Elevated platelet-associated IgG in the thrombocytopenia of septicemia.

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.     

Clinical significance of platelet-associated immunoglobulins in narcotic addicts with human immunodeficiency virus infection

Increased amounts of platelet-associated immunoglobulin G (PAIgG) have been reported in human immunodeficiency virus (HIV)-infected patients with thrombocytopenia. We have prospectively investigated PAIgG in 21 asymptomatic (group A; CDC stages IIA-IIB) and 9 symptomatic (group B: CDC stages IIIB-IV) HIV-infected narcotic addicts. In both groups only two subjects presented with a decreased platelet count. By competitive ELISA, we found a 1.8- and 2.3-fold greater total PAIgG (PAIgGtot) as measured on platelet lysates in group A and B, respectively; surface-bound IgG (PAIgGsurf) as measured on intact platelets was 2.5- and 3.0-fold greater in groups A and B, respectively, as compared to 36 controls. The ratio between PAIgGtot and PAIgGsurf was lower in HIV-infected addicts when compared to controls (P less than 0.05). This indicates that platelets from HIV-infected subjects not only have increased surface and internal pools of PAIgG, but also present with a distribution between these two pools that differs from that of normal platelets. In addition, levels of circulating immune complexes (CIC) were abnormally raised in 17/21 (81%) subjects of group A in 6/9 (66%) subjects of group B.     

A simplified micro ELISA procedure for the measurement of platelet-associated IgG (PAIgG)

A simplified micro ELISA procedure to measure platelet-associated IgG is described. The platelet-bound IgG first is extracted into the fluid phase by solubilizing washed platelets in 0.1% triton X-100. The solubilized IgG in the extract and IgG standards are incubated in microtiter wells previously coated with antihuman IgG. The IgG in the standards and extract bind to the solid phase antihuman IgG. The bound IgG then is measured by the addition of peroxidase labeled antihuman IgG and appropriate substrate. With this method platelets from normal controls were found to have 1.7 +/- 0.6 fg IgG/platelet (mean +/- SD). Platelets from patients with ATP had values that were two to seven times the control values. The relative advantage of this technic is discussed.     

Correlation between increased platelet-associated IgG and thrombocytopenia in secondary dengue virus infections

Although the public health impact of dengue is increasing rapidly, the mechanism of thrombocytopenia in this disease remains unknown. To elucidate this mechanism, the relationship between platelet-associated IgG (PAIgG) and platelet count in 53 patients in the acute phase of secondary dengue virus infection was investigated in a prospective-hospital-based study. A significant inverse correlation between the two parameters was found in these patients, while no correlation was observed in healthy volunteers. The low baseline platelet counts during the acute phase in 12 patients with secondary dengue virus infection significantly increased during the convalescent phase, while the increased PAIgG levels during the acute phase in these patients significantly decreased during the convalescent phase. Anti-platelet IgG autoantibody was detected rarely in the plasma of 53 patients with secondary dengue infection. The involvement of anti-dengue virus IgG was also shown in platelets from all of 8 patients in the acute phase of secondary dengue virus infection. These findings suggest that PAIgG formation involving anti-dengue virus IgG plays a pivotal role in the induction of transient thrombocytopenia during the acute phase of secondary dengue virus infection.     

Association of increased platelet-associated immunoglobulins with thrombocytopenia and the severity of disease in secondary dengue virus infections

Severe thrombocytopenia and increased vascular permeability are two major characteristics of dengue haemorrhagic fever (DHF). To develop a better understanding of the roles of platelet-associated IgG (PAIgG) and IgM (PAIgM) in inducing thrombocytopenia and its severity of disease in patients with secondary dengue virus infection, the relationship between the PAIgG or PAIgM levels and disease severity as well as thrombocytopenia was examined in 78 patients with acute phase secondary infection in a prospective hospital-based study. The decrease in platelet count during the acute phase recovered significantly during the convalescent phase. In contrast, the increased levels of PAIgG or PAIgM that occurred during the acute phase of these patients decreased significantly during the convalescent phase. An inverse correlation between platelet count and PAIgG or PAIgM levels was found in these patients. Anti-dengue virus IgG and IgM activity was found in platelet eluates from 10 patients in an acute phase of secondary infection. Increased levels of PAIgG or PAIgM were significantly higher in DHF than those in dengue fever (DF). An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with a high specificity. Our present data suggest that platelet-associated immunoglobulins involving antidengue virus activity play a pivotal role in the induction of thrombocytopenia and the severity of the disease in secondary dengue virus infections.     

ELISA for platelet-associated IgG, IgM, and C3, and their clinical application

The platelet-associated IgG (PAIgG) has been reported to elevate in the patients with idiopathic thrombocytopenic purpura (ITP) and other autoimmune diseases. However, low PAIgG levels have been often recognized in thrombocytopenia. We speculated about the increasing of other platelet-associated proteins in those patients, and tried to determine platelet-associated IgM (PAIgM) and platelet-associated C3 (PAC3) using a high sensitive competitive micro-ELISA as well as PAIgG. Our results showed the specific elevation of PAIgM and PAC3 in thrombocytopenia as well as the PAIgG level (p less than 0.01). Further, the weak correlations among these levels were found (PAIgG/PAIgM: n = 7, correlation coefficient (r) = 0.55, PAIgG/PAC3: n = 73, r = 0.61, PAIgM/PAC3: n = 56, r = 0.39). We discussed on the possibility that the PAIgM and PAC3 also could be an indicator for the platelet injury and may cause the short platelet life span resulting thrombocytopenia as well as PAIgG.     

Circulating immune complexes and platelet IgG in various diseases

Correlation between platelet associated IgG (PAIgG), platelet count, and plasma polyethylene glycol (PEG) precipitable IgG immune complex (IC) like material was tested in normal subjects and patients with immune thrombocytopenia (ITP), systemic lupus erythematosus (SLE) and various types of liver disease. Elevated IC were observed in 27% and 22% of ITP and recovered ITP, respectively. A significant inverse correlation between platelet count and PAIgG was demonstrable in the ITP group. A significant direct correlation between platelet count and IC was found only in SLE patients. Impaired reticuloendothelial cell (RE) Fc receptor function in SLE patients is suggested as a possible explanation for the data. If receptor function was normal in SLE patients, lower IC levels and lower platelet counts would have been expected.     

Thrombocytopenia associated with the induction of neonatal tolerance to alloantigens: immunopathogenic mechanisms

BALB/c mice rendered tolerant to alloantigens by neonatal injection of semi-allogeneic (C57BL/6 x BALB/c)F1 spleen cells develop a thrombocytopenia in association with an autoimmune lupus-like syndrome. The possible mechanisms involved in the thrombocytopenia were investigated. The development of thrombocytopenia was first detected at 3 weeks of age coinciding with the start of the other autoimmune manifestations and was always related to a state of tolerance and B cell chimerism. There was a significant increase of megakaryocytes in bone marrow and spleens from thrombocytopenic tolerant mice and radiolabeled platelets from these mice were more rapidly eliminated from the bloodstream than normal platelets when injected into normal recipients. A significant correlation between the spleen weight and the decrease of the circulating platelets was observed, although some mice with severe thrombocytopenia had only a moderate spleen enlargement. Thrombocytopenia significantly correlates with the levels of platelet-associated IgG (PAIgG) but not with anti-single-stranded DNA antibodies or circulating immune complexes. Platelets from mice with high levels of PAIgG had a shorter life-span when injected into normal mice than those from mice with low or normal PAIgG. The possibility that PAIgG are partially due to antibodies reacting specifically with platelet membrane components was analyzed. First, F(ab')2 Ig fragments from tolerant mice were shown to bind to normal platelets, in contrast to F(ab')2 Ig fragments from normal mice. Second, some monoclonal antibodies produced by hybridomas derived from tolerant mice reacted in vitro with platelets and induced a transient thrombocytopenia after i.v. injection into normal mice. These data suggest that the thrombocytopenia observed in tolerant mice is the result of a peripheral hyperdestruction of platelets associated with (a) hypersplenism, (b) nonspecific fixation of immunoglobulins, probably as immune complexes and (c) with autoantibodies reacting specifically with platelets. It may represent an interesting model for human chronic idiopathic thrombocytopenia.     

Microplate enzyme immuno-assay for detection of platelet antibodies

A simplified and sensitive enzyme immuno-assay employing microtiter plates as the solid phase carrier for detection of circulating platelet antibodies and bound antiplatelet IgG has been described. The assay was done using fresh as well as frozen platelets and commercially available peroxidase labeled anti-human IgG. The sensitivity of the enzyme immuno-assay was found similar or superior to that of the platelet suspension immunofluorescence test and superior to the lymphocyte cytotoxicity test and the platelet complement fixation test. The use of platelets frozen in the wells of the microtiter plates (stored for up to 17 months at -20 degrees C without loss of antigenicity) facilitates the performance of the test. In addition the small volumes of sera and platelets needed in using microtiter plates makes the assay particularly suitable in testing platelets from thrombocytopenic patients. In one of 31 sera from normal Zwa-negative donors a weak anti-Zwa (P1A1) was found only detectable by enzyme immuno-assay. Alloantibodies were found in 14 of 23 patients suffering from non-haemolytic transfusion reactions. All of three patients with post-transfusion purpura had anti-Zwa antibodies in serum. Anti-Zwa antibodies were demonstrated in the sera from all of 9 Zwa-negative mothers, who had given birth to children with alloimmune neonatal thrombocytopenia. In 8 of 12 Zwa-positive mothers alloantibodies of other specificities were found in the serum. In one case the antibody was a known anti-Baka. Six of 7 patients with autoimmune thrombocytopenic purpura had values of platelet-bound IgG exceeding the normal range and circulating platelet antibodies were seen in 4 patients.     

Treatment of septic thrombocytopenia with immune globulin

Thrombocytopenia frequently complicates systemic infection and results from multiple possible mechanisms. We and others have demonstrated that platelet-associated IgG (PAIgG) levels are elevated in the majority of patients with septic thrombocytopenia. Corticosteroids may be undesirable as a treatment for thrombocytopenia for patients with severe infection because of their potential for suppressing the immune response. We hypothesized that septic thrombocytopenia is, in most cases, an immune disorder analogous to idiopathic thrombocytopenic purpura (ITP) which might respond to intravenous gamma-globulin as a treatment for increasing the platelet count in this disorder. Intravenous immune globulin (IVIG), 400 mg/kg daily for 3 days, was administered in a randomized double-blind placebo-controlled trial. Twenty-nine patients who developed thrombocytopenia during a documented, septic episode were studied. Patients with disseminated intravascular coagulation (DIC), hypersplenism, or drugs known to cause thrombocytopenia were excluded. Elevated PAIgG levels were documented in 52% of evaluable patients. Mean platelet counts in the IVIG group rose from 43K at study entry to 178K (411% rise) by Day 9. In the placebo group platelets rose from 51K to 125K (261% rise;P = 0.02). Seventy-seven percent of the IVIG group had a minimum peak rise of 35K, vs 56% of the placebo group. Three patients in the placebo group had a serious bleeding episode, vs one in the IVIG group. The use of IVIG to treat septic thrombocytopenia not associated with DIC leads to a more rapid, more sustained, and greater increase in platelet count than placebo. Its use is recommended in the septic patient who is bleeding or is likely to need invasive or surgical procedures.     

Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection.

BACKGROUND. Isolated thrombocytopenia accompanied by increased amounts of platelet-associated antibody is a common manifestation of human immunodeficiency virus (HIV) infection, and the thrombocytopenia often improves with zidovudine. It is not clear whether the mechanism of HIV-related thrombocytopenia primarily involves autoimmune destruction of platelets or reduced platelet production by megakaryocytes. METHODS. We studied the survival of 111In-labeled autologous platelets and performed platelet imaging in 24 men with isolated HIV-related thrombocytopenia (16 who received no treatment and 8 who received zidovudine). We also studied 20 HIV-infected men with normal platelet counts (10 who received no treatment and 10 who received zidovudine) and studied 12 healthy seronegative men as controls. RESULTS. Mean (+/- SD) platelet survival was significantly decreased in both the untreated and the zidovudine-treated patients with HIV-related thrombocytopenia (to 92 +/- 33 and 129 +/- 44 hours, respectively; both P < 0.001), as compared with the normal controls (198 +/- 15 hours). Mean platelet survival was also significantly decreased in the HIV-infected patients with normal platelet counts (untreated, 162 +/- 23 hours, P < 0.01; zidovudine-treated, 166 +/- 35 hours, P < 0.05). Imaging studies, however, revealed no evidence of increased clearance of autologous platelets in the liver or spleen in any of these groups. Mean platelet production was significantly depressed in the untreated patients with thrombocytopenia (23,000 +/- 11,000 platelets per cubic millimeter per day, P < 0.001) as compared with the healthy controls (45,000 +/- 6,000 per cubic millimeter per day). Mean platelet production was significantly increased, however, in the men treated with zidovudine, both in those with thrombocytopenia (60,000 +/- 31,000 platelets per cubic millimeter per day, P < 0.01 vs. controls) and in those without thrombocytopenia (68,000 +/- 22,000 per cubic millimeter per day, P < 0.01). CONCLUSIONS. Although there was a moderate reduction in platelet survival in HIV-infected persons, these patients, regardless of platelet counts, also had decreased production of platelets, possibly due to viral infection of the megakaryocytes. Zidovudine appears to improve platelet production.     

Determination of heparin-induced IgG antibody by fluorescence-linked immunofiltration assay (FLIFA)

A fluorescence-linked immunofiltration assay (FLIFA) was developed for the determination of heparin-induced IgG in heparin-induced thrombocytopenia (HIT) type II patients. Protein A was immobilized on a nitrocellulose membrane to bind heparin-induced IgG of HIT type II patients. Fluorescein-5-isothiocynate (FITC)-heparin was added to platelet factor 4 present in normal serum to form the neo-antigen which was captured by heparin-induced IgG. The heparin-induced IgG was quantified by the relative fluorescence intensity (RFI) of bound FITC-heparin. Values were expressed as a RFI ratio (RFI patient / RFI normal) and were 1.965+/-0.413 in HIT type II patients (n = 36) and 1.064+/-0.162 in healthy controls (n = 50, p<0.0001). The intra- and inter-assay coefficients of variation were 4.9 and 10.4%, respectively. The heparin-induced IgG FLIFA will be useful in individual and epidemiological studies in patients during treatment with heparin. The FLIFA technique offers an alternative, rapid and sensitive methodological approach for studies on the interaction between antigen-antibody or ligand-receptor.     

Decision analysis for use of platelet aggregation test, carbon 14-serotonin release assay, and heparin-platelet factor 4 enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia

The value of the platelet aggregation test, carbon 14-labeled serotonin release assay (SRA), and heparin-platelet factor 4 enzyme-linked immunosorbent assay (H-PF4 ELISA) for the diagnosis of heparin-induced thrombocytopenia was evaluated by studying blood samples from 100 patients with suspected heparin-induced thrombocytopenia, and categorized into 4 clinical groups: unlikely (n = 22), possible (34), probable (36), and definite (8) thrombocytopenia. Results of the platelet aggregation test were positive in 40 of 44 patients with probable or definite heparin-induced thrombocytopenia (sensitivity 91%) and in 5 of 22 unlikely to have heparin-induced thrombocytopenia (specificity 77%). The SRA exhibited sensitivity of 88% and negative predictive value of 81%, close to those values for the platelet aggregation test; specificity and positive predictive value were 100%. The sensitivity of the heparin-PF4 ELISA was 97%, with specificity 86%, and a positive correlation was recorded between the level of antibodies to H-PF4 and clinical score (P = 0.66). When ELISA was used with the platelet aggregation test or SRA, positive predictive value and specificity were 100% when both tests yielded positive results, and negative predictive value was 100% when both tests yielded negative results. A biologic flow chart was designed that presented a choice based on the results of the platelet aggregation test or SRA in association with ELISA, and enabled more accurate and specific identification of heparin-induced thrombocytopenia.     

Diagnostic value of measurement of RNA in platelets by fluorescence flow cytometry]

We analyzed the RNA in platelets by fluorescence flow cytometry after staining with thiazole orange(TO) in whole blood samples from hematologically normal subjects and patients with thrombocytopenia. The percentage of TO-positive platelets and their mean fluorescence channel number in 32 control subjects were 6.2 +/- 2.5% (mean +/- SD) and 6.9 +/- 0.7, respectively. In 11 patients with idiopathic thrombocytopenic purpura, the percentage of fluorescently labeled platelets was significantly elevated (p less than 0.05) to 21.5 +/- 14.3%. By contrast, the proportion of positively stained platelets in 14 patients with thrombocytopenia due to impaired platelet production did not significantly differ from that of the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (p less than 0.05). In both patient groups, the mean fluorescence channel numbers of TO-positive platelets were significantly elevated to 16.1 +/- 16.8 and 6.9 +/- 0.7, respectively (p less than 0.05, 0.005). We conclude that flow cytometric analysis of platelets after staining with TO provides information on the thrombopoietic activity in thrombocytopenic disorders. The main advantages of this method for clinical use are its simplicity and the rapidity.     

Platelet-associated IgG in hepatitis and cirrhosis

Increasing evidence is accumulating which indicates that immunological abnormalities contribute to the development of liver disease and its signs and symptoms. Platelet-associated IgG (PAIgG) levels were quantified in 42 patients with biopsy-proven liver disease of various etiologies to determine the relationship of thrombocytopenia to immunologic abnormalities in these disorders. Five of six nonthrombocytopenic patients with acute viral hepatitis B had elevated PAIgG. Six of ten patients with chronic active hepatitis had elevated PAIgG and thrombocytopenia. In contrast, only one of six patients with chronic persistent hepatitis had elevated PAIgG. Nine of ten patients with alcoholic hepatitis had elevated PAIgG; seven of the nine were thrombocytopenic. Seven of ten alcoholic patients with cirrhosis had elevated PAIgG; six of seven were thrombocytopenic. Thus the increase in PAIgG may be present without thrombocytopenia in acute liver injury, while patients with chronic persistent hepatitis do not usually exhibit this abnormality. Severe chronic active liver disease is accompanied by thrombocytopenia and an increase in PAIgG levels.     

Immune thrombocytopenia. Use of a Coombs antiglobulin test to detect IgG and C3 on platelets

We applied a radiolabeled Coombs antiglobulin test to the diagnosis and management of immune thrombocytopenia in adults and children. This assay substantiated that the majority of patients with idiopathic thrombocytopenic purpura have increased levels of IgG on their platelets. Platelet-associated C3 was elevated in a subset of these patients, some of whom had normal levels oet-associated IgG, thus suggesting a role for C3 in the pathogenesis of the thrombocytopenia. The assay enabled us to anticipate a change in clinical status, establish the presence of immunologic remission after splenectomy and propose several mechanisms by which corticosteroids act. Platelets from a patient with the post-transfusion-purpura syndrome also carried increased IgG, indicating a role for IgG antibody or IgG-containing immune complexes in the destruction of host platelets in this disease. The radiolabeled Coombs test provides a general means to help diagnose, manage and study immune platelet disorders.     

Thrombocytopenia in systemic lupus erythematosus: association with antiplatelet and anticardiolipin antibodies

Serum platelet bindable immunoglobulin G (SPbIgG) and anticardiolipin antibodies (ACA) have been assayed in SLE patients with (group A, n = 8) and without thrombocytopenia (group B, n = 8). Moreover, we studied the binding of serum IgG on platelet proteins using a Western blotting procedure. Increased levels of SPbIgG were found in all thrombocytopenic patients and in five cases of group B. A binding of serum IgG on platelet proteins (80 and 53 kDa) was seen in seven of eight thrombocytopenic patients and in only one case of group B. ACA were present in the serum of four patients in each group and a significant inhibition of ACA IgG by entire washed platelets was achieved in only two severe thrombocytopenic cases. These data suggest that participation of ACA to the platelet destruction can be evoked for a few SLE patients, whereas recognition of platelet protein determinants by lupus autoantibodies is necessary to the thrombocytopenia.     

Platelet Kinetics in Canine Ehrlichiosis: Evidence for Increased Platelet Destruction as the Cause of Thrombocytopenia

A significant (P < 0.025) increase in the mean platelet diameter occurred in five Ehrlichia canis-infected dogs when platelet numbers decreased to 100,000/mul or less. Maximal incorporation of [(75)Se]selenomethionine into platelets of six uninfected dogs was 0.080 +/- 0.019% (mean +/- standard error) and occurred 5 to 6 days after dosage, whereas maximal incorporation was 0.036 +/- 0.004% within 2 to 3 days after dosage in seven chronically infected dogs that had thrombocytopenia. Analysis of the [(75)Se]selenomethionine curves yielded a platelet lifespan of 9 days in uninfected dogs versus 4 days in chronically infected dogs. Thus, megakaryocyte maturation and/or platelet release occurred at an accelerated rate in infected dogs, whereas increased destruction of newly produced labeled platelets diminished their number of peripheral blood. [(51)Cr]sodium chromate-labeled platelet survival was exponential, with a half-life of approximately 1 day in two dogs at 2 to 4 days postinfection and three chronically infected dogs. Platelet survival time was 8 days and rectilinear in four uninfected dogs. Platelet recovery was 39.43 +/- 2.86% in infected dogs as compared with 68.2 +/- 10.72% in uninfected dogs. Whole-body scans of one dog prior to and 7 days after infection showed that labeled platelets were destroyed primarily in the spleen. It is concluded that the thrombocytopenia in E. canis-infected dogs is the result of increased platelet destruction which begins within a few days after infection.     

Differentiation between Bernard-Soulier syndrome and immune thrombocytopenia by immunostaining of peripheral blood.

Peripheral blood smears from seven patients with Bernard-Soulier syndrome were examined by an immunocytochemical staining procedure using a monoclonal antibody specific for platelet glycoprotein Ib. No platelet staining was observed except for very slight staining of the large sized platelets from one of the patients. Application of the assay to blood smears from 12 patients with immune thrombocytopenia showed that their peripheral platelets stained normally, so the assay can be used to differentiate between immune thrombocytopenia and Bernard-Soulier syndrome and to confirm a diagnosis of the syndrome.     

Clinical significance of simultaneous measurement of reticulated platelets and large platelets in idiopathic thrombocytopenic purpura

Frequencies of reticulated platelets (RP) and large platelets (LP) among circulating platelets can now be simultaneously determined using the R-3000 automated reticulocyte counter (Sysmex, Kobe, Japan) equipped with special software. We measured frequencies of RP and LP in patients with idiopathic thrombocytopenic purpura (ITP. acute type n = 5; chronic type n = 39), and healthy normal controls (n = 20). In ITP patients, the platelet-associated IgG (PAIgG) level was also determined. Both RP and LP were significantly higher in chronic ITP patients than those in normal volunteers, and interestingly, the LP in acute ITP was significantly lower than that in chronic ITP although there was no significant difference in RP between acute and chronic ITP. Furthermore, we analyzed the changes in both RP and LP during the clinical course of ITP to monitor the therapeutic effect in 2 patients. An elevation of RP with a steep slope prior to a decrease in the platelet count level was observed. The RP significantly correlated with the PAIgG level. Simultaneous measurement of RP and LP may be helpful for the diagnosis of chronic ITP, for the differentiation of acute from chronic type and for the control of the efficacy of management in ITP, since RP seems to reflect the disease activity of ITP.