PAIgG by flow cytometry compared to MAIPA.     

Disparities in estimates of IgG bound to normal platelets

Estimates of the number of IgG molecules bound to normal platelets have ranged from several hundred to several tens of thousands. The lower estimates were generated from direct binding assays and stoichiometric assumptions. The higher values derive from competitive binding assays, in which platelet-associated IgG (PAIgG) is calculated from a standard curve using soluble IgG standards. Using a kinetic-ELISA (enzyme-linked immunosorbent assay) antiglobulin assay, we measured normal platelet IgG to be 21,200 +/- 9,400 molecules per platelet when a competitive assay and soluble IgG standards were used. Direct measurement of bound antiglobulin by kinetic-ELISA and stoichiometric assumptions yielded a measurement of 259 +/- 117 IgG molecules per platelet. Soluble IgG and PAIgG are not comparable in their ability to bind anti-IgG. Disparities in estimates of normal PAIgG are probably due to methodological differences. The estimate most likely to be correct is several hundred IgG or less per normal platelet.     

The value of platelet-associated IgG in predicting the efficacy of splenectomy in autoimmune thrombocytopenia

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.