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.     

The place of intravenous immunoglobulin (IgG i.v.) therapy in thrombocytopenia

Summary Acute ITP following infection in children has a high rate of spontaneous remission. Since the platelet count is not uncommonly profoundly depressed, ITP is associated with a mortality of around 1%. The aim of treatment in ITP is to prevent bleeding. As a rule, this can only be achieved by normalizing the platelet count. Intravenous immunoglobulin offers certain advantages over conventional steroid therapy. Moreover, two out of three patients with chronic ITP respond to IgG i.v. The characteristic features of ITP in pregnancy, thrombocytopenia in babies born to mothers with ITP and neonatal isoimmune thrombocytopenia are discussed.     

High-dose intravenous IgG in the management of pregnancy in women with idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) may develop during pregnancy or affect later pregnancies, causing serious risks of bleeding to the mother and fetus. High-dose intravenous immunoglobulin (IGIV) has caused an immediate and predictable rise in platelet count during the infusion in both adults and children with chronic or acute ITP. The rapid rise in platelet counts may be important in preparing pregnant women with ITP for surgery or delivery. We report our experience in managing two women at weeks 29 and 37 week of gestation who required splenectomy and/or cesarean section. Both patients demonstrated an increase in platelet counts, underwent surgery without excess bleeding, and had normal infants with normal platelets, and with mild thrombocytopenia at delivery.     

Monocyte-platelet interaction in immune and nonimmune thrombocytopenia

Platelets from 24 patients with immune thrombocytopenia resistant to standard therapy (refractory ITP), 35 patients with nonimmune thrombocytopenia (non-ITP), and 32 normal donors were studied in regard to platelet surface-bound IgG (PBIgG) and the ability of these platelets to be bound by human monocytes in vitro (monocyte-platelet rosette assay). Fourteen (58%) of the platelet samples from refractory ITP patients but none (0%) from the non-ITP or control donors had PBIgG greater than 800 molecules IgG/platelet. Seventeen of 24 (71%) of the ITP patients had platelets which demonstrated increased monocyte- platelet rosette formation [rosette index (RI) greater than 2], whereas only four (11%) of the non-ITP patients had such platelets. There was a direct correlation between PBIgG and rosette index for the platelets from resistant ITP patients. There was no correlation of severity of thrombocytopenia with PBIgG or rosette index. Monocyte-platelet interaction in the presence of elevated PBIgG is mediated through the monocyte Fc-receptor. Platelets from five of ten refractory ITP patients with PBIgG less than 800 molecules IgG/platelet had increased rosette formation. Monocyte-platelet interaction in the absence of increased PBIgG may be due to small amounts of platelet surface IgG which are still able to mediate monocyte Fc-receptor interaction or to alternate membrane receptor interaction through the monocyte C3 receptor. Our data underscore the pathophysiologic relevance of monocyte/macrophage-mediated interaction in immune platelet destruction syndromes.     

Platelet apoptosis in paediatric immune thrombocytopenia is ameliorated by intravenous immunoglobulin

To evaluate the role of intravenous immunoglobulin (IVIg) in platelet apoptosis in paediatric immune thrombocytopenia, we investigated the platelets of 20 paediatric patients with acute immune thrombocytopenia (ITP), before and after IVIg treatment. Healthy children with platelet counts in the normal range and children with thrombocytopenia due to chemotherapy were enrolled as controls. All ITP patients presented with platelet counts <20 × 10⁹/l and bleeding symptoms. Markers of apoptosis, including activated caspase‐3, ‐8 and ‐9, phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), as well as platelet‐derived microparticle formation, were analysed by flow cytometry. After IVIg treatment, platelet counts increased to >20 × 10⁹/l in all patients. ITP patients had significantly increased proportions of platelets with activated caspase‐3, ‐8 and ‐9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation. Except for ΔΨm, these markers for apoptosis were reduced by IVIg treatment. Platelets of children with thrombocytopenia after chemotherapy also demonstrated increased microparticle formation and decreased ΔΨm, but no activation of caspases 3, 8 and 9 or PS exposure. In conclusion, in acute paediatric ITP, enhanced platelet apoptosis is seen at diagnosis that normalizes after IVIg treatment.     

Endothelial alterations in a canine model of immune thrombocytopenia

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.     

Characterization of platelet-reactive antibodies in children with varicella-associated acute immune thrombocytopenic purpura (ITP)

Biochemical analyses were performed on blood samples obtained from two children (P1, P2) who presented with acute immune thrombocytopenic purpura (ITP) following a recent varicella zoster virus (VZV) infection. Patient sera had antibodies that were reactive with normal blood-group O platelets as measured by flow-cytometric assay. Western blot analysis of electrophoretically separated normal blood-group O platelets under reducing and non-reducing conditions demonstrated that these sera were reactive with platelet antigens of ∼50 and ∼110 kD, respectively. These 50/110 kD antigens were not reactive with seven sera from acute ITP patients whose illness was not preceded by VZV infection, with serum from a patient with a prior history of VZV and no thrombocytopenia, nor with normal healthy control sera. VZV antibodies (IgG and IgM), isolated from patient sera by affinity chromatography using immobilized purified VZV glycoproteins, were found to bind to gel-filtered autologous platelets and with normal blood-group O platelets, as analysed by flow cytometry. No binding was observed using antibodies similarly prepared from healthy volunteer sera. To investigate their ability to sensitize platelets to complement activation, affinity-purified VZV antibodies were incubated with platelets and then with purified complement components C1 and ¹²⁵I-labelled C4. Platelets reacted with VZV-specific antibodies from the two patients and showed increases of 2.3–2.4-fold of platelet-surface deposition of ¹²⁵I-C4b, compared to controls. These data provide evidence that virus-specific antibodies occurring in children with varicella-associated acute ITP cross-react with normal platelet antigens, and may contribute to platelet clearance.     

Phase II trial of methylprednisolone pulse therapy in childhood chronic thrombocytopenia

Nineteen children with chronic idiopathic thrombocytopenia (ITP) were treated with a single intravenous injection of methylprednisolone (MP), 15 mg/kg/day, for 3 consecutive days. The 3-day pulses gave rise to a positive and fast therapeutic response with increase of the platelet count in about three quarters of the patients. The platelet count remained above 50 X 10(9)/1 for more than 1 month in 10 children. Eight out of them still presented a safe platelet count (greater than 50 X 10(9)/1) 4 months after the onset of the therapy. The MP therapy improved the platelet count more in the older children and possibly in the females. No severe side effects were observed. Our results suggest that this therapeutic approach could be useful in the management of acute bleeding episodes in children with chronic ITP.     

ITP患儿血小板内cAMP的研究

本文用放免法测定了38例正常儿及47例 ITP 患儿血小板内 cAMP 值。发现无论急、慢性患儿血小板内 cAMP 值均显著增高,其增高与血小板功能呈负相关,与血小板计数无相关性。急性患儿 PAIgG 增高,血小板内 cAMP 亦增高,二者之间呈正相关,而慢性患儿则无此相关性。并对 cAMP 的增高与 ITP 的发病机制进行了阐述。     

Interstitial lung disease (ILD) and severe ITP

Introduction: Platelets play an important role in inflammatory and immune responses. We report interstitial lung disease (ILD) developing during the acute phase of severe thrombocytopenia in 3 patients with severe refractory ITP.

Methods and Results: We identified 3 cases with severe ITP who developed ILD in the course of refractory chronic ITP. The thrombocytopenia was severe in all cases. ILD was an incidental finding at the presentation and often misdiagnosed as lung infections. ILD was documented by lung biopsy in cases 1 and 2, supplemented by serial chest X-rays and/or CAT scan. As the ITP improved, ILD regressed in case 1, persisted in case 2, and progressed to advanced pulmonary fibrosis in case 3.

Conclusion: We report an association of ILD with severe refractory ITP. ILD was detected in acute phase of platelet destruction, suggesting that platelet destruction may have triggered inflammation in the lung, leading to ILD.     

Possible lower rate of chronic ITP after IVIG for acute childhood ITP an analysis from registry I of the Intercontinental Cooperative ITP Study Group (ICIS)

In children, one-third of immune thrombocytopenic purpura (ITP) patients follow a chronic course. The present study investigated whether treatment with intravenous immunoglobulin (IVIG) at the time of diagnosis of ITP is of prognostic significance, using data from 1984 children entered in Registry I of the Intercontinental Cooperative ITP Study Group. A matched pairs analysis compared children with thrombocytopenia (platelet count <150 × 10⁹/l) 6 months following diagnosis with children whose platelet count was normal 6 months after diagnosis. It was found that children initially treated with IVIG were more likely to have a normal platelet count 6 months after diagnosis than children not receiving IVIG (odds ratio 1·81; 95% confidence interval: 1·25–2·64). This result was independent of age, gender, country of origin, platelet count at diagnosis or infection preceding the diagnosis of ITP. In a similar analysis, comparing children with a platelet count <50 × 10⁹/l 6 months after diagnosis with children whose platelet count was ≥50 × 10⁹/l at that time point, the former group was less often treated with IVIG than with steroids ( P  = 0·02). Prospective studies are required to further explore this potential effect of IVIG.     

Circulating thrombopoietin level in chronic immune thrombocytopenic purpura

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean±SD) in ITP patients was mildly elevated (1.86±1.17 fmol/ml) compared to that in normal subjects (0.76±0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35±6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.     

Recent Advances in the Treatment of Chronic Refractory Immune Thrombocytopenic Purpura

We define chronic refractory immune thrombocytopenic purpura (ITP) as ITP with persistent thrombocytopenia following treatment with glucocorticoids and splenectomy. Chronic refractory ITP is uncommon, occurring in fewer than 10% of all adult patients with ITP diagnoses. The goal of treatment is only to achieve a safe platelet count with minimal treatment-related risk. A safe platelet count may be considered to be as low as 10,000/microL, because the risk for major bleeding in otherwise healthy subjects is great only when the platelet count is less than 10,000/microL. Observation without specific treatment is appropriate for patients with moderate thrombocytopenia and no clinically important bleeding symptoms. For patients with chronic refractory ITP who require treatment, there is no consensus for what therapies to use or the sequence in which to use them. For patients with severe and symptomatic thrombocytopenia, the use of anti-CD20 (rituximab) and immunosuppressive agents, alone or in combination, may be most effective. The mechanism of all current therapies is to decrease the accelerated platelet destruction brought about by immunosuppression. An alternative approach, the stimulation of platelet production with thrombopoietic agents, has been successful in investigational studies and may provide a new management option.     

Isolated thrombocytopenia in patients infected with HIV: treatment with intravenous gammaglobulin

Isolated thrombocytopenia occurs frequently in patients infected with HIV. Studies of mechanisms of thrombocytopenia and clinical response to therapy suggest that the thrombocytopenia is often antibody mediated (ITP). The best approach to treatment of these patients is uncertain in that the routine modalities (steroids, splenectomy, vinca alkaloids) that are used to increase the platelet count in patients with classic ITP are known to be immunosuppressive. We report here the results of intravenous gammaglobulin (IVGG) treatment of 22 patients with HIV-related acute and chronic ITP who had severe thrombocytopenia and bleeding symptoms. Only one patient had an opportunistic infection at the time of treatment. Eight patients were homosexual, eight had hemophilia, three were i.v. drug abusers, two children had congenital acquisition of HIV, and one was the wife of an HIV + i.v. drug abuser. The average pretreatment platelet count was 22,000/microliter (hemophiliacs were treated at higher platelet counts than were the other patients), and the mean peak platelet count measured on days 5 to 8 was 182,000/microliter. Nineteen of 22 patients had peak platelet counts greater than 50,000/microliter following IVGG and 17/22 had peak counts greater than 100,000/microliter. After the initial infusions, all but three refractory patients could maintain adequate platelet counts with IVGG alone infused no more often than once every 2 weeks. The outcomes for the 22 patients after multiple maintenance IVGG infusions were remission, 5; stable without therapy, 1; maintenance, 13; and refractory, 3. The eight hemophiliacs with ITP responded better than did the eight homosexual ITP patients; their mean peak platelet count was 227,000/microliter versus 142,000/microliter in the homosexuals. In summary, patients with HIV-related ITP without opportunistic infections responded well to IVGG, with peak platelet counts comparable to those of ITP patients not infected with HIV. IVGG may be a useful therapy of ITP in HIV+ patients, since it appears to be less immunosuppressive than are conventional therapies, and none of the 22 HIV+ patients developed an opportunistic infection while receiving IVGG alone.     

High dose dexamethasone as an alternative rescue therapy for active bleeding in children with chronic ITP: clinical and immunological effects

High-dose dexamethasone (HD-DXM) is debated as a second-line therapy for chronic Immune thrombocytopenia (ITP) in children. The aim of this study is to evaluate the efficacy and safety of HD-DXM as an emergency therapy in uncontrolled bleeding in children with chronic ITP and to assess its immunological effect on dendritic cells (DCs) percentage and their co-stimulatory markers CD86 and CD83.

Totally, 20 children previously diagnosed as chronic ITP were enrolled in this study and all admitted to hospital with uncontrolled bleeding. Patients received HD-DXM as a single daily dose for 4 days. Blood samples were withdrawn from patients just prior to HD-DXM therapy and on day 5 to evaluate the platelet count and for flowcytometric analysis of DCs. Daily assessment of bleeding severity was performed. The platelet counts significantly increased in patients after 5 days of initiation of therapy compared with platelet count before therapy (p-value = 0. 0002). Control of bleeding observed in (90%), complete response (CR) documented in (50%), response (R) documented in (40%), and no response (NR) documented in (10%) of patients. The time to respond was raging from 1 to 3 days and minor complication recorded in two patients. Both plasmacytoid DCs and myeloid DCs percentage and their expression of co-stimulatory markers, CD86 and CD83 decreased significantly after HD-DXM therapy. Conclusion: short course of HD-DXM as a rescue therapy seems to be an effective alternative emergency treatment for uncontrolled bleeding in chronic ITP children especially in nations with limited resources.     

Refractory chronic immune thrombocytopenic purpura in a child with acute lymphoblastic leukemia

Immune thrombocytopenic purpura (ITP) has been associated with several hematologic malignancies such as Hodgkin and non-Hodgkin lymphomas and chronic lymphocytic leukemia, but it is rare in children with acute lymphoblastic leukemia (ALL). Here, we report a 7-year-old girl with chronic ITP during early intensive phase of chemotherapy for ALL. She underwent splenectomy because thrombocytopenia had persisted even after treatment with intravenous immunoglobulin (IVIG), steroids, vincristine, rituximab, and anti-D antibody. After splenectomy, her platelet count had recovered, and maintenance therapy could be resumed with a support of IVIG. To our knowledge, this is the first child case of chronic ITP during chemotherapy for ALL and splenectomy was effective in this patient.     

Efficacy and safety of anti-D for treatment of adults with immune thrombocytopenia

This study was planned to assess the response of anti-D in patients with immune thrombocytopenia (ITP). Twenty adults (8 males: 12 females) with a median age 33.5 years (19–59 years) were included. Nine patients had newly-diagnosed ITP, 6 had persistent ITP and 5 had chronic ITP. The overall response rate was 65%. Patients with newly diagnosed ITP showed response rates of 77% (7/9), persistent ITP had response rates 50% (3/6) and patients with chronic ITP had response rates of 60% (3/5). The median time to response was 3 days (1–11 days). There was no correlation of response with age, sex, severity of bleeding, presenting platelet counts, ABO blood group or prior steroid or IVIG response.     

Platelet-associated IgG in immune thrombocytopenic purpura

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.     

Antiplatelet antibodies and their correlation with clinical findings in childhood immune thrombocytopenic purpura

The demonstration of antiplatelet antibodies (PAIgG, PAIgM) and decreased detection of platelet surface antigens (CD41, CD61, CD42b) in children with immune thrombocytopenic purpura (ITP) have a diagnostic role. This study was conducted to determine whether these parameters differed in acute and chronic ITP. Chronic ITP was defined as thrombocytopenia persisting for more than 6 months from the onset of illness. A total of 80 subjects were divided into three groups: group 1 included 39 patients with acute ITP; group 2 included 31 patients with chronic ITP, and group 3 included 10 healthy children. At diagnosis, blood samples were obtained for platelet count, mean platelet volume, plateletcrit and platelet distribution width along with platelet surface antigens and antiplatelet immunoglobulins. We found that platelet surface antigens were significantly decreased in both acute and chronic ITP when compared to the control group (p = 0.001). In contrast, PAIgG was increased in acute and chronic ITP patients compared to the control group. PAIgM was significantly higher in acute ITP. We conclude that decreased platelet surface antigens and increased antiplatelet antibodies are observed in both acute and chronic ITP. In patients with chronic progress, a relatively lower level of PAIgM can be identified.     

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.