Immune Thrombocytopenic Patients Research Articles

Use of dapsone in chronic/refractory immune thrombocytopenic patients: A single center experience

Background: Dapsone is a second-line therapy for immune thrombocytopenia (ITP). It is cost-effective, with a response rate comparable to other drugs used as second-line therapy, such as azathioprine, danazol, cyclophosphamide, cyclosporine, vincristine, rituximab, and eltrombopag.
 Material and Methods: This retrospective study analyzed ten adult patients who presented to our hematology division outpatient clinic between March 2013 and July 2021, was diagnosed with chronic/refractory ITP, did not respond to first-line therapy, and used dapsone.
 Results: Eight (80%) patients were female, and 2 (20%) were male. The median age was 50 (range, 24-64) years. The mean pre-treatment platelet value was 12.8x109/L (range: 4-22.1x109/L). The median duration of symptoms before dapsone treatment was 60 (6-360) months. The median number of treatments received before dapsone was 4 (range: 3-6). All patients were routinely treated with oral dapsone 50 mg for two weeks, followed by 100 mg. The median time to treatment response was 39 (range: 14-90) days. The response rate was 60% (complete response 40%, partial response 20%). Asymptomatic anemia was observed as a side effect in only one patient.
 Conclusions: Based on these results, it can be speculated that dapsone is an effective, inexpensive, and well tolerated
 treatment option. Considering the economic status of developing countries, it seems very attractive
 to use dapsone as the second-line therapy for chronic/refractory ITP. To the best of our knowledge, this is the
 first study in Turkey on the use of dapsone for chronic/refractory ITP.

Gene mutations in the PI3K/Akt signaling pathway were related to immune thrombocytopenia pathogenesis.

Immune thrombocytopenic (ITP) is an autoimmune bleeding disease with genetic susceptibility. Twenty newly diagnosed active primary ITP patients who had not been treated with glucocorticosteroids, immune globulin or immunosuppressants prior to sampling were enrolled in this study. Bone marrow blood mononuclear cells were used for whole exome sequencing to further elucidation the variant genes of ITP. High-molecular-weight genomic DNA was extracted from freshly frozen bone marrow blood mononuclear cells from 20 active ITP patients. Next, the samples were subjected to molecular genetic analysis by whole-exome sequencing, and the results were confirmed by Sanger sequencing. The signaling pathways and cellular processes associated with the mutated genes were identified with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The results showed that there were 3998 missense mutations involving 2269 genes in more than 10 individuals. Unique genetic variants including phosphatase and tensin homolog, insulin receptor, and coagulation factor C homology were the most associated with the pathogenesis of ITP. Functional analysis revealed these mutation genes mainly affect Phosphatidylinositol 3 kinase/serine/threonine kinase B signaling pathways (signal transduction) and platelet activation (immune system). Our finding further demonstrates the functional connections between these variant genes and ITP. Although the substantial mechanism and the impact of genetic variation are required further investigation, the application of next generation sequencing in ITP in this paper is a valuable method to reveal the genetic susceptibility.

A comparative study between light transmission aggregometry and flow cytometric platelet aggregation test for the identification of platelet function defects in patients with bleeding.

BackgroundPlatelet aggregation studies using conventional light transmission aggregometry (LTA) have several disadvantages and require strict pre-analytical measures for reliable results. We aimed to examine the utility of flow cytometric platelet aggregation (FCA) assay in detecting platelet function defects (PFDs) in patients with a history of bleeding symptoms.MethodsSixty-four participants (24 patients and 40 healthy controls) were included in this study. LTA and FCA assay were performed simultaneously in patients and healthy controls. In the FCA assay, two portions of platelets from the same individual were labeled separately with CD31-FITC and CD31-PE. After mixing and stimulation with agonists, the double-colored platelet aggregates were visualized using a flow cytometer. The results generated using the two techniques were compared and correlated.ResultsThe patients’ median age was 17 years (range, 3‒72 yr) with a male-to-female ratio of 11.7. There was substantial agreement between LTA and FCA assay in detecting a PFD (k=0.792). Four patients showing a Glanzmann thrombasthenia-like pattern on LTA exhibited an abnormal FCA. A functional defect in collagen binding was detected on the FCA assay conducted in two immune thrombocytopenic patients with severe bleeding.ConclusionFCA assay can be used to identify functional defects in platelets, with potential applications in thrombocytopenic individuals. It also facilitates the diagnosis of inherited bleeding disorders with platelet defects.

Endothelial Activation and Immune Thrombocytopenia: An Engagement Waiting for Consolidation.

Immune thrombocytopenia (ITP) appears to be a heterogeneous disease. In some patients, autoimmunity may be associated with an inflammatory process, and in other patients, low platelets may interfere with other aspects of the coagulation system. Either may predispose to thrombosis or bleeding. Further investigation of the interactions of platelets, with inflammatory cytokines and endothelial biomarkers, may help us to better understand the disease, and to recognize those patients at risk of bleeding, or conversely thrombosis. The aim of this work is to estimate von Willebrand factor (vWF) and vascular cellular adhesion molecule (V-CAM) serum levels in adult immune thrombocytopenic patients (ITP) and to decipher their possible clinical correlates. Eighty adults (≥ 18 years) were enrolled in the study; naive newly diagnosed 40 patients with primary ITP (according to the ASH 2019) and 40 sex and age-matched healthy controls, all groups are subjected for complete blood count (CBC), liver, and renal function tests, ESR, CRP, V-CAM, and VWF-Ag by enzyme-linked immunosorbent assay (ELISA). There was a highly statistically significant difference between case and control as regards to the mean level of VWF-Ag and V-CAM. vWF and V-CAM could serve as biomarkers for endothelial alterations and should be investigated as a predictor of thrombocytopenic bleeding and tailor patient management accordingly.

SARS-COV-2 infection in a patient with Evans syndrome: A silent enemy or an ally?

Introduction. During the current outbreak of Coronavirus disease 2019 (COVID-19), the way to manage patients with autoimmune diseases remains elusive due to limited data available. Case report. We presented a case of a COVID-19 positive 20-year-old female with prior history of Evans syndrome. The patients remained asymptomatic even though she had been treated with immunosuppressants (prednisolone and azathioprine) together with romiplostim. Moreover, her course of infection was accompanied by thrombocytosis, although her platelet count was mostly be-low the reference range before the infection. The patient was monitored vigilantly, with special regard to platelet count and signs of thrombotic events. Conclusion. Platelet count monitoring and romiplostim administration should be performed more cautiously in chronic immune thrombocy-topenic patients infected by SARS-CoV-2.

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan.

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet lifespan in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from patients with immune thrombocytopenia, which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleavage of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of an ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment of the production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia.

Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients.

Immature platelet fraction measurement is influenced by platelet size and is a useful parameter for discrimination of macrothrombocytopenia

BackgroundReticulated platelets (RPs) as measured using flow cytometry are useful parameters of thrombopoiesis; however, difficulties remain with standardization between laboratories. On the other hand, immature platelet fraction (IPF) measurement, as determined using an automated hematology analyzer, is simple, reproducible, and displays a good correlation with RP, although specific factors may affect its value. We previously noticed that a small proportion of patients exhibit extremely high IPF values that do not correlate with flow cytometrically measured RP.ObjectivesWe investigated the mechanism of the aberrant increase in IPF values of different types of macrothrombocytopenia.Patients/methodsIPF, RP, and other platelet indexes were analyzed using samples from 15 congenital macrothrombocytopenic patients from 12 families, 150 immune thrombocytopenic patients, and 27 normal individuals. We further monitored the change in IPF values and morphology during platelet agglutination.ResultsIPF values were about five times higher in MYH9 disorders (IPF 48.6 ± 1.9%) and about twice as high in other macrothrombocytopenias (IPF 18.4 ± 2.1%) than in immune thrombocytopenic patients with similar platelet counts (IPF 9.2 ± 0.3%). We then examined changes in IPF values during ethylenediaminetetraacetic acid- and macroglobulinemia-induced platelet agglutination. The IPF value significantly increased in a time-dependent manner along with the formation of platelet clumps and was strongly influenced by a few tiny platelet aggregates.ConclusionsThese results suggested that IPF values are influenced by platelet size. Furthermore, IPF could be a useful and convenient parameter for screening of macrothrombocytopenia, which presents with a disproportionately high IPF value.

Frequency of Helicobactor pylori infection among adult patients with chronic immune thrombocytopenia: A Pakistani Perspective

Objective: Many studies have reported an association between chronic immune thrombocytopenia and Helicobactor pylori infection. Significant improvement in platelet count is seen after eradication therapy. We looked for the frequency of H. pylori infection in immune thrombocytopenia and clinico-hematological features. Material and Method: A prospective case-control study was conducted from January 2009 to December 2011. One hundred and ten chronic immune thrombocytopenic patients and 40 control subjects were enrolled. H. pylori infection was documented by H. pylori stool antigen enzyme immunoassay method. Results: H. pylori infection in immune thrombocytopenic patients was seen in 45 cases (40.9%) and in 24 (60%) controls; not significant statistically. No significant differences were seen in H. pylori positive and negative patients with respect to maternal characteristics and clinical features except for mean platelet count, which was lower in H. pylori negative group (P = 0.035). Conclusion: Unlike many related studies, we found no significant difference between the frequencies of cases versus controls. Degree of thrombocytopenia appears more marked in H pylori negative patients.

Study of serum prolactin in primary immune thrombocytopenic patients

Objectives The aims of this work were to study serum prolactin (PRL) levels in patients with primary immune thrombocytopenia (ITP) and to investigate its possible correlation with disease activity and manifestations. Background ITP is a disorder characterized by immune-mediated accelerated platelet destruction and suppressed platelet production. Hyperprolactinemia (HPRL) has been described in many autoimmune diseases such as systemic lupus erythematosus. Patients and methods The study was carried out on 40 cases of primary ITP patients (group I) and 50 healthy controls (group II). PRL was measured directly in the serum samples by VIDAS PRL kits using the ELFA technique for all patients and controls. Results Moderate HPRL (serum PRL 30-200 ng/ml) was present in eight (20%) of primary ITP patients, but was not present in any of the 50 controls. Among 22 patients with platelet count below 30 000/μl, eight (36.4%) patients had HPRL and 14 (63.6%) patients had normal PRL levels. HPRL was associated with lower platelet counts. Conclusion This study shows that HPRL is present in 20% of patients with primary ITP. Also, patients with HPRL have a lower platelet count than patients with normal PRL levels.

Expression and significance of vitamin D and its receptor mRNA in the peripheral blood of initial immune thrombocytopenic patients

To investigate the effect of vitamin D in pathogenesis and clinical treatment of patients with immune thrombocytopenic (ITP), ELISA was used to detect the level of 25-hydroxylate vitamin D3[25(OH)D3] and 1,25-dihydroxyvitamin D3[1,25(OH)2D3] in peripheral blood from 45 ITP patients and 30 normal controls. Vitamin D receptor (VDR) mRNA expression was detected by RT-PCR. The results showed that the levels of 25(OH)D3 (10.6 ± 7.7 ng/ml) and 1,25(OH)2D3 (69.9 ± 29.0 pg/ml) in peripheral blood of the initial ITP patients were lower than those in peripheral blood of normal controls (13.7 ± 9.1 ng/ml, 87.3 ± 19.9 pg/ml) (P < 0.05). The expression of VDR gene obviously increased in peripheral blood of initial ITP patients (1.588 ± 0.127), as compared with that in peripheral blood of normal controls (1.055 ± 0.734) (P < 0.05). It is concluded that vitamin D level and its receptor expression may play an important role in ITP, and vitamin D and its similarities may be a new agent to treat patients with ITP.

The Immature Platelet Fraction Is Sensitive to the Platelet Size and a Useful Parameter for Screening for Macrothrombocytopenia

Abstract 1097The immature platelet fraction (IPF) is a useful parameter indicating thrombopoietic activity to differentiate the causes of thrombocytopenia. We previously reported that the percentage of IPF (%IPF) is negatively correlated to the platelet count among ITP patients, and not among myelodysplastic syndrome (MDS) patients. We also noticed that some MDS patients exhibited extremely high %IPF values, which were dissociated from the percentages of reticulated platelets (%RP) measured by flow cytometry. Such discrepancies were also observed in hereditary macrothrombocytopenias, which are sometimes difficult to be distinguished from ITP, because ITP also exhibits increased number of reticulated platelets in a slightly larger size. Once misdiagnosed, a hereditary macrothrombocytopenia patient might be subjected to an invasive treatment such as splenectomy. In order to avoid such mistreatments, a clear marker to differentiate macrothrombocytopenia is desperately needed. In this study, we investigated the IPF values of 16 individuals from 12 families with various hereditary macrothrombocytopenia in order to clarify whether the IPF could be a useful marker to distinguish macrothrombocytopenia from ITP, and examined the IPF during EDTA aggregation and cold-storage to elucidate how platelet size may affect the IPF value.The IPF values were about 5 times higher in MYH9 disorders (%IPF 48.0 ± 1.8) and about 1.5 times higher in other macrothrombocytopenias (%IPF 17.0 ± 2.2) than immune thrombocytopenic patients with similar platelet counts (%IPF 9.3 ± 0.4). These results suggested that the platelet size can affect the IPF value. However it still remains the possibility that some factors specific to these macrothrombocytopenias other than the platelet size might make an influence on the IPF, because the characteristic of large platelets in hereditary macrothrombocytopenia has not been fully understood, and no one knows whether large platelets are functionally identical to normal platelets except for the size. In order to exclude the possibility, we next examined the changes of IPF values during EDTA aggregation and cold-storage. The IPF was significantly increased during storage in a time dependent manner along with forming platelet clumps. The IPF was strongly influenced by a few tiny platelet aggregates rather than other platelet indices, such as mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW).In conclusion, the IPF is susceptible to the platelet size, and could be a useful parameter for screening of macrothrombocytopenia from ITP. Disclosures:No relevant conflicts of interest to declare.

Treatment outcome of immune thrombocytopenia

Treatment of immune thrombocytopenia is sometimes difficult and needs personal setting. According to evidence-based guidelines, corticosteroids are suggested for first-line treatment. In case of corticosteroid ineffectiveness, second-line therapeutic options (splenectomy, immunosuppressive drugs and, recently, thrombopoietin-mimetics) may result in beneficial therapeutic effect. The aim of the authors was to examine the clinicopathological data, disease course, treatment results, and the effectiveness of novel drugs in patients with immune thrombocytopenia. The authors retrospectively analysed the files of 79 immune thrombocytopenic patients (26 males and 53 females) diagnosed and treated at the hematologic in- and outpatient units of the Markusovszky Hospital, County Vas, Hungary between January 1, 2000 and December 31, 2011. Remission rates, disease-free and overall survivals in response to corticosteroids (first-line treatment), after splenectomy (in cases when corticosteroids proved to be ineffective) and following second-line treatment were analysed. Survival curves were constructed using statistical software programs. Of the 79 patients during a median follow-up of 66 months (min. 3, max. 144 months), 28 patients receiving first-line corticosteroids achieved complete remission and remained in a prolonged disease-free condition (35.4%; median disease-free survival 75.5 months; min. 2, max. 140 months). Thirty-eight patients underwent splenectomy after ineffective treatment with corticosteroids or other immunosuppressive (48.0%; median disease-free survival 94.2 months; min. 6, max. 136 months). Surgical complications occurred in 2 cases, while postoperative and late infections were absent. Five patients died but death was not related to immune thrombocytemia. Second-line treatment was applied in 13 patients (16.4%) and among these patients relapse of immune thrombocytopenia after splenectomy was observed in 6 patients. Favourable effects of both conventional (immunosuppressive) and novel treatments (rituximab, thrombopoietin-mimetics) were also detected. More than two-thirds of patients with immune thrombocytopenia responded to corticosteroids or to splenectomy and achieved prolonged disease-free remission. Novel drugs (rituximab, thrombopoietin-mimetics) applied only in few cases produced also favourable results in patients not responding to corticosteroids and splenectomy.

Hepatotoxicity During Eltrombopag Administration Might Be Due to Necrosis of Hepatocytes

Abstract 2228 Background:Eltrombopag is an oral thrombopoietin receptor agonist for the treatment of immune thrombocytopenic patients who are refractory to the medications including corticosteroid. In RAISE study (The Lancet 2011: 377; 393–402), 79% patients in the eltrombopag group responded to treatment at least once during the study. However, 7% of eltrombopag-treated patients had increase of alanine aminotransferase (ALT) concentration and 4% of total bilirubin. The mechanism of eltrombopag-induced hepatobiliary toxicity remains unknown. In this study, we evaluated the effects of eltrombopag on hepatocytes using HepG2, human hepatocellular carcinoma cell line. Method: Cell proliferation was analyzed by MTT assay. Analysis of apoptosis/necrosis was analyzed by flow cytometry assay using Annexin V and propium iodide (PI) (Annexin-/PI-, viable cells; Annexin+/PI-, early apoptosis cells; Annexin+/PI+, late apoptosis and necrosis cells; Annexin-/PI+, late necrosis cells). Reduced glutathione was measured by DTNB colorimetric method. Murine hepatoma cell line Hepa 1–6 and murine normal liver derived cell line NCTC clone 1469 were also used in this study. Results: HepG2 was incubated with eltrombopag for 72 hrs and then MTT assay was performed. Figure 1a showed the growth inhibition curve of HepG2. HepG2 growth was suppressed by eltrombopag in a dose dependent manner (Figure 1a: without NAC, 12.5 μM 44.3 ± 8.7 %). In the addition of N-acetylcysteine (NAC) canceled the inhibitory effect (Figure 1a: with NAC 10mM: 60.4 ± 22.3 % at 12.5μM eltrombopag, p<0.05). To investigate whether this suppression was due to apoptosis or necrosis, we used PI/ Annexin V assay using flow cytometry. Figure 1b showed that each cell fraction after 72 hrs incubation with eltrombopag. PI positive and PI negative fraction was markedly increased (0μM: 0.7 ± 0.4%, 12.5 μM: 8.0 ± 6.0% p<0.01, 25μM: 39.6 ± 12.2% p<0.001). To confirm that the inhibitory effects of eltrombopag might be due to necrosis, we used IM-54, which is inhibitory molecule against oxidative stress induced necrosis, in MTT assay and PI/ Annexin V assay. As shown in Figure 2 a and 2 b, IM-54 canceled the inhibitory effects of eltrombopag in MTT assay (IM-54 0μM: 18.3 ± 7.5 %, IM-54 10 μM: 33.9 ± 10.6 % at eltrombopag 25μM, p<0.01) and PI/Annexin V assay (IM-54 0μM: 48.3 ± 12.4 %, IM-54: 10 μM 74.2 ± 8.3 % in PI/Annexin V double negative fraction at eltrombopag 25μM, p<0.01). The addition of Caspase-Inhibitor III (Boc-D-FMK) did not cancel the growth inhibition by eltrombopag in MTT assey. We measured GSH concentration of HepG2 cells. After the treatment of eltrombopag for 72 hrs, GSH decreased at 12.5μM of eltrpmbopag, however IM-54 restored the GSH concentration (83 mM at IM-54 0μM and eltrombopag 0 μM, IM-54 0 μM: 42.0 mM and IM-54 10μM: 83.3 mM at eltrombopag 12.5μM). Similar data were obtained in the study using Hepa 1–6 and NCTC clone 1469. Conclusion: Basic structure of eltrombopag is similar to that of 3-bipheyl-carboxylic acid, which is a strong oxidizing agent. Taken together, eltrombopag have an oxidative stress on hepatocytes to result in hepatotoxicity. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone marrow reticulin and collagen content in patients with adult chronic immune thrombocytopenic purpura: A Danish nationwide study

We quantified and differentiated reticulin and collagen content in bone marrow specimens from chronic immune thrombocytopenic (ITP) patients and examined the correlation between some clinical characteristics and the fibrosis grading. Through the Danish National Patient Registry, we identified 378 patients with chronic ITP from 1997 until 2007. Of these, 253 (67%) had undergone at least one bone marrow biopsy, and we retrieved the bone marrow specimens from 187 (74%). We graded the bone marrow content of reticulin and collagen according to the Thiele scale (Grade 0-3). We also retrieved information on patients' clinical characteristics. We examined the prevalence of bone marrow fibrosis grading > 0 by patients' age (≤ 75 years and > 75 years), sex, platelet count at baseline (< 30 × 10⁹/L, and ≥ 30 × 10⁹/L), splenomegaly, hepatomegaly, and medications. In total 75 chronic ITP patients (40%) had a bone marrow grading >0. Of these, 72 (39%) had Grade 1 reticulin fibers present. Only three patients (< 2%) had collagen fibers present: two had Grade 2 and one had Grade 3. The prevalence of bone marrow grading > 0 was lower in patients aged > 75 years than ≤ 75 years (prevalence ratio = 0.64, 95% CI: 0.36-1.15) and lower in men than women (prevalence ratio = 0.70, 95% CI: 0.45-1.09), while a baseline platelet count ≥ 30 × 10⁹/L was associated with a higher prevalence of grading > 0 (prevalence ratio = 1.24, 95% CI: 0.81-1.86). Thus, bone marrow reticulin and collagen content in chronic ITP patients may be associated with some clinical characteristics.

Differences in Platelet α-granule Release between Normals and Immune Thrombocytopenic Patients and between Young and Old Platelets

The risk of serious bleeding in patients with immune thrombocytopenic purpura (ITP) appears to be less than in comparably thrombocytopenic patients with megakaryocytic hypoplasia. It has been proposed that this difference is due to enhanced hemostatic activity of young platelets, which are increased in the circulation during ITP. We examined alpha-granule release in reticulated platelets (RP), which are thought to be the youngest circulating platelets, and in older non-reticulated platelets (non-RP) in normal human controls and ITP patients. Normal controls had a mean RP of 7%, compared with 42% in ITP patients. The mean concentration of thrombin receptor agonist peptide (TRAP) causing 50% of control RP to express CD62P (EC50) was 0.82+/-0.08 microM (SEM), significantly higher than the TRAP CD62P EC50 for RP in ITP, 0.57+/-0.06 microM (p = 0.04). Similarly, the TRAP EC50 for non-RP in controls, 0.84+/-0.09 microM, was significantly higher than in ITP, 0.56+/-0.07 microM (p = 0.03), suggesting that all platelets in ITP have an enhanced alpha-granule threshold response to TRAP compared with controls, while RP and older platelets within each patient group have similar threshold sensitivities to TRAP. By contrast, high-dose TRAP caused RP to express twice as much mean and total CD62P as non-RP in both ITP patients and controls (p <0.05 for both comparisons). We conclude that compared with controls, all platelets in ITP are primed to undergo alpha-granule release to TRAP, while in both ITP and controls, the newly circulating, reticulated platelets have the potential to contribute greater amounts of CD62P surface ligand compared with older platelets (non-RP) after stimulation. Both the increased RP% and enhanced platelet response to agonist in ITP may contribute to maintenance of hemostasis despite thrombocytopenia.

Immune thrombocytopenia and Fc receptor-mediated phagocyte function

The response to intravenous immunoglobulin treatment (IVIG) is thought, in part, to be due to blockade of Fc receptor for IgG in the mononuclear phagocyte system (MPS). We have studied this by measuring splenic clearance of heat-damaged and IgG antibody-coated red cells in immune thrombocytopenic patients receiving IVIG. Clearance of heat-damaged red blood cells (HDRBC) labelled with technetium 99MM was measured in eight patients before and after a 5-day infusion of IVIG. In six of the eight there was an enhanced clearance of HDRBC post IVIG. Clearance studies were performed in eight rhesus-D-positive patients using autologous red cells coated with anti-D and labelled with 99MTc. The time taken for the radioactivity at 3 min to fall to 50% (T1/2) was calculated. The T1/2 increased in seven of the eight patients from a mean of 78.5 min to 137.1 min, a prolongation of 74.6%. In addition, we have examined the effect of IVIG on Fc-mediated phagocytosis by neutrophils. Platelet phagocytosis by patients' neutrophils was measured using opsonized autologous platelets in nine patients. There was a marked reduction in platelet phagocytosis as measured by formazan production in all patients at day 4-5 when compared with the preinfusion value, and in half of these this reduction was still evident at further studies, 1 week after the end of the infusion period. The platelet count rose in parallel with the fall in platelet phagocytic ability of the neutrophils, reaching a peak at 7 days post infusion, and fell with the recovery of phagocytic function in weeks 2 and 3. These findings suggest that IVIG has a marked effect on Fc-mediated phagocytosis.

Detection of elevated anti-idiotypic antibody levels in immune thrombocytopenic patients expressing antiplatelet antibody [letter

Detection of elevated anti-idiotypic antibody levels in immune thrombocytopenic patients expressing antiplatelet antibody [letter

Measurement of platelet surface-bound IgG by a monoclonal 125I-anti-IgG assay.

We have described the use of a monoclonal 125I-labeled anti-IgG (125I-MA) to assay IgG antibody displayed on the surface of platelets from normal and immune thrombocytopenic patients and reported levels of IgG 10-100-fold lower than previous studies. This report describes the immunologic characteristics of the 125I-MA and the assay for surface IgG. The 125I-MA has a high binding affinity for surface-displayed IgG (2.22 X 10(9) M-1), reacts equally well with all four subclasses of IgG and not at all with IgM or IgA. In our assay, the binding of 125I-MA was found to be greater than or equal to 99% specific for IgG (no nonspecific association of 125I-MA with platelets) and the binding ratio of 125I-MA to IgG displayed on the cell surface was 0.91 (close to unity). Finally, platelet lysates were found to contain large amounts of IgG protein (39,597 +/- 27,418 molecules/platelet) as compared to surface-displayed IgG (124 +/- 86 molecules/platelet). This assay has excellent characteristics for quantitation of IgG on platelets and the discrepancy with other techniques may, in part, be due to intentional or inadvertent lysis of platelets during assay conditions.

Measurement of Platelet Surface-Bound IgG by a Monoclonal 125I-Anti-IgG Assay

We have described the use of a monoclonal l25I-labeled anti-IgG (125I-MA) to assay IgG antibody displayed on the surface of platelets from normal and immune thrombocytopenic patients and reported levels of IgG 10-100-fold lower than previous studies. This report describes the immunologic characteristics of the 125I-MA and the assay for surface IgG. The 125I-MA has a high binding affinity for surface-displayed IgG (2.22 x 10^9 M^-1), reacts equally well with all four subclasses of IgG and not at all with IgM or IgA. In our assay, the binding of 125I-MA was found to be ≥99% specific for IgG (no nonspecific association of 125I-MA with platelets) and the binding ratio of 125I-MA to IgG displayed on the cell surface was 0.91 (close to unity). Finally, platelet lysates were found to contain large amounts of IgG protein (39,597±27,418 molecules/platelet) as compared to surface-displayed IgG (124±86 molecules/platelet). This assay has excellent characteristics for quantitation of IgG on platelets and the discrepancy with other techniques may, in part, be due to intentional or inadvertent lysis of platelets during assay conditions.