Immune Thrombocytopenic Purpura Group Research Articles

The diagnostic accuracy of mean platelet volume in differentiating immune thrombocytopenic purpura from hypo-productive thrombocytopenia: A systematic review and meta-analysis.

Thrombocytopenia is defined as a decreased number of platelets in the circulating blood as a result of hypo-proliferation in marrow or peripheral destruction of platelets. Several diagnostic methods have been proposed to discriminate the underline cause of thrombocytopenia. Recent studies showed that mean platelet volume (MPV) could be used for differential diagnosis of immune thrombocytopenic purpura (ITP). Thus, we aimed to investigate the diagnostic accuracy of MPV for differential diagnosis of ITP from hypo-productive thrombocytopenia. This study was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines (PRISMA). The study protocol was registered on PROSPERO with the reference number CRD42023447789. Relevant published studies that were published up to April 10, 2023, in peer-reviewed journals were searched on electronic different databases. The methodological quality of the included studies was appraised using the quality assessment of diagnostic accuracy studies 2 (QADAS-2) tool. The pooled weight mean difference (WMD) of MPV between the ITP group and hypo-productive group was analyzed using a random-effects model meta-analysis. Relevant data were extracted using a Microsoft Excel spreadsheet and analyzed using STATA 11.0 and Meta-disc 1.4 software. Publication bias was evaluated using Deek's funnel plot asymmetry test. A total of 14 articles were included in this systematic review and meta-analysis. The comparison of MPV between groups revealed that the pooled mean value of MPV increased significantly in ITP patients compared to patients with hypo-productive thrombocytopenia (WMD = 2.03; 95% CI, 1.38-2.69). The pooled sensitivity and specificity of MPV in differentiating ITP from hypo-productive thrombocytopenia were 76.0% (95% CI: 71.0%, 80.0%) and 79.0% (95% CI: 75.0%, 83.0%), respectively. The summary positive likelihood ratio (PLR) and negative likelihood ratio (NLR)using the random effects model were 3.89 (95% CI: 2.49, 6.10) and 0.29 (95% CI: 0.18, 0.46), respectively. MPV can be used to discriminate ITP from hypo-productive thrombocytopenia. It can possess large advantages as it is noninvasive, simple, quick, inexpensive, easy to perform, reliable, and routinely generated by automated cell counters.

Cell Death Markers in Children with Immune Thrombocytopenic Purpura: A Preliminary Study

Immune thrombocytopenic purpura (ITP) is an autoimmune disease with possible dysregulation of the apoptotic pathways. We aimed to evaluate the possible role of some apoptotic markers (caspase 3, caspase 8 and BCL2) in the pathogenesis and course of ITP. We investigated some apoptotic markers (caspase 3, caspase 8 and BCL2) using the flow cytometry in 60 children with newly diagnosed ITP, 20 children with chemotherapy-related thrombocytopenia (CRT) and 20 healthy children. We also assessed the effects of intravenous immunoglobulin (IVIG) and methyl prednisolone therapies on the platelet apoptosis in children with newly diagnosed ITP. We demonstrated significantly higher values of caspase 3 in the newly diagnosed ITP group than control and CRT groups, and non-significantly higher values of caspase 8 in the ITP group than the healthy group. After IVIG treatment, the platelet count increased in all patients, and there was a significant decrease in caspase 3 and caspase 8 levels while BCL2 level increased. Regarding methylprednisolone treatment, there was a significant decrease in BCL2 and caspase 8 levels while caspase 3 levels did not significantly decrease. There is a possible role of the caspase dependent cell death pathway of the platelets in the occurrence of newly diagnosed ITP. There is heterogeneity in the apoptotic changes of newly diagnosed ITP children who received IVIG versus those who received methylprednisolone.

Immunological markers changes in pediatric immune Thrombocytopenic Purpura

Background Individuals with immune thrombocytopenic purpura (ITP) have cellular immune responses that show the characteristics of tolerance mechanisms that have broken down. Objectives This study aimed to assess the percentage of regulatory T cells (Tregs) and CD4 lymphocytes that express CXCR6 in untreated newly diagnosed ITP in pediatric patients and their correlation with each other, the platelet (PLT) count and other PLT indices. Patients and methods A case–control study was carried out that included 40 pediatric patients with ITP, and 20 age-matched and sex-matched healthy controls were selected from Benha Children Hospital hematology clinic. Assessment of CD4+, CD25+, and FoxP3+as markers of Treg cells and also expression of the CXCR6 receptor on CD4+ T lymphocytes to evaluate the percentage of Tregs and CD4/CXCR6 +T lymphocytes were investigated by flow cytometry. Results There was a marked reduction in CD4/CXCR6 and Tregs (P=0.02 and 0.001) in the ITP group versus the control group. In contrast, there was a marked increase in CD4/CD25+cells (P=0.002) in the ITP group compared with the control group. There was a considerable reduction in the PLT count and the hemoglobin concentration in the ITP group compared with the control group (P=0.000 and 0.01), while there was a significant increase in large platelet cell ratio and platelet distribution width in the ITP group in comparison to the control group (P=0.04 for both of them); also, there were insignificant differences in total leukocytic count and mean platelet volume in the ITP group in comparison to the control group (P=0.28 and 0.77). Conclusion There was a significant decrease in the concentration of CD4+ cells that express CXCR6+ and Treg percentage in ITP patients as compared with the controls, which may explain the defect in immune self-tolerance in ITP patients.

CHARACTERISTIC OF TIM-3 EXPRESSION IN T CELL SUBSETS IN PATIENTS WITH IMMUNE THROMBOCYTOPENIC PURPURA

T cell dysregulation is a common event involved in immune thrombocytopenic purpura (ITP). Recent findings indicated that aberrant expression of immune checkpoints may be related to autoimmune disease pathogenesis as well as in ITP. However, the distribution of Tim-3 and its co-expression with exhausted phenotype in T cell subsets remain unclear. In this study, the frequency of T-cell immunoglobulin mucin 3 (TIM-3) expression and co-expression with CD244 and CD57 in CD3+, CD4+ and CD8+ T cells were evaluated in peripheral blood (PB) from 16 cases with ITP and 21 healthy individuals (HI). Overall, the frequency of Tim-3+CD3+, CD4+ and CD8+T cells in ITP is similar to HI. However, Tim-3+CD244+CD8+ and Tim-3+CD57+CD8+T cells were increased in ITP in compared with HI group. While the percentage of CD244+ and CD57+CD3+, CD4+ or CD8+T cells was similar between ITP and HI groups. Moreover, the Tim-3+CD244+CD3+ and Tim-3+CD244+CD8+ T cells in newly diagnosed ITP group were higher than that in relapsed ITP group, similar trends showed in Tim-3+CD57+CD3+T cells. Obviously, relative higher Tim-3+T cells in most T cell subsets were detected in a case with acute ITP with viral infection. The distribution of Tim-3+ T cells in different subsets was also compared between PB and bone marrow (BM). High trends of Tim-3+T cells was detected in BM group. In conclusion, we firstly characterized T cell immune disorder with higher level of Tim-3+exhausted+CD8+T cells in ITP, the effect and mechanism of such alteration in ITP is needed further investigation.

Mean Platelet Volume, Platelet Distribution Width, and Platelet Count, in Connection with Immune Thrombocytopenic Purpura and Essential Thrombocytopenia.

To examine the kinetic characteristics of platelet (PLT) destruction and thrombopoiesis by using mean platelet volume (MPV) and platelet distribution width (PDW). Using the ADVIA2120i instrument, we measured PLT counts, MPV, and PDW in 153 healthy individuals, 35 patients with immune thrombocytopenic purpura (ITP), and 48 patients with essential thrombocytopenia (ET). In the ITP group, the MPV and PDW were higher than those values in healthy individuals. In the ET group, the MPV was lower than in the ITP group and in healthy individuals, and the PDW was lower than in the ITP group. When the ITP group was subdivided (PLT counts <45 × 103/µL vs ≥ 45 × 103/µL), the MPV and PDW tended to be higher in patients with PLT counts less than 45 × 103 per µL. When patients with ET were subdivided (PLT counts <770 × 103/µL vs ≥770 × 103/µL), the MPV and PDW were lower in patients with PLT count of 770 or greater × 103 per µL. In ITP, the overall PLT composition varies, and PLT sequestration is nondiscriminatory. In ET, PLTs quickly shrink and remain small, resulting in a high proportion of small-sized PLTs.

Association of IL-17A and IL-17F Gene Polymorphisms with Acute Immune Thrombocytopenia in Egyptian Children

Background: IL-17 is an inflammatory cytokine that plays a crucial role in many autoimmune diseases. Aim: To investigate the association of IL-17A rs2275913 and IL-17F rs763780 gene polymorphisms with acute immune thrombocytopenic purpura (ITP) in Egyptian children. Patients and methods: We examined 80 patients (male/female, 33/47; median age, 7 years old) diagnosed with acute ITP and 55 healthy controls (male/female, 28/27; median age, 7 years old). Genotyping was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: In the acute ITP group compared to control, statistical analysis of the genotype frequencies (GG, AG, AA) of the IL-17A rs2275913 polymorphism and its alleles (A, G) showed no significant difference between the two groups (p > 0.05). Interestingly, the IL17A rs2275913 GG genotype was associated with early recovery (p = 0.04). As regard the genotype frequencies of the IL-17F rs763780 polymorphism, there was statistical significant difference in the TT and TC genotype frequencies between the case and control groups (p = 0.001 and p = 0.003, respectively). The number of IL-17F rs763780 T alleles was significantly higher in acute ITP patients as compared with children in the control group (p Conclusion: The present findings indicate that the IL-17 polymorphism IL-17F rs763780, but not IL-17A rs2275913 may be associated with a higher risk of acute ITP in Egyptian children.

Vitamin D receptor polymorphisms in immune thrombocytopenic purpura.

Vitamin D receptor (VDR) polymorphisms have been studied in immune-mediated disorders, but not yet in immune thrombocytopenic purpura (ITP). We investigated whether VDR variants were associated with ITP in children. The study included 44 children with a diagnosis of ITP and 100 healthy controls. Five VDR polymorphisms (Cdx-2, FokI, BsmI, ApaI and TaqI) were genotyped and used to evaluate the association of VDR variants with ITP. The distribution of the three Cdx-2 genotype groups (GG, GA, and AA) was significantly different between ITP patients and controls (P = 0.025); the homozygous GG genotype of Cdx-2 was overrepresented in ITP patients. The frequency of the A allele of Cdx-2 was significantly different between patients and controls (P = 0.01). The A allele of Cdx-2 was associated with a decreased risk of ITP (OR, 0.343; 95% CI: 0.150-0.782). No statistically significant difference was found between the ITP group and control group for Fok1, Bsm1, Apa1, and Taq1 polymorphisms (P > 0.5). There appears to be an interaction between the Cdx-2 variant of VDR and childhood immune thrombocytopenia.

Fcγ Receptor Gene Polymorphisms in Childhood Immune Thrombocytopenic Purpura

Immune thrombocytopenic purpura (ITP) is a common hematological disorder in the childhood, and it is one of the most common forms of autoimmune disease in pediatric patients. The ITP basis is a primary dysfunction of the immune system. This study aimed to analyze the genetic polymorphisms of the Fcγ receptors IIA and IIIA. The genetic polymorphisms of the Fc receptors γIIA (131H/R) and γRIIIA (158V/F) were analyzed by polymerase chain reaction-restriction fragment length polymorphism technique. Odds ratio and 95% confidence interval were calculated by χ(2) test. Homozygous polymorphic genotype for the FcγRIIIA was significantly more frequent among patients compared with controls (odds ratio = 0.27; 95% confidence interval, 0.09-0.80; P = 0.03). There was no statistical difference between the ITP group and the controls in the analysis of combinations of alleles of the high-affinity Fc receptor, but the ITP individuals with this combination had a lower duration of disease (P = 0.01). Genetic polymorphisms in immune system genes can be important for ITP pathogenesis and disease outcome.

The Association of Oxidant Status and Antioxidant Capacity in Children With Acute and Chronic ITP

This study was undertaken to investigate oxidant and antioxidant systems in patients with immune thrombocytopenic purpura (ITP). With this purpose, we measured the levels of serum malondialdehyde (MDA), total antioxidant capacity (TAC), total oxidant status (TOS), and other oxidative stress parameters. Fifty-two pediatric patients with ITP (25 acute, 27 chronic) and 21 healthy children were included in the study. Patients with acute ITP were studied, before and after, methylprednisolone treatment. Hemoglobin, hematocrit, platelet count, and TAC were statistically significantly lower in patients with acute ITP before treatment than those in the control group (P<0.05). In addition, in this group, MDA, TOS levels, and OSI (oxidative stress index) were found to be higher than those in the control group. In chronic ITP group, although hemoglobin hematocrit, platelet counts, and TAC levels were statistically significantly lower than those in the control groups,the mean platelet volume, MDA, TOS, and OSI were found to be statistically significantly higher (P<0.05). Platelet count and mean platelet volume values were statistically significantly lower in patients with acute ITP before treatment than after treatment (P<0.05). We also found a positive correlation between thrombocyte count and TAC, in patients with acute ITP before treatment (r: 0.601, P<0.001) and acute ITP after treatment (r: 0.601, P<0.001) and chronic ITP (r: 0.601, P<0.001). A negative correlation was found between thrombocyte count and serum MDA levels, in patients with acute ITP before treatment (r: -0.356, P<0.001) and acute ITP after treatment (r: -0.356, P<0.001) and chronic ITP (r: -0.356, P<0.001). We also found a negative correlation between thrombocyte count and serum OSI, in patients with acute ITP before treatment (r: -0.494, P<0.001) and acute ITP after treatment (r: -0.494, P<0.001) and chronic ITP (r: -0.494, P<0.001). A negative correlation was found between thrombocyte count and TOS, in patients with acute ITP before treatment (r: -0.470, P<0.001) and acute ITP after treatment (r: -0.470, P<0.001) and chronic ITP (r: -0.470, P<0.001). In conclusion, increased MDA, TOS and OSI, and decreased TAC levels were found in patients with acute and chronic ITP. On the basis of these findings, we suggest that free oxygen radicals may have an effect on the structural and functional damage of platelets, and on the mechanism of thrombocytopenia in both, acute and chronic ITP.

Alemtuzumab and Rituximab in the treatment of refractory autoimmune Cytopenias.

Abstract 4458 IntroductionThe combination of rituximab and alemtuzumab have been used to treat CLL. This combination is feasible without excessive toxicity. Both antibodies have been used as monotherapy for the treatment of autoimmune diseases, including autoimmune hemolytic anemia (AHA) and Immune thrombocytopenic purpura (ITP). ObjectiveEvaluate the efficacy and safety of the combination of low dose alemtuzumab and rituximab in patients with AHA and ITP. Material and methodspatients diagnosed with AHA (clinical and biochemical data of hemolysis) or ITP (less than < 50 K/μl) who failed or relapsed after first line therapy (steroids) were included. Basal lymphocyte count and immunoglobulin levels were assessed. Patients received alemtuzumab 10 mg SC on days 1,2 and 3 followed by rituximab 100 mg IV on days 4,11,18 and 25. Complete response (CR) was defined as normal hemoglobin in the absence of hemolysis and platelet count >150K/μl; and partial response (PR): increase of 2 g/dl of hemoglobin and platelets 50-149K/μl The effect of the antibodies combination was evaluated by lymphocyte B, T and NK count 8 and 24 weeks after their administration. Prophylactic bactrim, acyclovir and itraconazole were given for the first 2 months. ResultsWe included 13 patients, and eleven were evaluable, 5 patients in the AHA group and 6 in the ITP group, demographic data is shown on table 1. For AHA the 5 patients achieved CR with a median follow up of 37 weeks; only 1 patient relapsed after the 49th week of follow up. For the ITP group 2 patients achieved CR, 2 patients PR and 2 were classified as non responders. Alemtuzumab and rituximab combination quickly depleted B lymphocites falling to zero or near zero at the 8th week determination, with a sustained decrease at 24th week, in which levels did not return to pre-treatment levels. ConclusionsThese initial results indicate that low dose alemtuzumab and rituximab combination is safe and demonstrate promising activity for AHA and ITP refractory patients including durable complete responses and as a steroid spring therapy.Table 1DiagnosisAgeSexResponseFollow up (weeks)ITP26FNR44ITP16FNR52ITP71FPR54ITP33FCR39ITP20FPR36ITP25FCR15AHA60FCR34AHA71FCR54AHA31FCR37AHA31MCR36AHA67MCR44 Disclosures:Off Label Use: alemtuzumab for refractory autoimmune cytopenias rituximab for refractory autoimmune cytopenias.

Elevated Complement (C) and IgG Bound Microparticles (MP) in Immune Thrombocytopenic Purpura (ITP) and Hemolytic Anemia (HA).

Abstract 1313Poster Board I-337 BackgroundCell derived microparticles (MP) are shed during cell activation and apotosis and MP profiles reflect the status of cell disturbances in various forms of pancytopenias. It was demonstrated that when antibodies fix complement (C) on the membrane, red cells evade C mediated lysis by shedding MP with bound C. We analyzed C and IgG bound to MP to gain insight on the underlying mechanisms of cell destruction. We measured C1q fragment and IgG on cell-derived (MP) in plasmas of patients with ITP, hemolytic anemias (HA) and thrombosis (TBS). Methods(1) Patient population. Consenting patients consisted of 18 TBS, 14 ITP and 6 HA (5 AIHA and 1 TTP), as well as 20 normal controls (NC). (2) Flow cytometry. MP were centrifuged from 1 mL fresh (not frozen) platelet-poor plasma (PPP), washed twice with saline, resuspended in 100 μL, then incubated with fluorescent mAb to C1q and IgG, then analyzed by flow cytometry. In addition, MP in the PPP were analyzed for MP from RBC (RMP) using marker glycophorin A, and MP from platelets (PMP) by CD42b. Values were considered elevated if >2SD above the mean of NC. Control values were (mean ±SD per μL): C1q+ MP = 536 ±151; IgG+ MP = 5,542 ±2,081; RMP = 823 ±246; PMP = 7,520 ±2,084. ResultsWe observed significant elevation of C1q+ and IgG+ MP in patients with ITP (2-3 fold) and hemolytic anemias (6-10 fold) but not in those with thrombosis. These findings indicate that complement mediated cell destruction or disturbance in these disorders is frequent. (1). The ITP group consisted of 2 subgroups, one of which had elevated C1q+ and IgG+ MP, the other not, and these subgroups also differed in RMP. Specifically, six ITP with high C1q+ MP also had high RMP (1,899 ±682 /μL) and PMP (16,602 ±4,216 /μL) while those with normal C1q+ MP had normal RMP (504 ±186 /μL) and PMP (3,472 ±1196 /μL). This suggests that platelet destruction in ITP can proceed via C in some but not all cases, probably depending on the autoantibodies. We have previously reported high RMP in ITP. These findings suggest subclinical C mediated hemolysis in a subset of ITP. (2) In HA patients, all 6 had elevated C1q+ MP (3,934 ±1,419 /μL, p<0.001) as compared to normal controls (536 ±151 /μL). The mean in HA was nearly 6-fold greater than NC. The HA group also had higher IgG+ MP, with mean counts about 10-fold higher than NC (61,531 ±20,733 vs. 5,542 ±2,081 /μL, p<0.001). Furthermore, the HA patients also had elevated RMP (2,191 ±635 /μL, p<0.01). This suggests that C-mediated destruction of RBC is a major mechanism in HA. (3) Linear regression analysis showed that C1q+ MP is well correlated with IgG+ MP (R = 0.84, p<0.0001) and RMP (R = 0.79, p<0.001). (4) The TBS group did not show higher levels of any of the measures assayed.It is widely believed that phagocytosis is the mechanism of cell destruction in ITP and AIHA. Our findings support the concept that complement (C) -mediated platelet or red cell destruction play an important role and is common in these disorders. Assay of C and IgG on MP can provide new insight to underlying mechanisms of immune mediated platelet and red cell destruction. DisclosuresNo relevant conflicts of interest to declare.

Splenectomy for immune thrombocytopenic purpura

The therapeutic effects of splenectomy in 126 patients with various hematological disorders managed from 1982 to 2007 at Sheri Kashmir Institute of Medical Sciences, Srinagar (Jammu & Kashmir), India, were studied. Immune thrombocytopenic purpura (ITP) was the main indication for therapeutic splenectomy among all the hematological disorders i.e., 53 patients (42%). The ITP group is the main study group for this paper; mean age was 30 years (range 7-66), and the male to female ratio was 1.03:1. The mean platelet count in the pre-operative period was 30,650/mm3 (range 4000-85,000). All patients presented with thrombocytopenia i.e., platelet count of 50,000/mm3, age <50years, no concomitant disease and disease of shorter duration.

Increased Procoagulant Cell-Derived Microparticles (MP) in Splenectomized Patients with ITP: New Prediisposing Factors for Thrombosis.

INTRODUCTION: Splenectomy has been widely employed in the treatment of immune thrombocytopenic purpura (ITP) and various other disorders. Increased life threatening infection is well recognized but the long-term safety of splenectomy has not yet been delineated. Splenectomy has been associated with increased incidence of cardiovascular death [Lancet , 1977; 2 (8029):127–9] and ischemic small vessel disease leading to recurrent TIA and cognitive impairment [Thromb Res , 2000; 107: 337–44]. Recent studies have demonstrated that procoagulant MP play an important role in the pathogenesis of thrombosis and cardiovascular complications (JTH , 2005; 3: 884–7). In the present study, we investigated profiles of cell-derived microparticles (MP) and thrombotic events in ITP patients with / without splenectomy.METHODS: Fifty-seven patients meeting diagnostic criteria of ITP were studied. Excluded were those with HIV, viral hepatitis, or lupus anticoagulant. Fifteen had splenectomy (ITPs) while 42 had not (ITPns). The ITPs group comprised 11F, 4M, while ITPns group had 28F, 14M. The 2 groups were comparable in sex, ages and platelet counts. In the latter, 9 failed to undergo remission, having active ITP (ITPs-A) while the other 6 were in remission (ITPs-R) following splenectomy. In the ITPns group, 23 responded poorly to therapies (ITPns-A) while the remaining 19 were in remission (ITPns-R). CBC and platelet counts, blood chemistry and PT, aPTT were measured. Flow-cytometry was employed to assay cell-derived MP from platelets (PMP) by anti-CD41, from leukocytes (LMP) by anti-CD45, from red cells (RMP) by anti-glycophorin, and from endothelial cells (EMP) by anti-CD31+/anti-CD41-. All patients were followed and incidence of thrombotic events was evaluated.RESULTS: Data are summarized in Table 1. Mean values of EMP, LMP, PMP and RMP were all significantly higher except PMP in ITPs compared to ITPns: RMP (p=0.002), EMP (p=0.008), LMP (p=0.03). In addition, aPTT was significantly shortened in ITPs vs ITPns (p=0.0059), as well as in ITPs-A vs. ITPns-A (p=0.0025). In the ITPs-R group, the aPTT was shorter than ITPns-R but did not reach statistical significance. Thrombotic events were recorded in 6 of the 15 ITPs group (40%) but in only 4 of the 42 ITPns (10%), p<0.001. Of the 6 ITPs with thrombosis, 5 had TIA and one DVT. Of the 4 with ITPns, 3 had TIA and 1 DVT.CONCLUSIONS: (i) Cell-derived MP, which are known to be procoagulant and implicated in the pathogenesis of thrombosis and inflammation, are significantly elevated, (ii) along with significantly shortened aPTT and (iii) thrombotic events were more prevalent in those without spleen compared to those with spleen among ITP patients. These observations support the hypothesis that spleen is the main site of clearance of procoagulant MP, and that accumulation of procoagulant MP may increase risk of thrombotic complications following splenectomy. A larger-scale study is warranted to clarify the long term safety of splenectomy in ITP and other disorders.Table 1ITPs (no spleen)ITPns (with spleen)p valuePatients; n=1542EMP7303230.008PMP16147109950.11LMP181012380.03RMP277717590.002aPTT22.525.70.006Thrombosis40%10%0.002

Autoimmune thrombocytopenia: flow cytometric determination of platelet-associated autoantibodies against platelet-specific receptors

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. We here describe a feasible diagnostic method using the commonly available technique of flow cytometry. An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls. The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads. The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8-31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7-1.2), and in the healthy controls 1.0 (range 0.7-1.3). The average assay coefficient of variation was 0.218 [95% confidence interval (CI) 0.213, 0.221]. The difference between the ITP patients and both groups was highly significant (P < 0.001), using a stringent non-parametric analysis. A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R2 = 0.71, 95% CI 0.39, 0.53). The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033. We conclude that the present FCM assay is clinically useful for routine diagnosis and follow-up of ITP.

Prospective evaluation of the immunobead assay for the diagnosis of adult chronic immune thrombocytopenic purpura (ITP)

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-induced platelet destruction. To better define the role of antigen-specific assays in adult chronic ITP, we prospectively measured platelet-associated autoantibody against either glycoprotein (GP) IIb/IIIa or GPIb/IX in 282 patients with chronic ITP and 289 patients with thrombocytopenia of other causes. We divided chronic ITP into four subgroups: presplenectomy, mild (platelet count >30 000 micro (L-1) requiring no therapy), presplenectomy, severe (platelet count <30 000 micro L(-1) requiring therapy but not splenectomy), postsplenectomy, remission (postsplenectomy partial or complete remission without further therapy) and postsplenectomy refractory (required therapy after splenectomy failure). Positive results: total ITP group, 55.4%; presplenectomy, mild, 31.1%; presplenectomy, severe, 42.6%; postsplenectomy, remission, 50.0%; and postsplenectomy, refractory, 87.8%. In addition, the degree of positivity increased with the severity of the patient's disease. The assay had a minimum specificity of 84.4% if clinical factors, consistent with immune thrombocytopenia, were not considered in patients with thrombocytopenia associated with other diseases. However, if clinical factors consistent with immune thrombocytopenia were considered and only patients with questionable immune thrombocytopenia and patients 'lost to follow-up' were included in the false-positive group the specificity was 93.1%. We conclude that the presence of immune thrombocytopenia is highly probable if the immunobead assay is positive and that antigen-specific assays are diagnostically useful in adult chronic ITP.

Reticulated platelet counts in the diagnosis of acute immune thrombocytopenic purpura.

Bone marrow aspiration is often performed to diagnose childhood acute immune thrombocytopenic purpura (ITP) because no non-invasive investigation to confirm the diagnosis is routinely available. Reticulated platelets (RPs--young platelets characterized by a high RNA content--increase with increased platelet production and may be useful in the diagnosis of ITP. To assess the role of RP counts in distinguishing ITP, we compared counts from 15 consecutive patients with ITP with counts from 20 patients with acute lymphoblastic leukemia (ALL), 10 with aplasia, and 27 healthy normal children. Whole blood in edetic acid (EDTA) was labelled with a platelet-specific monoclonal antibody and incubated with thiazole orange (TO). A standard gate was set to achieve a fluorescence value of 1.3 +/- 0.5% for control lyophilized platelets. Patients with ITP had a mean (+/- 1 standard deviation) RP level of 32.9 +/- 10.2%; patients with ALL, 6.6 +/- 3.1%; patients with aplasia, 3.4 +/- 2.0%; and normal patients, 7.9 +/- 2.9%. The difference in RPs between the ITP group and the ALL, aplasia, and normal groups was highly significant (p < 0.0001 each), with no significant difference between the non-ITP groups (p = 0.12). Measuring RPs by this simple whole-blood cytometric technique discriminated very well between the acute ITP and non-ITP groups. This test has a strong positive predictive value and may prove very useful in the assessment of childhood acute ITP and the screening of candidates for bone marrow aspiration.

Original Article: Electrophoretic Properties of Platelets from Normal, Thrombotic and ITP Patients by Doppler Electrophoretic Light Scattering Analysis.

At fixed pH and ionic environment platelet net charge depends on the composition of surface glycoproteins and phospholipids. It is believed that this composition changes, becoming more negative in the course of activation, and that certain thrombotic disorders such as transient ischemic attacks (TIA) may be associated with platelet activation. To investigate this hypothesis we measured the electrophoretic mobility (U) of normal platelets, activated normal platelets and platelets from patients with TIA and immune thrombocytopenic purpura (ITP). Normal platelets gave U = -0.91 ± 0.05 pm cmh, while normal platelets activated by ADP gave -1.25 and by collagen -1.70. TIA patients (n = 22) had mean U = -1.14 ± 0.10 and the ITP patients (n = 37) had U= -1.07 ± 0.13. The U of both patient groups differed significantly from normal controls, P < 0.001. Indeed, only 2 of the 22 TIA cases had U within 2 SD of the control value. The ITP group included 10 patients with TIA-Like symptoms; the mean U of this subgroup was identical to that of the TIA group without ITP. We conclude that U is a useful measure for research on platelet abnormalities and may even be useful as a routine clinical tool. This work was greatly facilitated by the use of an automated instrument, the DELSA 440, allowing determination of U of a given sample in as little as 5 min.

Use of splenic ultrasound: a new wave for immune thrombocytopenic purpura.

To examine whether a therapeutic dose of ultrasound waves, when directed through the thoracic wall to the spleen, would significantly affect the platelet count in patients with stable immune thrombocytopenic purpura (ITP). Continuous ultrasound at 1 W/cm2 spatial average-time average (SATA) intensity for up to one minute/5 cm2 treatment field was well tolerated in 13 patients with ITP and one with non-Hodgkin's lymphoma. Five healthy controls were also similarly treated. Peak platelet increments occurred four to eight hours after ultrasound treatment in the ITP group (n = 16 treatments). The mean peak platelet increment was 6.25 x 10(9)/l with a 5% confidence interval of the mean (95% CI) of 3.32 to 8.93 x 10(9)/l (p = 0.0004). The mean peak platelet increment of normal controls was 6.6 (n = 5; 95% CI = -2.3 to 15.5; p = 0.21) and for sham treated patients it was 0.66 (n = 11; 95% CI = -1.5 to 2.8; p = 0.60). There was a significant inverse correlation between patient age in the ITP group and peak platelet increment (r = -0.60; p = 0.015). Splenic ultrasound is a novel approach to the treatment of ITP, and may find a place in its diagnosis or management.

Elevated Common Acute Lymphoblastic Leukemia Antigen Expression in Pediatric Immune Thrombocytopenic Purpura

Bone marrow examination is often performed in thrombocytopenic children to distinguish immune thrombocytopenic purpura (ITP) from acute leukemia. We describe a patient with thrombocytopenia and 50% common acute lymphoblastic leukemia antigen (CALLA) positivity in his marrow who was subsequently shown to have ITP. CALLA (CD10) is a surface antigen found in early B-lymphocytes and is elevated in most cases of childhood acute lymphoblastic leukemia (ALL). This case prompted us to prospectively study the frequency of immature lymphocyte populations in children with ITP. Fourteen patients with acute ITP and five with other conditions were studied. The two groups were comparable with respect to age: ITP mean, 4.3 (range 0.3-15.5) years; control mean, 5.8 (0.6-13.8) years. The ITP group had a significantly higher percentage of CD10 positive bone marrow lymphocytes (p = 0.007). Five of the 10 patients younger than 4 years of age in the ITP group had CD10 levels of greater than 30%, which is in the leukemic range, whereas none of the control patients had a CD10 levels of greater than 17% (p = 0.003). There was good correlation between CD10 positivity and B4 positivity indicating that both of these markers arise from the same population of immature B-lymphocytes. None of the ITP patients who were older than 4 years had a CD10 level of greater than 30%. We conclude that it is common to have an increase in the proportion of immature lymphocytes in the marrow of young children with ITP. The cause of this increase in CD10 positive cells is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies Against Platelet Membrane Glycoproteins in Children With Acute and Chronic Immune Thrombocytopenic Purpura

The autoimmune nature of chronic immune thrombocytopenic purpura (ITP) in adults is widely accepted. In contrast, the pathogenetic mechanism in acute and chronic ITP in children is not known. In this report, we studied 39 children with destructive thrombocytopenia, 15 patients with acute ITP and 24 patients with chronic ITP. Platelet autoantibodies to platelet glycoprotein IIb/IIIa were detected in 14 of 24 patients (58.3%) in the chronic ITP group and in four of 15 (26.7%) with acute ITP. Binding ratios (+/- SD) of positive patients were significantly greater (P = .01) in chronic ITP (8.0 +/- 9.1) when compared with those of acute ITP where the binding ratios were only slightly above the normal range (1.9 +/- 0.4). The results show that autoantibodies against platelet glycoproteins are present in the majority of children with chronic ITP confirming the autoimmune nature of this disorder. The minimal elevation seen in the positive children with acute ITP suggests a different pathogenetic mechanism. These data suggest that this approach may be useful in differentiating acute from chronic ITP patients.