Absolute Immature Platelet Fraction Research Articles

Immature platelet fraction levels predict the development of prolonged thrombocytopenia after haematopoietic stem cell transplantation

Prolonged thrombocytopenia (PT) is a serious complication after haematopoietic stem cell transplantation (HSCT). PT has been suggested to be associated with an increased platelet transfusion requirement and poor outcomes after transplantation. Due to the complex mechanism of PT development, it is difficult to diagnose in the early post-transplant period. Our study aimed to identify an early predictive marker for PT after HSCT. Previous studies showed that the clinical utility of immature platelet fraction (IPF) predicts platelet recovery after chemotherapy and successful engraftment. However, the relationship between IPF and PT after HSCT remains unclear. Fifty-two patients with malignant haematological diseases who underwent HSCT were included in the study. We observed the kinetics of recovery of haematological parameters after transplantation and performed receiver operating characteristics (ROC) curve analysis using data from the 52 HSCT patients. The days to rise and peak of IPF, absolute IPF count (A-IPF) and highly fluorescent IPF (H-IPF) were almost synchronised in all patients, at day 10 and day 15, respectively. The begin to rise levels of IPF, H-IPF and A-IPF were all significantly lower in the PT group than in the good engraftment (GE) group (p=0.0016, p=0.0094, p=0.0086, respectively). The peak levels of IPF were significantly lower in the PT group than the GE group (p=0.0036). However, the peaks of H-IPF and A-IPF were not statistically significant between the two groups (p=0.3383, p=0.0887, respectively). The area under the ROC curve (AUC) of IPF rise was 0.739 (95% CI 0.583-0.896; p<0.05) and the cut-off value was 3.5%, while the AUC of IPF peak was 0.800 (95% CI 0.637-0.962; p<0.01) and the cut-off value was 8.0%. In conclusion, early low levels of IPF predict the development of PT after HSCT. These findings may help improve the management and treatment strategies for PT after HSCT.

Abstract 1034: Cardiometabolic Risk Factor Burden Positively Associates With Platelet Immaturity And Size

Background: Cardiometabolic risk factors including, obesity, diabetes and hyperlipidemia contribute to increased cardiovascular disease (CVD). While platelets are involved in the pathogenesis of CVD, the relationship between risk factor burden on platelet indices remains uncertain. Indices of platelet size and immaturity can serve as surrogates for platelet hyperreactivity. Hypothesis: We hypothesized that greater cardiometabolic risk factor burden is associated with increased markers of platelet immaturity and platelet size. Methods: Blood was collected from CVD-free adults and platelet parameters including platelet count, mean platelet volume (MPV), percent immature platelet fraction (IPF), and absolute immature platelet fraction (AIPF) measured by CBC. Participants were stratified by number of cardiometabolic risk factors (diabetes, obesity (BMI &gt;=30 mg/kg 2 ), hyperlipidemia (LDL &gt;=130 mg/dL))We calculated median (IQR) values of platelet indices across categories of risk factor burden. We calculated a p-for-trend across risk factor groups via linear regression. Results: Among 141 participants, the mean age was 50.5 ± 14.8 years, 42% were male, 26% Black, 18% Hispanic, 50% had diabetes, 35% obesity and 53% hyperlipidemia. Overall, 14%, 48%, 28%, and 11% had 0, 1, 2 and 3 risk factors, respectively. No association was found between platelet count and risk factor burden. In contrast, risk factor burden was associated with increasing platelet size (p-for-trend 0.0047), percent IPF (p-for-trend 0.0086), and AIPF (p-for-trend 0.0005; Table ). Conclusions: Greater cardiometabolic risk factor burden is associated with increased platelet size and immaturity. These findings offer insight into potential mechanisms linking risk factor burden with CVD.

Performance of the Sysmex XN-V hematology analyzer in determining the immature platelet fraction in dogs: A preliminary study and reference values.

Immature platelets (IPs) are newly formed platelets released into circulation that have been demonstrated as good markers of thrombopoiesis. Although many flow cytometric and fully automated-based methods are available, the latest Sysmex XN-V hematology analyzer for veterinary use is equipped with a specific fluorescent platelet channel (PLT-F) that detects platelets using a platelet-specific dye. The aims of this study were to evaluate the performance of the Sysmex XSN-1000 V in determining the IPF (immature platelet fraction) and other selected PLT-F channel parameters and to propose IPF reference intervals (RIs) for canine blood samples. Canine EDTA blood samples were analyzed on the Sysmex XN-1000 V to assess linearity, imprecision, carryover, stability, and the effect of platelet clumping on selected platelet parameters from the PLT-F channel. We also reported the de novo generated RIs for the IPF in dogs. Imprecision was acceptable (CV <10%) for all parameters except for the absolute IPF values (IPF#), in which the reproducibility was 12.15% for the normal-low concentration samples. Linearity and carryover were excellent for all variables. Relative IPF values (IPF %) and IPF# remained stable for both storage conditions for up to 48 hours; however, a nonsignificant progressive increase in these parameters was observed from 12 hours at 4°C. We observed a statistical increase in IPF% and IPF# and a statistically significant decrease in PLT-F counts after intentional in vitro platelet aggregation. RIs were generated for all reference samples (n = 69) and for samples with (n = 25) or without (n = 44) platelet clumps. The performance of the new PLT-F channel-derived variables for dogs was excellent. Specific RIs for IPF should be used when platelet aggregates are present.

Immature Platelet Fraction and Its Kinetics in Neonates.

Thrombocytopenia is a common abnormality encountered in the neonatal period, and immature platelet fraction (IPF) may be an informative indicator of thrombopoiesis; however, data on IPF in neonates are scarce. To define reference intervals (RIs) and factors affecting IPF in neonates, we measured the IPF of 533 consecutive neonates. With a multiple regression analysis of 330 newborns with normal platelet counts at birth, premature delivery, neonatal asphyxia, intrauterine infection, chromosomal abnormalities, and respiratory disorders were identified as independent factors for IPF%. The RIs of IPF% and absolute IPF value in neonates were determined to be 1.3% to 5.7% and 3.2 to 14.5×10 9 /L, respectively. On day 14 after birth, IPF% increased to twice the value at birth and thereafter returned to the previous value on day 28. Reticulocyte counts, in contrast, were the lowest at day 14. IPF% was increased in 16 thrombocytopenic patients with various clinical conditions, especially those with immune-mediated thrombocytopenia. IPF in neonates may be evaluated essentially based on the same RIs as in adults, although some precautions must be taken when evaluating IPF in neonates in the first 2 weeks of life. IPF may be useful for evaluating thrombopoiesis and thrombocytopenia in neonates.

Immature platelets in patients with Covid-19: association with disease severity

Abstract Background Coronavirus disease 2019 (Covid-19) is associated with a high incidence of thromboembolic events, both venous and arterial. Currently, there are no clinical or laboratory markers to guide risk-stratification or antithrombotic therapy in Covid-19 patients. Circulating immature platelets represent a population of hyper-reactive platelets, which are associated with arterial thrombotic events. Objectives To assess whether the proportion of immature platelets in the circulation is associated with disease severity in patients with Covid-19 Methods This prospective study evaluated consecutive patients with COVID-19 admitted with various degrees of disease severity, as determined by the standard Covid-19 severity Score. Disease severity was evaluated during hospitalization. Immature platelet fraction (IPF) absolute number and percentage were measured on admission and at additional time points during the hospital course using the SysmexXN-3000 auto-analyzer. The maximal values of IPF% and absolute IPF was analyzed according to disease severity. Results A total of 136 consecutive patients with Covid-19 were recruited. Mean age was 60±19 years for patients with mild and moderate disease and 69±14 years for patients with severe disease, 52% with mild and moderate disease and 48% with severe disease were woman, 11% with mild and moderate disease and 20% with severe disease with concurrent cardiovascular disease The median of IPF% was higher in the severe COVID-19 group compared to patients with mild or moderate disease [4.2 (IQR 2.73–6.45) vs 5.8 (IQR 3.9–8.7), P=0.01, Figure 1)]. The median of IPF absolute number was also significantly higher in patients with severe disease comparing to patients with mild or moderate disease (4.2 (2.85–6.1) vs 5.1 (IQR 3.65–7.35), P&amp;lt;0.0001, Figure 2]. Conclusions Patients with severe Covid-19 have a higher level of IPF in the circulation than patients with mild or moderate disease. IPF may serve as a reliable prognostic marker for in-hospital disease severity in patients with Covid-19 Funding Acknowledgement Type of funding sources: None. Figure 1. IPF% (median, 95% confidence interval)Figure 2. IPF# (median, 95% confidence interval)

Effects of Analyzer Detection Method, Ethnicity, and Reference Individuals on Reference Interval of Immature Platelet Fraction.

Immature platelet fraction (IPF) is a new biomarker for thrombopoiesis and inflammation. However, the reference interval (RI) is wildly discrepant among published reports. This study aimed to establish the RI of IPF for a population in Taiwan and evaluate the effects the detection method of the analyzer, ethnicity, and reference individuals have on the RI of IPF. The RI of absolute IPF (A-IPF) and IPF% were established with healthy subjects from the outpatient services of the Health Management Department of Taichung Veterans General Hospital between January 1, 2015 and March 1, 2016. These values were used along with published reports for meta-analysis. A-IPF (109/L) and IPF% of Taiwanese were 6.9 - 7.6 and 3.1 - 3.4, respectively. Significant differences were found when performing paired comparisons of the RI of A-IPF and IPF% published in reports. For A-IPF, there was only one paired comparison with a significant difference (Z > 1.96) across 6 reports. Thus, the contribution of the factors examined on the RI of IPF cannot be determined. For IPF%, there were 8 paired comparisons with significant differences across 10 reports. The discrepancy rates of RI for IPF% were 41.2%, 50.0%, and 25.0% with the difference of reference individuals, the analyzer method, and ethnicity, respectively. The RIs of Taiwanese for A-IPF and IPF% were established. Furthermore, the analyzer detection method and the reference individuals contribute to the discrepancy of the RI for IPF% and should be considered cautiously when the value of IPF is interpreted.

Immature platelet fraction (IPF) as a predictive value for thrombopoietic recovery after allogeneic stem cell transplantation

We consecutively examined the utility of measurements of percentage of immature platelet fraction (IPF%) and absolute IPF number (A-IPF) in predicting thrombopoietic recovery in 15 adult patients who underwent allogeneic hematopoietic stem cell transplantation (allo-SCT). Four patients were excluded from the evaluation due to insufficient data. Platelet count and IPF were measured by Sysmex XN-1000 (XN), a newer generation analyzer. First, we confirmed that platelet count measured by XN was more accurate than by XE-2100 (XE). IPF measurement was effective to predict the recovery in 7 of the 11 patients examined. Moreover, IPF measurement, especially IPF% measurement, suggested accelerated platelet turnover in two patients who failed to achieve platelet recovery by day 60. In addition to IPF%, A-IPF showed a complementary role on the prediction of thrombopoietic recovery. The increase in IPF% was only transient, while A-IPF values showed lasting increase during platelet recovery. In two patients (cases 6 and 7) an increase in A-IPF, but not in IPF%, was observed during platelet recovery. Our data suggest that IPF% and A-IPF measured by XN are useful for the prediction of thrombopoietic recovery and the assessment of pathogenesis of thrombocytopenia in patients after allo-SCT.

Effects of Anti-Glycoprotein Antibodies on Response of Immune Thrombocytopenia Patients to Thrombopoietin Receptor Agonists and on Megakaryocytes Viability

BackgroundImmune Thrombocytopenia (ITP) is a bleeding disorder due to a combination of increased platelet destruction and reduced production, often secondary to anti-platelet/megakaryocyte antibodies.The presence of antibodies to glycoproteins (GP) IIb/IIIa (integrin αIIbβ3) and GPIb/IX, detected in majority of ITP patients, may correspond to different responses to treatment, i.e., anti-GPIb is associated with more severe disease, and less responsive to intravenous immunoglobulins and steroids.Thrombopoietin Receptor Agonists (TPO-RA) increase platelet production by stimulation of megakaryopoesis. Predictors of response to TPO-RA and influence of antibody profile on response are currently unknown. In our previous study we investigated Absolute Immature Platelet Fraction (A-IPF) prior to TPO-RA treatment and did not find a correlation between A-IPF, anti-GP antibodies, and platelet counts.The aims of this study were to further investigate:1. The role of anti-GP antibodies in response to TPO-RA;2. Effect of patients' antibodies on megakaryocyte (MK) viability, maturation, apoptosis and formation of proplatelets (in vitro);3. The influence of patients' clinical characteristics on response to TPO-RA.Materials and Methods91 patients with persistent or chronic ITP, were treated at Weill Medical College of Cornell University until January 2015 with TPO-RAs: 52 patients received eltrombopag, 22 romiplostim and 17 avatrombopag. Serum samples of 84 patients were analyzed for the presence of anti-GP by MAIPA assay as previously described.Patients with baseline platelet counts less than <30x109/L were defined as responders to TPO-RA if the average of their six median monthly platelet counts was ≥50x109/L and doubled from average baseline counts (prior to TPO-RA). Patients with baseline platelet counts 30-50x109/L were responders if the average platelet count was ≥75x109/L.MKs were derived from human umbilical cord blood stem cells as previously described. Cells were grown using SFEM medium, adding on day 0 of culture 50 ng/ml recombinant TPO and aliquots of serum of ITP patients or healthy controls. The percentages of immature (CD41+/CD42-), mature (CD41+/CD42+), viable and apoptotic MKs were analyzed by flow cytometry on day 12. Apoptosis was analyzed by measuring Mitochondrial Outer Membane Potential (MOMP) and Phosphatidyl Serine (PS) externalization. MKs were considered apoptotic if they had positive staining for PS externalization, viable if positive for MOMP, and dead if positive for 7-Aminoactinomycin D (7AAD).Proplatelet formation by MKs was analyzed by microscopy.Statistical analysis using unpaired T-test and Pearson correlation test were performed.ResultsNinety-one patients were included, 40 male (44%) and 51 female (56%), with a median age of 37.4 years (range 2-87). Median duration of ITP before TPO-RA treatment was 8 years (range 0.3-45).The 18/91 (19.8%) non-responders to TPO-RA were not different from the 73/91 responders in age, gender, number of prior treatments, duration of ITP, and past splenectomy. The presence of either or both anti-GP antibodies was correlated with average lower platelet counts on TPO-RA: 82.3 x109/L versus 123x109/L in patients without detected antibodies ("neither") (p=0.003). However, the response to TPO-RA was not influenced by the type of antibody: in patients with anti-GPIb the average platelet count was 76.1x109/L, and with anti-GPIIb/IIIa 80.7x109/L (Figure 1). In culture, excess dead MKs were found in anti-GPIb group and antiGPIb&antiGPIIb/IIIa group compared to "neither" group (p=0.0013 and p=0.027 respectively) and comparing antiGPIb&antiGPIIb/IIIa to control (p=0.0025). We did not observe changes in the degree of MK apoptisis or in MK maturation in the presence of serum antibodies. In cultures treated with serum of patients having anti-GPIb, less proplatelets were detected comparing to control (p=0.044) or to "neither" (p=0.0039).We conclude that patients with anti-GP antibodies respond less to TPO-RA, however there is no difference in response to TPO-RA between patients having anti-GPIb and anti-GPIIb/IIIa, unlike responses to other treatment modalities (e.g., steroids or immunoglobulins). TPO-RA could be a preferable treatment option in ITP patients having anti-GPIb. [Display omitted] DisclosuresOff Label Use: Eltrombopag, romiplostim and avatrombopag are a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. In some preliminary studies these medicines found as safe and effective treatment option in children and adolescents. Bussel:amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Novartis: Consultancy, Research Funding; Genzyme: Consultancy; BiologicTx: Research Funding; Ligand: Consultancy, Research Funding; Eisai: Consultancy, Research Funding; Shionogi: Consultancy, Research Funding; momenta: Consultancy; Protalex: Consultancy; Symphogen: Consultancy.

Numerical analysis of in vivo platelet consumption data from ITP patients.

BackgroundNumerical methods have recently allowed quantitative interpretation of in vivo murine platelet consumption data in terms of values for the random destruction rate constant (RD), intrinsic lifespan (LS), and the standard deviation of ln LS (SD), as well as the platelet production rate (PR) and age distribution (AD). But application of these methods to data obtained in thrombocytopenic patients is problematic for two reasons. First, such data has in all cases been obtained with radiolabeled platelets, and uptake of the radio-isotope by long lived cells complicates the analysis. Second, inferred values of the platelet production rate (PR) and random destruction rate (RD) are difficult to interpret, since increased RD can occur either as a cause or a consequence of thrombocytopenia.MethodsWe used a numerical method to analyze in vivo platelet consumption data from a series of 41 patients with immune thrombocytopenic purpura (ITP). An additional parameter, the fraction of labeled long-lived cells (LL), was evaluated concurrently with RD, LS, and SD. To provide a basis for interpreting these values, we used an iterative interpolation process to predict their response to different pathophysiologic mechanisms. The process also generates predicted effects on the widely used immature platelet fraction (IPF).ResultsOptimal parameter value sets were identified in 76 % (31 of 41) of the data sets. 27 of 31 ITP patients showed no substantial homeostatic increase in platelet production, with the remaining 4 showing both augmented platelet consumption and a compensatory increase in PR. Up to 1/3 of the patients showed the degree of increased RD expected to result from reduced thrombopoiesis only. “Jacknife” resampling yielded CV values of <0.5 in over 75 % of the evaluable data sets. Predicted platelet age distributions indicate that interpretation of the IPF and absolute IPF (aIPF) is a complex function of platelet count. We found, counter-intuitively, that reduced PR can increase the IPF, and increased RD can reduce the aIPF.ConclusionsOur findings support the feasibility of using numerical analysis to quantitatively interpret in vivo platelet consumption data, to identify likely etiologies of thrombocytopenias, and to assess the utility of IPF measurements in that context.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-015-0034-4) contains supplementary material, which is available to authorized users.

Application of immature platelet fraction absolute immature platelet fraction and thrombelastograph on assessment of bleeding risk in patients with immune thrombocytopenia

目的探讨未成熟血小板比例（IPF）、未成熟血小板绝对值（A-IPF）和血栓弹力图（TEG）在原发免疫性血小板减少症（ITP）患者出血倾向评估中的价值。方法采用ITP-BAT出血评分系统对271例ITP患者进行出血评分及出血程度分级，并行IPF、A-IPF检测，其中125例行TEG检测，分析ITP患者出血程度与IPF、A-IPF、TEG各指标的相关性。结果在271例ITP患者中，不同疾病分期和性别患者的出血程度差异无统计学意义（P>0.05）；儿童以轻度出血为主，与成人出血程度差异有统计学意义（P<0.05）；出血程度和血小板计数呈负相关（P<0.001）。在所有患者、PLT<30×109/L患者以及PLT<30×109/L儿童患者中，血小板计数与IPF呈负相关（P<0.05），与A-IPF、血栓最大幅度（MA）值呈正相关（P<0.05）。在所有患者及PLT<30×109/L患者中，出血程度和IPF呈正相关（P<0.001），与A-IPF、MA值呈负相关（P<0.001）。在PLT<30×109/L儿童患者中，出血程度与IPF、A-IPF、MA值均无相关性（P>0.05）。ROC曲线分析显示IPF、A-IPF、MA值评估ITP患者出血风险效能较好，ROC曲线下面积分别为0.745、0.744、0.813（P<0.001）。多因素分析显示IPF和MA值是预测ITP患者出血倾向的独立因素，综合诊断ROC曲线下面积0.846（P<0.001），优于单一指标。结论IPF、A-IPF和MA值能够准确评估ITP患者的出血风险，可以作为治疗的参考指标和疗效的观察指标。

Response to TPO-Receptor Agonists: Role of Immature Platelet Fraction and Anti-GP1b

Background: Thrombocytopenia in Immune Thrombocytopenia (ITP) results from a combination of increased platelet destruction and reduced production, both often secondary to anti-platelet antibodies. Absolute immature platelet fraction (A-IPF) is a measure of young reticulated platelets in peripheral blood, and therefore provides an assessment of platelet production. Decreased platelet production in patients with ITP has been addressed by the recently developed Thrombopoietin Receptor Agonists (TPO-RA), which stimulate megakaryopoiesis and thereby, in responders, increase platelet production to a level which overcomes platelet destruction. The majority of ITP patients will respond to these agents, but which ones will respond is not known.Aims: To exploreA) whether baseline A-IPF levels are associated with platelet response to TPO–RA, andB) whether antibodies to platelet glycoproteins 1b or β3 influence the platelet response to TPO-RA.Methods: Platelet counts and A-IPF values were collected from patients treated with TPO-RA (n=91) at Weill Medical College of Cornell University until 2013. All available counts were included, and the median count for each month was determined pre-treatment, and at 1, 2, 3, 4, 5, and 6 months. Patients were divided by whether (n=20) or not (n=71) they often received rescue therapy (i.e. IVIG and/or daily steroids >10mg).The 71 patients who received minimal or no rescue treatment were deemed responders if the average of their six median monthly platelet counts doubled from baseline and was >50x109/L. Based on pretreatment A-IPF, patients were stratified into three cohorts: low (0-25), middle (26-40), and high (41+). Clinical variables such as age, splenectomy status, duration of ITP, and gender were also studied. The 20 “rescue” patients were similarly analyzed.The 84 patients with available sera had their antibody levels measured to GP1b and β3 by Elisa by Dr. Ni. Samples were sent to the lab and analyzed without knowledge of response status or associated A-IPF level. Patients were divided into four categories for analysis of their antiplatelet antibodies: positive for anti-ß3 antibody, positive for anti-GP1b, positive for both, or negative for both.Fisher’s exact, student t-, and chi-square tests were used to analyze differences in response to TPO-RA among patient groups, A-IPF levels, and anti-platelet antibodies.Results:71 Patients with Minimal Rescue TherapyFifty-nine of the 71 patients responded to TPO-RA. There was no significant difference in A-IPF values for responders and non-responders (p=0.95; Figure 1). A significant positive correlation was observed between increase in A-IPF over the 6-month period and response rate to TPO-RA (p=0.009). Age, gender, duration of ITP, and splenectomy status neither correlated with response rate, nor trended with A-IPF cohorts. [Display omitted] 20 Patients with Rescue TherapyFor the 20 patients who often received rescue therapy, low response rates were seen in the low A-IPF cohort and high response rates in the higher A-IPF cohorts. The percent of responders to treatment was higher in patients with chronic ITP than with newly diagnosed and persistent ITP (p= 0.04). Age, gender, and splenectomy status showed no relationship with response to TPO-RA. In comparison with the 71 patient group, the 20 patients presented lower response rates in all groups, with the exception of females.Anti-platelet AntibodiesThere was a weak correlation between A-IPF and anti-GP1b antibody levels (R=0.225), demonstrating that antibodies to GP1b may have a specific impact on platelet production.Patients who did not have detectable antibody to either GP1b or β3 responded better to TPO-RA than patients who tested positive for both Anti-GP1b and Anti-ß3 (p=0.003). The strongest correlation was observed between level of anti-GP1b and response to TPO-RA (p<0.001; Figure 2). [Display omitted] Conclusion:Rather than baseline A-IPF levels or clinical variables, the best predictor of good response to TPO-RA therapy was the absence of anti-GP1b and anti- β3 platelet antibodies, especially anti-GP1b. An explanation for the dominant effect of antibodies to GP1b is that stimulating megakaryopoiesis with TPO-RA would not effect a platelet increase as these antibodies may block proplatelet extension, preventing platelet release into the circulation. This may also explain the effect of anti-GP1b on response to steroids and IVIG. DisclosuresOff Label Use: Eltrombopag is a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. Use in children and adolescents will be discussed.. Bussel:Amgen: Equity Ownership; GSK: Equity Ownership; Amgen: Membership on an entity’s Board of Directors or advisory committees; GSK: Membership on an entity’s Board of Directors or advisory committees; Amgen: Research Funding; GSK: Research Funding; Sysmex: Research Funding.

Classical Pathway of Complement Activation in ITP: Inhibition By TNT003, a Novel C1s Inhibitor

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet antibodies mediate accelerated platelet clearance from circulation and also inhibit platelet production, resulting in thrombocytopenia. Activation of the classical pathway (CP) of complement is associated with a variety of immune disorders involving the presence of autoantibodies. The role of the complement system in ITP is poorly understood.Methods: Plasma samples (0.32% sodium citrate) from patients with chronic ITP (n=55) were evaluated for their ability to activate the CP of complement. The 55 patients consisted primarily of adult but also of pediatric patients with ITP, undergoing various treatment regimens. The most common included IVIG, rituximab, and especially thrombopoietic agents (eltrombopag, romiplostim). Almost all patients included in the analysis had chronic ITP, defined as ITP lasting > 12 months. The complement activating capacity (CAC) of patient plasma was evaluated with a previously described in vitro assay ( Peerschke et al., Brit J Haematol, 2009) that measures complement activation on immobilized, fixed heterologous platelets using an ELISA approach with monoclonal antibodies to C1q, C4d, iC3b, and C5b-9. CAC represents assay optical density readings normalized to reference normal plasma pool. A CAC of >1.5 was considered indicative of enhanced complement activation, based on reference ranges established for plasma from healthy volunteers. Patient CAC values were correlated with platelet count. The ability of TNT003 to block in vitro complement activation was assessed relative to an isotype matched control. TNT003 is a mouse monoclonal antibody (IgG2a) that targets the CP-serine protease C1s.Results: A statistically significant (p=0.042) inverse correlation was noted between C4d deposition and platelet count in the 55 ITP patient samples tested. Heightened classical complement pathway activation was demonstrated in 7 of 55 patients (~13%) with ITP as evidenced by increased C4d deposition. 6 of the 7 patients with increased C4d deposition had platelet counts <100k/mcL, and 5 patients had platelet counts <50K/mcL. There was a non-significant trend for higher C4d levels on platelets and lower AIPF (absolute immature platelet fraction, equivalent to platelet reticulocytes). TNT003 (100 mcg/ml) inhibited C4 activation by 44 + 43% in ITP plasma in vitro. Inhibition of downstream complement activation, iC3b and C5b-9 deposition, was 72% + 17 % and 82% + 14% (mean + S.D.), respectively. Similar results were obtained using 10 mM EDTA, a known inhibitor of complement activation.Conclusions: The heterogeneity of patient responses to different treatment modalities in ITP support the concept of different immune mechanisms contributing to thrombocytopenia. Our data demonstrate classical complement pathway activation in a subgroup of patients with ITP, and further present the first evidence of CP complement inhibition by a novel C1s inhibitor in this setting. Failure to completely block C4 activation in ITP plasma in vitro by either of TNT003 or EDTA, suggests the presence of preformed, circulating C4d containing complement complexes in patient plasma. The ability of TNT003 to more completely inhibit C3 activation and C5b-9 assembly downstream of C4 in the in vitro assay system is consistent with direct activation and inhibition of complement at the platelet surface. Thus, TNT003 may mitigate enhanced platelet clearance by RES via inhibition of complement mediated platelet opsonization by C3b and platelet lysis by C5b-9. Further studies are required to evaluate the impact of TNT003 on thrombocytopenia in ITP. DisclosuresPeerschke:True North Therapeutics: Research Support Other. Panicker:True North Therapeutics: Employment. Bussel:True North Therapeutics: Research Support Other.

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia.

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A-IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A-IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10(9) /l and <30 × 10(9) /l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10(9) /l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10(9) /l, and children with PC <60 × 10(9) /l and <30 × 10(9) /l. A-IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10(9) /l, all children and children with PC <30 × 10(9) /l (r = -0·37; r = -0·34; r = -0·44; r = -0·60) versus ABS with PC (r = -0·36; ns; r = -0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A-IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A-IPF and ABS can be incorporated into routine or acute visits.

Steady Increment of Immature Platelet Fraction Is Suppressed by Irradiation in Single-Donor Platelet Components during Storage

Circulating immature platelet fraction (IPF) reflects real-time thrombopoiesis and correlates with platelet recovery from thrombocytopenic presentations. To understand the dynamics of IPF in platelet transfusions, we quantified the %-IPF in single-donor platelet components (SDP) during prolonged storage. %-IPF significantly increased from baseline by day 5 post-donation. Absolute IPF counts (A-IPC) had similar significant increments. However, gamma-irradiation suppressed the increments of %-IPF and A-IPC by >50%. Ultrastructural analysis of SDP units at day 10 showed well preserved morphology of immature platelets. Our findings suggest that IPF might actively expand ex-vivo and may have a longer shelf life than their mature counterparts. Closer study of IPF may be of critical clinical importance for transfusion practices.

Characterization Of The Immature Platelet Fraction (IPF) Parameters In Children With Immune Thrombocytopenia

BackgroundImmune thrombocytopenia (ITP) is characterized by impaired megakaryocte production and maturation, as well as premature platelet destruction. The effect of this disease on platelet production is not clear. The Immature Platelet Fraction (IPF) is a novel parameter available on the Sysmex XE-Series Hematology analyzers. It can be expressed as both IPF percent (IPF %) and absolute IPF count (AIPF#). It has been shown that IPF% is higher in Immune Thrombocytopenia (ITP) than in the normal population, and is an indicator of bone marrow platelet production or thrombopoiesis. ObjectivesTo characterizes the IPF parameters in pediatric patients with ITP at diagnosis and during different phases of the disease (newly diagnosed, persistent, and chronic). MethodThis is a retrospective, single institution study performed at a tertiary care pediatric hospital. We reviewed the medical charts and electronic databases of all patients with ITP who had measured IPF parameters between June 2011 and June 2013. The standard definitions and terminology of the International Working Group were used.Patient age, phase of ITP at the time of the blood test (newly diagnosed, persistent, and chronic), initial platelet count, IPF%, AIPF#, and type of therapy given (including observation alone) were recorded. We then compared the platelet count, IPF%, and AIPF# between the ITP phases at the time of initial CBC and looked at the relationship between the platelet count and IPF%.Comparisons between groups were performed using Kruskal-Wallis tests or Mann-Whitney tests, as appropriate. Association between measured blood parameters was measured using the Spearman correlation coefficient. Results•We included 47 patients; mean age was 9.4 years (1-18). At the time of the study,16 (34%) patients had newly diagnosed ITP (0-3 Months), 6 (12.8%) had persistent ITP (3-12 Months), and 25 (53.2%) had chronic ITP (>12 Months)•The initial platelet count was lower in newly diagnosed patients at 6x109/L (IQR 2-8), compared to patients with persistent ITP (16x109/L, IQR 7-24.5) and chronic ITP (30x109/L, IQR 17.5-53.5) (p=0.000).•IPF% was significantly higher in newly diagnosed ITP at 23.2% (IQR 19.6-27.8) compared to persistent ITP (17.4%, IQR 10.6-23.5) and chronic ITP (12.8%, IQR 7.3-17.1, p=.001) (figure 1A). In contrast, AIPF# was significantly lower in newly diagnosed ITP (1.1x109/L, IQR 0.44-1.5) compared to persistent ITP (1.7x109/L, IQR 1.1-4.4) and chronic ITP (3.5x109/L, IQR 2.0-4.5, p<0.001) (figure 1B).•IPF% correlated negatively with platelet counts for all patients (P=0.001) and this correlation was significantly more prominent in chronic ITP (figure 2). ConclusionIPF parameters may be useful in understanding the mechanism of ITP through the evolution of the disease. From the time of diagnosis of ITP through progression to chronic ITP, there is a steady decrease in IPF% and increase in AIPF# .The high IPF% in the newly diagnosed setting suggests that megakaryocytes are still active, whereas the lower IPF% in chronic ITP indicates inhibition of megakaryopoiesis or a different underlying pathophysiology. This may have important clinical implications when considering TPO receptor agonist therapy. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Utility Of The Immature Platelet Fraction In Predicting Recovery With Or Without Treatment In Pediatric Immune Thrombocytopenia

BackgroundThe Immature Platelet Fraction (IPF) is a novel parameter available on the Sysmex XE-Series Hematology analyzers. It can be expressed as both IPF percent (IPF %) and absolute IPF count (AIPF#). It has been shown that IPF% is higher in Immune Thrombocytopenia (ITP) than in the normal population, and correlates with bone marrow platelet production or thrombopoiesis. ObjectivesTo evaluates the utility of the IPF parameters to predict treatment response or recovery in pediatric ITP patients. MethodThis is a retrospective, single institution study performed at a 165 bed tertiary care pediatric hospital (Children's Hospital of Eastern Ontario, Ottawa, Canada). We reviewed the medical charts and electronic databases of all patients with ITP who had measured IPF parameters between June 2011 and June 2013. The standard definitions and terminology of the International Working Group were used.Patient age, phase of ITP at the time of the study (acute, persistent, and chronic), initial platelet count, IPF%, AIPF#, and type of therapy given (including observation alone) were recorded. For patients who responded to treatment or had spontaneous recovery without treatment, we analyzed the platelet count, IPF% and AIPF# on subsequent CBCs, then looked at the course of IPF% during platelet count recovery.Comparisons between groups were performed using Kruskal-Wallis tests or Mann-Whitney tests, as appropriate. Association between measured blood parameters was measured using the Spearman correlation coefficient. Within-patient Spearman correlations were also used to study the association between IPF% and platelet counts during recovery. The combined significance of these associations was determined using Stouffer's method. Results•47 patients were included, median age was 9.3 years (Interquartile range (IQR) 4.9-13.3). 16 (34%) patients had newly diagnosed ITP (0-3 Months), 6 (12.8%) had persistent ITP (3-12 Months), and 25 (53.2%) had chronic ITP (>12 Months) at the time of the study.13 patients (27.6%) received treatment and responded (complete response (CR) in 10 patients or response (R) in 3 patients), 11 (23.4%) had recovery without treatment, and 23 (49%) had no recovery.•The initial platelet count was lower in patients who responded to treatment at 8x109/L (IQR 3-8.5) compared to patients who recovered without treatment (21x109/L, IQR 6-30) and those who had no recovery (24x109/L, IQR 9-59) (p=0.008).•AIPF# was lower in patients who responded to treatment at 1.3x109/L (IQR 0.7-1.5) compared to those who had spontaneous recovery of platelet counts without treatment (i.e recovery without treatment) (1.8x109/L, IQR 1.3-3.1)) and those who had no recovery (3.4x109/L, IQR 1.6-4.3) (p= 0.03) (figure 1). IPF% was higher in patients who responded to treatment at 19.4% (IQR 13.8-25) compared to patients who had no recovery (16.4%, IQR9.7 -23)) (p=0.052); there was no significant difference between patients who recovered without treatment and who had no recovery.•IPF% on initial CBC correlated positively to platelet counts on subsequent CBCs (p=0.034 at CBC # 2), indicating that patients with higher IPF% recovered faster.•For patients who responded to treatment and who recovered without treatment the IPF% dropped very significantly with platelet recovery (combined p = 0.04 and 0.001 respectively) (figure 2A&B). [Display omitted] [Display omitted] [Display omitted] ConclusionIPF measurements are easily available parameters that are useful in the management of ITP in pediatric patients. This study suggests the use of AIPF# as a predictive factor for recovery in pediatric ITP patients with and without treatment, and IPF% as a predictive factor for early recovery. Disclosures:No relevant conflicts of interest to declare.

Low immature platelet fraction suggests decreased megakaryopoiesis in neonates with sepsis or necrotizing enterocolitis

In order to conclude on the megakaryopoietic activity during thrombocytopenia in sepsis or necrotizing enterocolitis (NEC), we analyzed the immature platelet fraction (IPF). Serial measurements of platelet counts and IPF in neonates with blood culture-proven late-onset sepsis (n=21) or surgical NEC (n=12) at T0: prior to the diagnosis of sepsis/NEC; T1: at diagnosis; T2: days 3 to 5 after onset; T3: days 8 to 12 after onset. In parallel to declining platelet counts, the median absolute IPF significantly decreased between T0 and T2 in neonates with sepsis or NEC. We found a significant positive correlation between the platelet count and absolute IPF (r=0.71; P<0.001). In patients with low IPF (<2 per nl), the platelet count did not subsequently increase. Neonates with NEC who died exhibited significantly lower IPF compared with survivors (P<0.05). Low absolute IPF values during the course of neonatal sepsis/NEC suggest suppression of megakaryopoietic activity.

Establishment of reference interval for immature platelet fraction

Immature platelet fraction (IPF) is a parameter for reticulated platelets. A high percentage IPF (%-IPF) is indicative of consumptive or recovering thrombocytopenic disorders in contrast to a low %-IPF seen in aplastic states. Absolute IPF (A-IPF) specifically reflects the number of immature platelets in circulation. This study aimed to establish reliable reference intervals for %-IPF and A-IPF. Except outliers, platelet counts and IPF were determined in 2152 healthy individuals (1252 men and 900 women) and 133 umbilical cord blood from healthy full-term neonates using XE-2100 hematology analyzer (Sysmex, Kobe, Japan). The reference intervals for %-IPF and A-IPF were defined using nonparametrical percentile methods according to the Clinical and Laboratory Standard Institute (CLSI) guideline. Platelets,%-IPF, and A-IPF all showed nonparametrical distributions. In total individuals, the reference intervals for %-IPF and A-IPF were 0.5-3.3% (0.5-3.1% in men; 0.5-3.4% in women) and 1.25-7.02 × 10(9) /L (1.30-6.80 × 10(9) /L in men; 1.21-7.15 × 10(9) /L in women), respectively. The reference intervals for %-IPF and A-IPF in umbilical cord blood were 0.7-3.8% and 1.93-9.7 × 10(9) /L, respectively. This study provides the reference interval for IPF, including %-IPF and A-IPF, according to the CLSI guideline. These results could be used as fundamental data for clinical use as well as future researches.

Successful Discontinuation of Eltrombopag Treatment in Patients with Chronic ITP

AbstractAbstract 1085 Introduction:Patients with immune thrombocytopenia (ITP) commonly experience platelet increases while under treatment with thrombopoietin-receptor agonists (TPO-RA). Anecdotal evidence suggests certain patients have been able to discontinue TPO-RA and still maintain platelet counts above baseline without additional treatment. This prospective ongoing study was designed to investigate the frequency and characteristics of patients exhibiting sustained responses after electively discontinuing eltrombopag (a TPO-RA) without substituting additional therapy. Methods:Enrolled patients were required to have ITP defined by consensus guidelines (Provan, Blood, 2009) for at least 6 months and must have been taking eltrombopag for a minimum of 4 months prior to starting this observational study. Rescue therapy is permitted once in the first 4 weeks off eltrombopag. The primary response endpoint was prospectively defined as a platelet count of ≥30,000/μl and ≥20,000/μl above initial baseline for 6 months off eltrombopag without intervening treatment (other than rescue). The secondary endpoint was being stably off therapy at 4 weeks after discontinuing eltrombopag. All patients who meet the 2 inclusion criteria (ITP for 6 months and eltrombopag for 4 months) are eligible for the study. Results:Fifteen patients are currently enrolled. Ages range from 3 to 86 years, median 55 years, with 10 females. There are 5 responders of 5 months or more (3 females, 2 males) and 10 non-responders (7 females, 3 males). Four of 5 responders have been off therapy for 6 months or more; the fifth “responder” has been off therapy for 5 months, with platelet counts ≥195,000/μL (figure 1). Responders are aged 19–86 years and have had ITP for 5–34 years; all had been on eltrombopag for > 2 years. Two responders were splenectomized and all had 3 or more prior therapies. One responder and 9 non-responders received rescue treatment in the first 4 weeks. One patient was a responder at 4 weeks, but she lost her response before reaching 6 months off-treatment. Factors not significantly associated with response were: age, duration of ITP, duration of eltrombopag therapy, splenectomy status, number of prior ITP treatments, bleeding history, and platelet count at the time that eltrombopag was discontinued. However, a lower absolute immature platelet fraction (AIPF) value at cessation of eltrombopag was seen in responders (fig 2, p=0.022). AIPF data for 1 non-responder is not available. An AIPF of 900 × 10/μl at the time of discontinuation of eltrombopag exceeds the AIPF of all 5 responders, but also of 3 of 9 non-responders. Conclusions:A substantial fraction of patients with ITP treated with eltrombopag, approximately 1/3, appear able to discontinue eltrombopag treatment and nonetheless maintain an at least adequate platelet count indefinitely (at the very least 6 months). Similar preliminary data has been reported with romiplostim (EHA and ASH abstracts).AIPF values (platelet retics) may better predict a patient's likelihood of successfully stopping therapy than other variables such as those listed above. [Display omitted] [Display omitted] Table 1:Patient DataRespondersBaseline AIPF570670750880890Age (years)5852711926SexMMFFFDuration of ITP (years)55341114Duration of TPO-RA Therapy (years)2.5422.53Number of Prior ITP Treatments (before Eltrombopag)35836Splenectomy Status (yes/no)NNYNYSignficant Bleeding History (yes/no)NNNYNPlatelet Count When Stopping Eltrombopag (×10⋀3/μl)6194182181663Non-RespondersBaseline AIPF8601090650120015006401000x12501450Age (years)5658688622741837510SexFMFFFFMFMFDuration of ITP (years)172318>202.522122127.5Duration of TPO-RA Therapy (years)4432.661.58.832.530.462.33Number of Prior ITP Treatments (before Eltrombopag)47254103726Splenectomy Status (yes/no)NYNNNYNYNNSignficant Bleeding History (yes/no)YYNNYNYYYYPlatelet Count When Stopping Eltrombopag (×10⋀3/μl)12312150101223128257892607 Disclosures:Bussel:Amgen: Family owns Amgen stock Other, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; GlaxoSmithKline: Family owns GSK stock, Family owns GSK stock Other, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Portola: Consultancy. Off Label Use: The use of romiplostim in pediatric patients was examined in this study.

The beta 1 tubulin R307H single nucleotide polymorphism is associated with treatment failures in immune thrombocytopenia (ITP)

Predictive biomarkers are needed in immune thrombocytopenia (ITP). Single nucleotide polymorphisms (SNPs) in beta 1 tubulin are potential candidates, as beta 1 tubulin is integral for platelet production and function, and SNPs in beta 1 tubulin have been associated with distinct phenotypes in platelets. We investigated the most prevalent beta 1 tubulin SNP (R307H) as a biomarker in patients with ITP via a retrospective chart review. Allelic frequencies between a group of 191 ITP patients and a healthy control group showed no difference, suggesting no direct aetiological role for the SNP in ITP. However, over similar periods of follow-up, both heterozygote and homozygote minor allele ITP patients were treated with significantly more treatment modalities and had significantly higher risk of failure to immune-modulatory therapies [relative risk (RR) = 1·5, 95% confidence interval (CI) = 1·1-2·1; P = 0·01]; with rituximab, in particular, ITP patients with the SNP experienced a 58% failure rate (RR = 1·6, 95%CI = 1·03-2·5; P = 0·04). Analysis of the absolute immature platelet fraction (A-IPF) as a marker of platelet production showed that SNP patients had significantly higher median A-IPFs compared to non-SNP patients when complete responses were achieved using immune modulatory therapies. The data suggest that the beta 1 tubulin R307H SNP has potential for use as a biomarker in ITP and may affect platelet turnover.