Quantitative Antiglobulin Consumption Research Articles

Complement (C3) binding to platelets in autoimmune thrombocytopenia.

In view of conflicting reports on the occurrence of complement binding to platelets in idiopathic autoimmune thrombocytopenic purpura (AITP) we performed measurements of platelet bound C3 in patients with AITP who had elevated levels of platelet bound IgG. Using a quantitative antiglobulin consumption technique 38 out of 42 patients were found to have fixed abnormally large amounts of C3 to their platelets, and a significant positive correlation between the amounts of platelet bound IgG and C3 was shown to exist. In additional experiments antibody eluates were prepared from AITP platelets and were shown to cause the fixation of C3 to normal donor platelets in vitro. Taken together these findings strongly suggest that the C3 binding in AITP is specifically related to the disease process.

An autoanalyzer test for the quantitation of platelet-associated IgG.

A new quantitative antiglobulin consumption (QAC) test for the measurement of platelet-associated IgG is described. In this test washed platelets are incubated with anti-IgG at a final dilution of 1:2 million. The unneutralized fraction of anti-IgG remaining in solution is then measured with an Autoanalyzer and soluble IgG is used for calibration. The dose-response curves depicting the percent neutralization of anti-IgG by platelets and by soluble IgG were compared in detail and found to be nearly identical, indicating that platelet-associated IgG can be accurately quantitated by this method. The mean IgG values were 2,287 molecules/platelet for normal adults and 38,112 molecules/platelet for ITP patients. The Autoanalyzer QAC test is a sensitive and reproducible assay for the quantitation of platelet-associated IgG.

An Autoanalyzer Test for the Quantitation of Platelet-Associated IgG

A new quantitative antiglobulin consumption (QAC) test for the measurement of platelet-associated IgG is described. In this test washed platelets are incubated with anti-IgG at a final dilution of 1:2 million. The unneutralized fraction of anti-IgG remaining in solution is then measured with an Autoanalyzer and soluble IgG is used for calibration. The dose-response curves depicting the percent neutralization of anti-IgG by platelets and by soluble IgG were compared in detail and found to be nearly identical, indicating that platelet-associated IgG can be accurately quantitated by this method. The mean IgG values were 2,287 molecules/platelet for normal adults and 38,112 molecules/platelet for ITP patients. The Autoanalyzer QAC test is a sensitive and reproducible assay for the quantitation of platelet-associated IgG.

Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to the platelet. The results of the indirect QAC test were 10-fold greater. The discrepancy appears to be due to the fact that there is a difference between the IgG-anti-IgG interaction when IgG is bound to a platelet and when it is in solution or bound to plastic nonspecifically or specifically. This difference results in a falsely high value for platelet-bound IgG when fluid-phase or plastic-bound IgG is used to calibrate the antiglobulin consumption test.

The measurement of platelet-associated IgG by a microplate quantitative antiglobulin consumption assay using automated through-the-well spectrophotometry.

A microplate modification of the quantitative antiglobulin consumption assay (QACA) has been developed for the measurement of platelet-associated IgG (PAIgG). This method employs an automated through-the-well absorbance spectrophotometer (microplate reader) to directly measure the hemolysis of target indicator cells (IgG-coated sheep erythrocytes) in an assay performed entirely in a 96-well microtiter plate. PAIgG measured by this method in 54 thrombocytopenic patients and 17 controls correlated highly (r = 0.94) with results obtained by the standard tube assay procedure. By substantially simplifying the performance of the QACA and increasing its sensitivity by nearly 1 order of magnitude, this method permits the rapid quantitative determination of PAIgG on platelets obtained from as little as 1 to 4 ml of whole blood.

Platelet antibody levels do not correlate with response to therapy in idiopathic thrombocytopenic purpura.

Platelet bound antibody was measured at presentation in 67 patients with idiopathic thrombocytopenic purpura by means of a quantitative antiglobulin consumption technique. The amounts of antibody present on the platelets of patients who failed steroid therapy were similar to those present on the platelets of patients who achieved a remission of their illness. It was also not possible on the basis of these data to identify those patients whose illness would remit after splenectomy. It is concluded that the pretreatment platelet antibody level does not assist in predicting the responses to treatment.

A quantitative determination for the detection of immunoglobulin (IgG) on the surface of platelets

A sensitive method for the quantitation of IgG on platelets had not been demonstrated until 1975, when Dixon, Rosse, and Ebbert described a quantitative antiglobulin consumption test useful in detecting platelet associated IgG (N Engl J Med 292:230, 1975). A modification of that technique has rendered the assay reproducible and removed the need for daily repetition of a standard IgG titration curve for quantitation. This modification utilizes 1-ethyl-3-3(dimethylaminopropyl)carbodiimide HCl (ECDI) (Sigma, E-7750), in place of chromic chloride, as a coupling agent for attaching IgG (Miles 64–145) to sheep cells (SRC), used as indicator cells. The ECDI consistently couples IgG to SRC and does not subject the SRC to sporadic spontaneous lysis, as does chromic chloride. This modification permits the detection of IgG on platelets (Direct Test), or in sera (Indirect Test) by incubation of a washed platelet pool with sera in vitro, and testing as in the Direct Test. Normal values of 0.01–1.56 and 0.14–1.6 femtograms (F) per platelet have been obtained for the Direct and Indirect Tests, respectively. In six cases of suspected ITP, values ranged 12.0–221.0 F and 2.9–37.6 F for the Direct and Indirect Tests, respectively. In conclusion, in disease states or other abnormal situations, quantities of IgG can be detected that are not usually present on the platelets of normal subjects.

Human IgG antigranulocyte antibodies: comparison of detection by quantitative antiglobulin consumption and by binding of 125I staph protein A.

The amount of IgG in the serum of patients with suspected immune neutropenia that binds to normal paraformaldehyde-fixed human granulocytes was measured simultaneously by a quantitative antiglobulin consumption assay and by binding of 125I-staphylococcal protein A (SPA). There was a significant linear relationship between the results of these two assays for the sera of 42 different patients. However, SPA binding appeared more sensitive than the quantitative antiglobulin assay for determining IgG antigranulocyte antibodies in serum. In a patient with Felty's syndrome who underwent splenectomy, the results of both assays on sequential serum samples correlated with clinical improvement. Thus, SPA binding appears to be a sensitive and reliable technique for measuring antigranulocyte antibodies, and there is a close correlation between antibody measured by antiglobulin consumption and those detected by SPA binding.

Influence of the amount of platelet-bound IgG on platelet survival and site of sequestration in autoimmune thrombocytopenia

Seventeen patients with idiopathic autoimmune thrombocytopenic purpura were studied in order to establish whether the amount of platelet-bound antibody influenced the rate and site at which these platelets were destroyed. Platelet-bound antibody was measured by a quantitative antiglobulin consumption technique, and platelet survival and sites of sequestration were determined by the use of 51Cr-labeled platelets and surface radioactivity measurements. A correlation significant at the 5% level was observed between the amount of platelet-bound antibody and the rate of platelet destruction. A highly significant correlation (p < 0.001) existed between antiplatelet antibody and spleen: liver surface radioactivity ratios, demonstrating that greater amounts of antibody bound to platelets result increasingly in their destruction within the liver.

Influence of the amount of platelet-bound IgG on platelet survival and site of sequestration in autoimmune thrombocytopenia

Seventeen patients with idiopathic autoimmune thrombocytopenic purpura were studied in order to establish whether the amount of platelet-bound antibody influenced the rate and site at which these platelets were destroyed. Platelet-bound antibody was measured by a quantitative antiglobulin consumption technique, and platelet survival and sites of sequestration were determined by the use of 51Cr-labeled platelets and surface radioactivity measurements. A correlation significant at the 5% level was observed between the amount of platelet-bound antibody and the rate of platelet destruction. A highly significant correlation (p less than 0.001) existed between antiplatelet antibody and spleen: liver surface radioactivity ratios, demonstrating that greater amounts of antibody bound to platelets result increasingly in their destruction within the liver.

Felty's syndrome: granulocyte-bound immunoglobulin G and splenectomy.

A quantitative antiglobulin consumption technique was used to measure immunoglobulin G (IgG) present on human granulocytes. Granulocytes from 50 normal subjects had less than 20 X 10(-14) g IgG per cell. Patients with granulocytopenia due to bone-marrow failure, patients with IgG multiple myeloma, patients with splenomegaly, and patients with rheumatoid arthritis without granulocytopenia had granulocyte-bound IgG within the range of normal. Four patients with rheumatoid arthritis, splenomegaly, and severe granulocytopenia (Felty's syndrome) had granulocyte-bound IgG between 30 and 220 X 10(-14) g IgG per cell. One of these patients underwent splenectomy, after which his granulocyte-bound IgG fell to normal. Seven additional patients with Felty's syndrome who had previously undergone splenectomy had normal levels of granulocyte-bound IgG. Thus quantitation of granulocyte-bound IgG appears to be useful in defining patients with immunologically mediated granulocytopenia. Studies of patients with Felty's syndrome who have undergone splenectomy suggest that the spleen may produce this neutrophile-bound immunoglobulin.