Plasma Interleukin-12 Levels as a Potential Biomarker of Fatigue and Quality of Life in Primary Immune Thrombocytopenia

Plasma Interleukin-12 Levels as a Potential Biomarker of Fatigue and Quality of Life in Primary Immune Thrombocytopenia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plasma Interleukin-12 Levels as a Potential Biomarker of Fatigue and Quality of Life in Primary Immune Thrombocytopenia Sarthak Wadhera, Arihant Jain, Aarushi Sahni, Rudra Narayan Swain, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7030456/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Fatigue significantly affects health-related quality of life (HRQoL) in patients with Immune Thrombocytopenia (ITP). The interplay of fatigue with cytokines in ITP remains poorly understood. Methods This study included 100 patients with persistent or chronic ITP who had not received ITP-directed therapy in the previous 8 weeks, along with 50 age-matched healthy controls. Fatigue and HRQoL were assessed using the Functional Assessment of Chronic Illness Therapy (FACIT) scale. Concurrently, plasma levels of IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, and IFN-γ were measured via ELISA. Results ITP patients reported significantly lower total FACIT scores [median 146 (range 59–160)] and fatigue subscale scores [median 45 (range 9–52)] compared to controls [155 (149–159) and 49 (44–52), respectively; both p < 0.001]. IL-12 levels were markedly elevated in patients (313.2 vs. 95.5 pg/mL; p < 0.001) and showed a significant negative correlation with FACIT scores (Spearman’s ρ = − 0.272, p = 0.007). Multivariate analysis revealed that female sex, longer disease duration among chronic ITP patients (> 24 months), and elevated IL-12 levels were independently associated with greater fatigue and poorer HRQoL. Conclusions ITP is associated with significant fatigue and HRQoL impairment, with IL-12 emerging as a potential immunologic marker linked to this symptom burden. Interleukin-12 Immune Thrombocytopenia HRQoL Figures Figure 1 Figure 2 Introduction Immune thrombocytopenia (ITP) is an acquired disorder characterized by immune-mediated destruction of platelets and their precursor cells ( 1 , 2 ). The disease follows a chronic course in a majority of adults, thereby impacting the health related quality of life (HRQoL) ( 3 ). Fatigue has been increasingly recognized as a manifestation of this disease, reported in up to 60% of patients with ITP ( 4 , 5 ). As fatigue is also commonly reported in the general population and is difficult to measure objectively, this symptom is often overlooked by the clinicians during treatment decision making. However, from the patient's perspective, fatigue is a possibly a significant determinant of social, physical, functional, and emotional domains of HRQoL ( 6 ). Despite the recognition of fatigue as a common symptom in patients with ITP, the biologic basis of fatigue in ITP has been poorly understood and studied. The complex interaction of pro-inflammatory cytokines and neuro-endocrine networks is thought to play a key role in the genesis of fatigue in most autoimmune disorders ( 7 , 8 ). Additionally, the presence of anemia, use of corticosteroids and immune-modulatory drugs, psycho-social factors and co-existent illnesses can also contribute to fatigue burden and impediments in HRQoL in patients with auto-immune disorders including ITP. T cell dysregulation plays a crucial role in the pathogenesis of ITP ( 9 – 11 ). Given that cytokines are crucial mediators of T cell-mediated cellular activities, the profiles of cytokines are likely to impact the variations in severity and symptom burden among patients with ITP. The present study aimed to evaluate the burden of fatigue and health-related quality of life (HRQoL) in patients with primary ITP, and to investigate their association with Th1-mediated cytokines (IL-2, IL-12), Th2-mediated cytokines (IL-4, IFN-γ), and Th17-mediated cytokines (IL-6, IL-17) ( 12 ). Methods In this single-centre case-control study, consecutive patients aged over 12 years with a confirmed diagnosis of persistent or chronic primary ITP presenting to our hospital in the period of January 2024 to December 2024, who had not received any ITP-directed therapy in the past 8 weeks based on physician discretion, were included. Patients with secondary ITP, hypothyroidism, diabetes mellitus, obstructive sleep apnoea, hemoglobin levels ≤ 10 gm/dL, patients on any form of ITP-directed therapy (e.g. prednisolone, azathioprine, or eltrombopag etc.) in previous 8 weeks were excluded from study. In addition, patients having any known psychiatric condition or major cognitive impairment hampering a self-reported HRQoL evaluation were excluded. The study was approved by the institute ethics committee (IEC-INT-2023/DM-1207) and all patients were provided with written informed consent or assent form from patients less than 18 years of age. The study was conducted in accordance with the principles of the Declaration of Helsinki. Study Procedures : The Functional Assessment of Chronic Illness Therapy (FACIT) questionnaire was administered by study investigators at the hospital in each participant’s preferred language (Hindi or English), with prior approval from FACIT.org, to assess fatigue burden and HRQoL. The FACIT scale is divided into five subscales: Physical Wellbeing (PWB), Social Wellbeing (SWB), Emotional Wellbeing (EWB), Functional Wellbeing (FWB), and Fatigue Subscale (FS) and each item is scored on a scale of 0–4. The Fatigue Subscale, consisting of 13 items, measures the impact of fatigue on cognitive, physical, and psychosocial functioning. The FS score ranges from 0–52 with higher values indicating less fatigue. A total of 100 consecutive patients of ITP meeting the eligibility criteria were recruited as cases. Information regarding the baseline demographic variables, clinical factors related to ITP and WHO bleeding score ( 13 ) at the time of study enrolment was gathered. In addition, 50 age-matched healthy voluntary blood donors coming to the hospital were selected as controls and underwent FACIT scoring prior to their blood donation. All controls were screened based on self-declarations of not having any major comorbidities and not being on any medication. As the presence of co-existing anxiety and depression can impact HRQoL, all study participants also underwent Generalised Anxiety Disorder 7 (GAD-7) scoring to assess the burden of anxiety and Patient Health Questionnaire 9 (PHQ-9) scoring for the assessment of depression ( 14 , 15 ). Cytokine profiling : For cytokine analysis, 10 ml of blood was collected from study participants (100 patients with persistent and chronic ITP and 50 age matched controls) in EDTA vials at the time of HRQoL assessment. The samples were centrifuged immediately at 2500 rpm for 10 minutes, after which the plasma was aliquoted and stored at -20°C. After thawing, the plasma samples were analysed for cytokine profiles (IL-2, IL-12, interferon gamma, IL-4, IL-10, IL-6, and IL-17) using a sandwich enzyme-linked immunosorbent assay (ELISA) (Diaclone, France). The absolute plasma concentrations of these cytokines were used for statistical analysis. Statistical analysis Data was expressed as percentages (%), mean ± SD, or median, as appropriate. The normality of quantitative variables was assessed using the Kolmogorov-Smirnov test. Continuous variables with a normal distribution were compared using the t-test, while non-normally distributed continuous variables were compared using the Mann-Whitney U test. Categorical variables were compared using the chi-square test. Exploratory correlation analysis using the Spearman coefficient was performed to assess the relationship between two continuous variables. Variables with a bivariate p-value ≤ 0.2 in the univariable analysis, along with those with possible clinical relevance, were included in the multivariable Cox regression model. A p value of ≤ 0.05 was taken to indicate statistical significance. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS®), version 20.0 (SPSS Inc., IBM Corp.). Results A total of 100 eligible patients were included in the study. The baseline characteristics are summarized in Table 1. Among the 100 patients, 53 were females and 47 were males. The median age of the participants was 33 years (range:13-61 years). Thirty-one (%) patients had persistent ITP, while the remaining 69 (%) had chronic ITP. The median duration of diagnosis was 25 months (range: 3-300 months). Of the 100 patients, 52 patients had history of bleeding manifestations. Among these, 13 (25%) had a WHO grade 3 bleed, and none had a WHO grade 4 bleed. Table 1 : Baseline demographic and disease characteristics of study patients with ITP: Characteristics Value (n=100) Age, years, median (range) 33 (13-61) Gender: Male, n (%) Female, n (%) 47 (47) 53 (53) Phase of ITP Persistent, n (%) Chronic, n (%) 31 (31) 69 (69) Duration of diagnosis (months), median (range) 25 (3-300) Receipt of Red blood cell transfusion in past, n (%) 23 (23) Haemoglobin, gm/dl, median (range) 12.4 (10.2-15.7) Total Leucocyte count (x 10 9 /L), median (range) 6.8 (4-14) Platelet counts (x10 9 /L), median (range)* At the time of study enrolment Nadir platelet count 45 (10-94) 15 (1-55) Mean platelet volume (MPV) (fl), mean 12.1 (7.8-14.2) Antinuclear antibody positivity, n (%) 7 (7) Patients experiencing history of bleeding event, n (%) Grade of bleed among bleeders, n (%) WHO grade 1 WHO grade 2 WHO grade 3 WHO grade 4 52 (52) 26 (50) 13 (25) 13 (25) 0 (0) Patients receiving ITP-directed therapy prior to enrolment, n (%) 53 (53) Median no of previous ITP directed therapies 1 (0-3) Previous splenectomy 0 (0) Duration from last therapy, median, months* 8 (2-98) HRQoL parameters in the study population : Details of the FACIT scores within the study population are presented in Supplementary Figures 1–5. The median total FACIT score among patients was 146 (range: 59–160), significantly lower than the control group’s median of 155 (range: 149–159) [p-value < 0.001]. Likewise, the median PWB, EWB, and FWB subscale scores were all significantly lower, highlighting the significant impact of ITP on HRQoL (Supplementary table 1). Fifty-two percent of patients reported feeling ill to varying degrees, while 66% reported a lack of energy. Only 30 % of patients did not report any bodily pain (Supplement Figure 1). Notably, 95% of patients reported receiving "very much" support from family, and 93% received "very much" support from friends, indicating strong social support within the community (Supplement Figure 2). Depression (PHQ-9 ≥5) was identified in 8% of ITP patients, while anxiety (GAD-7 ≥5) was observed in 7% of patients. Female patients had a significantly lower median total FACIT score compared to male patients (136 vs. 151.8; p < 0.001). There was no difference in median total scores between persistent and chronic ITP patients (146 vs. 146; p = 0.935), bleeders and non-bleeders (141.4 vs. 147.9; p = 0.42), or between patients with hemoglobin >12 g/dL and ≤12 g/dL (146 vs. 140.9; p = 0.23). Similarly, no significant difference was noted between those with platelet counts >50 ×10⁹/μL and ≤50 ×10⁹/μL (140.9 vs. 146.5; p = 0.14). However, among patients with chronic ITP, patients with disease duration >2 years had significantly lower median total FACIT scores compared to those with duration ≤2 years (139 vs. 150; p = 0.039) (Supplementary Figure 6). No significant correlation was observed between the total FACIT score and either age ( r = 0.067; p = 0.51) or nadir platelet count ( r = 0.062; p = 0.54). Fatigue subscale scores and predictors of fatigue : The median Fatigue Subscale Score (FSS) among patients was 45 (range: 9–52), significantly lower than the control group’s median of 49 (range: 44–52) [p-value < 0.001]. Seventy-seven percent of patients reported feeling fatigued as compared to 22% in controls, 58% experienced overall body weakness, and 38% described feeling listless and washed out. Nineteen percent had trouble starting tasks due to tiredness, while 69% struggled to complete them. Additionally, 62% reported reduced energy levels, and 37% found it challenging to perform their usual activities (Supplementary Figure 5). Fifty two percent patients had clinically significant fatigue, based on the cutoff of less than 44 (2 SD below the mean of healthy controls). The FSS showed a strong correlation with all domain sub-scores and the total FACIT score (Supplementary table 2). Female patients had a significantly higher fatigue burden as reflected by a lower median Fatigue Subscale Score (FSS) compared to males (40 vs. 48; p 12 g/dL and ≤12 g/dL (46 vs. 43; p = 0.19), bleeders and non-bleeders (43 vs. 45.5; p = 0.39), or between those with nadir platelet counts >50 ×10⁹/L and ≤50 ×10⁹/L (43 vs. 46; p = 0.95). However, within the chronic ITP subgroup, patients with a disease duration of >2 years exhibited significantly lower FSS scores compared to those with a duration ≤2 years (47 vs. 43; p = 0.044) (Figure 1). Additionally, no significant correlation was found between FSS scores and either age (r = 0.046; p = 0.65) or nadir platelet count (r = 0.07; p = 0.49). Table 2 : Determinants of Fatigue subscale and total FACIT score using linear regression. Fatigue subscale score Total FACIT score Univariable analysis Multivariable analysis Univariable analysis Multivariable analysis Beta coefficient P value Beta coefficient P value Beta coefficient P value Beta coefficient P value Female sex -0.447 <0.001 -0.441 <0.001 -0.421 <0.001 -0.49 24 months* -0.231 0.021 -0.218 0.012 -0.214 0.032 -0.275 0.02 Platelet count at recruitment 0.107 0.29 0.038 0.78 0.095 0.35 0.091 0.51 Nadir platelet count 0.070 0.49 0.245 0.09 0.062 0.54 0.31 0.061 Haemoglobin 0.183 0.069 0.018 0.85 0.174 0.083 0.020 0.83 Bleeder phenotype -0.122 0.23 -0.126 0.21 IL-2 levels 0.072 0.47 0.044 0.66 IL-12 levels -0.257 0.01 -0.277 0.001 -0.287 0.004 -0.305 0.01 IFN- ¡ levels 0.032 0.75 0.027 0.79 IL-4 levels 0.004 0.97 -0.015 0.88 IL-10 levels 0.108 0.29 0.10 0.33 IL-6 levels -0.112 0.27 -0.119 0.24 IL-17 levels 0.010 0.92 -0.007 0.94 Discussion Fatigue and impaired HRQoL are significant concerns for patients with ITP. This study confirms a high burden of fatigue and an impaired HRQoL in patients with persistent and chronic ITP, regardless of platelet count or bleeding symptoms. In addition, female sex, duration of chronic ITP, and IL-12 levels were independent predictors of fatigue. Several studies have suggested an alteration of Th2/Th1 cytokines in patients with ITP. A significant correlation between HRQoL and fatigue scores with IL-12 levels was observed, a novel finding not previously reported and suggesting a role of cytokine in the immune pathogenesis of fatigue in ITP. The current study utilised the FACIT score, with the median total FACIT score nearly 16 points lower than that of the control population, with a significant impact on quality of life across all domains: physical, emotional, functional, and social. Previous studies utilising SF-39 questionnaires in patients with ITP had also reported impaired HRQoL in majority of domains including fatigue ( 16 , 17 ). In the recent I-WISH survey, which included 1,507 ITP patients from 13 countries, 73% patients reported fatigue as the most significant symptom at diagnosis while only 30% of the ITP physicians reported hearing of fatigue from the patients. Additionally, fatigue was the primary symptom that patients wished to have resolved ( 18 ). In line with the I-WISH survey, fatigue was reported by 77% patients of chronic/persistent ITP patients who were currently not on any ITP directed therapy in the previous 8 weeks at the time of survey. These findings highlight that fatigue burden in ITP continues to be frequently under-estimated by the health care providers. Various clinical factors have been examined for their association with fatigue and/or HRQoL in ITP. Platelet count is often used as a surrogate marker for disease severity and has been variably correlated with fatigue in several studies. While Newton et al. found an association between fatigue and platelet count, with a cutoff of < 100 x 10 9 /L in patients with bleeding and < 30 x 10 9 /L in non-bleeding patients, a study by Sarpatwari et. al did not reveal any correlation of fatigue with platelet count ( 19 ). The I-WISH survey found no correlation between improvements in platelet count and the alleviation of fatigue. Similarly, no correlation of fatigue with platelet count was observed in the current study. The heterogeneity in the findings regarding the correlation of fatigue with platelet counts can partly be explained based on differences in methodology of assessment of fatigue as well as socio-cultural differences in the study populations. Moreover, these findings suggest that pathogenic mechanisms that contribute to the development of fatigue may persist even after improvements in platelet counts, atleast in a subset of patients. The immune basis of fatigue in autoimmune disorders is thought to be mediated by cytokines, resulting in neuroinflammation and alterations in neuroendocrine metabolism ( 20 ). IL-1β has been shown to positively correlate with fatigue in rheumatoid arthritis, while IL-1Ra has been linked to fatigue in Sjögren's syndrome and prostate cancer ( 21 – 23 ). Interferon gamma and TNF-α have also been positively correlated with fatigue in Sjögren's syndrome ( 24 ). IL-12 was also shown to have a positive correlation with fatigue in Rheumatoid arthritis ( 25 ). In the current study, out of 7 cytokines tested, IL-12 levels were associated with fatigue in addition to duration of ITP. IL-12 is mainly produced by macrophages and dendritic cells, contributing to the differentiation of naïve T cells into Th1 cells ( 26 ). The correlation of fatigue with IL12 levels suggest possible links of the Th1 pathway in the immune pathogenesis of fatigue in ITP ( 27 , 28 ). Several studies have suggested the role of IL12 in the development of chronic fatigue syndrome, and depression through its effects on serotonin metabolism ( 29 , 30 ). IL12 mediates increased metabolism of serotonin through the induction of enzyme Indoleamine 2,3-dioxygenase. Increased levels of IL-12 in patients of ITP may lead to decreased levels of serotonin, consequently resulting in an increased fatigue burden in patients with ITP ( 9 , 31 ). The case control approach with a limited sample size in the current study has several inherent drawbacks, particularly the inability to analyze the impact of treatments on the cytokine levels and fatigue and HRQoL scores. The impact of socio-cultural disparities, diet and environment on the measured HRQoL variables and cytokines was not analyzed and therefore the findings of the study need to be confirmed in larger number of patients across diverse ethnicities. Despite these drawbacks, the current study is one of the initial studies attempting to identify the cytokine biomarkers of fatigue in patients with ITP. The findings of the current study suggest the need to explore therapeutic strategies that improvise fatigue and the potential role of targeting inflammatory pathways to ameliorate fatigue in patients with ITP. Conclusion This study highlights the substantial burden of fatigue and impaired HRQoL in patients with persistent and chronic ITP, independent of platelet count or bleeding manifestations. Elevated plasma interleukin-12 levels demonstrated a significant negative correlation with fatigue and overall HRQoL scores. These findings suggest a potential immunological basis for fatigue in ITP, likely mediated via Th1 cytokine pathways, particularly IL-12. Declarations Acknowledgement : Authors thank the patients who participated in this study and their families and caregivers. The authors have no competing interests. Ethical Approval declaration : The study was Approved by the Institutional Ethics Committee, with letter number IEC-INT/23/DM-1207. Human Ethics and Consent to Participate declaration : All patients provided written informed consent, or assent in the case of those under 18 years of age, and the study was conducted in accordance with the principles of the Declaration of Helsinki. Funding information : Funding for the study was provided by Departmental Research Grant, PGIMER Chandigarh. Author Contribution SW, A Jain, AS and RNS wrote the manuscript. CP, AJ, RH, GP, AK and SG contributed to the study design. RA analysed plasma cytokines. RD and PM reviewed the manuscript and gave final approval. Data Availability The data will be made available by contacting the corresponding author on reasonable request. References Neunert C, Terrell DR, Arnold DM, Buchanan G, Cines DB, Cooper N, et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. 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Dendritic cells of immune thrombocytopenic purpura (ITP) show increased capacity to present apoptotic platelets to T lymphocytes. Exp Hematol. 2006;34(7):879-87. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile01.07.2025.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Sep, 2025 Reviews received at journal 10 Sep, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers invited by journal 05 Aug, 2025 Editor assigned by journal 03 Jul, 2025 Submission checks completed at journal 03 Jul, 2025 First submitted to journal 02 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Research","correspondingAuthor":false,"prefix":"","firstName":"Rekha","middleName":"","lastName":"Hans","suffix":""},{"id":496584831,"identity":"4d158271-96ad-43e9-95b7-a6d8b8cac6b0","order_by":8,"name":"Gaurav Prakash","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Gaurav","middleName":"","lastName":"Prakash","suffix":""},{"id":496584832,"identity":"5c99ae76-aeb7-422c-b9ee-c12e2d117e15","order_by":9,"name":"Alka Khadwal","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Alka","middleName":"","lastName":"Khadwal","suffix":""},{"id":496584833,"identity":"b0ab28b1-f1b1-4c06-a8b6-e463170176fd","order_by":10,"name":"Sandeep Grover","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Sandeep","middleName":"","lastName":"Grover","suffix":""},{"id":496584835,"identity":"c0166fe4-9500-428f-8706-1436d49f3676","order_by":11,"name":"Reena Das","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Reena","middleName":"","lastName":"Das","suffix":""},{"id":496584837,"identity":"0f9e6d84-025c-4c6b-9c90-54765ee94e58","order_by":12,"name":"Pankaj Malhotra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACAxDBAyKYmQ8ASQkZUrSwJYC08JCghYEHiY0PmLN3Jz5428aQJ+/O8/nVjRoLHgb2w0c34NNi2XN2s+HcNoZiw8O826xzjgEdxpOWdgOvw27kbpPmbWNI3NjMu804hw2oRYLHDL+W+2+3/4Zo4XlmnPOPGC03eLcxg7TMZ+ZhfpzbRoyWM7mbJeeckyg2YGYzY87tk+BhI+iX42c3fnhTZpMn33/48eecb3Vy/OyHj+HVAgUSCQYHGNgkQEw2IpSDQYJ8AwPzB2JVj4JRMApGwcgCALTbRTuQj2GGAAAAAElFTkSuQmCC","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":true,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Malhotra","suffix":""}],"badges":[],"createdAt":"2025-07-02 14:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7030456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7030456/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88757353,"identity":"9f89d42a-56f4-46f3-bf20-7f75ab7376dc","added_by":"auto","created_at":"2025-08-11 07:24:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":251737,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whisker plots illustrating median fatigue subscale score across various subgroups within the study population, including: (A) Males vs. Females, (B) Persistent vs. Chronic ITP, (C) Chronic ITP patients with a duration of \u0026gt;2 years vs. ≤2 years, (D) Hemoglobin \u0026gt;12 g/dL vs. ≤12 g/dL, (E) Bleeder vs. Non-bleeder phenotype, and (F) Platelet count at recruitment \u0026gt;50 × 10⁹/µL vs. ≤50 × 10⁹/µL.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7030456/v1/a39e525d10ffccda8dce63e6.jpeg"},{"id":88757322,"identity":"ae6ca229-79ee-404e-abac-548f5e43d2e8","added_by":"auto","created_at":"2025-08-11 07:24:23","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334448,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots depicting the correlation analysis between Interleukin-12 and various domains of the FACIT scale, including (A) FS score, (B) PWB score, (C) SWB score, (D) EWB score, (E) FWB score, and (F) Total FACIT score.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7030456/v1/34643368ed2cbc2cfb10aaf5.jpeg"},{"id":88758160,"identity":"7a1398be-e955-4710-8db9-30a1d76c4019","added_by":"auto","created_at":"2025-08-11 07:32:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1231324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7030456/v1/d27fcc5b-38b3-4e7c-a5ac-89d015bf290f.pdf"},{"id":88757319,"identity":"48b858ca-0ec1-4259-8be1-404a97e1f58d","added_by":"auto","created_at":"2025-08-11 07:24:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":674653,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile01.07.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7030456/v1/7d1b8a1f6c8293601d969498.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plasma Interleukin-12 Levels as a Potential Biomarker of Fatigue and Quality of Life in Primary Immune Thrombocytopenia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmune thrombocytopenia (ITP) is an acquired disorder characterized by immune-mediated destruction of platelets and their precursor cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The disease follows a chronic course in a majority of adults, thereby impacting the health related quality of life (HRQoL) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Fatigue has been increasingly recognized as a manifestation of this disease, reported in up to 60% of patients with ITP (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). As fatigue is also commonly reported in the general population and is difficult to measure objectively, this symptom is often overlooked by the clinicians during treatment decision making. However, from the patient's perspective, fatigue is a possibly a significant determinant of social, physical, functional, and emotional domains of HRQoL (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the recognition of fatigue as a common symptom in patients with ITP, the biologic basis of fatigue in ITP has been poorly understood and studied. The complex interaction of pro-inflammatory cytokines and neuro-endocrine networks is thought to play a key role in the genesis of fatigue in most autoimmune disorders (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Additionally, the presence of anemia, use of corticosteroids and immune-modulatory drugs, psycho-social factors and co-existent illnesses can also contribute to fatigue burden and impediments in HRQoL in patients with auto-immune disorders including ITP. T cell dysregulation plays a crucial role in the pathogenesis of ITP (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Given that cytokines are crucial mediators of T cell-mediated cellular activities, the profiles of cytokines are likely to impact the variations in severity and symptom burden among patients with ITP. The present study aimed to evaluate the burden of fatigue and health-related quality of life (HRQoL) in patients with primary ITP, and to investigate their association with Th1-mediated cytokines (IL-2, IL-12), Th2-mediated cytokines (IL-4, IFN-γ), and Th17-mediated cytokines (IL-6, IL-17) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eIn this single-centre case-control study, consecutive patients aged over 12 years with a confirmed diagnosis of persistent or chronic primary ITP presenting to our hospital in the period of January 2024 to December 2024, who had not received any ITP-directed therapy in the past 8 weeks based on physician discretion, were included. Patients with secondary ITP, hypothyroidism, diabetes mellitus, obstructive sleep apnoea, hemoglobin levels ≤ 10 gm/dL, patients on any form of ITP-directed therapy (e.g. prednisolone, azathioprine, or eltrombopag etc.) in previous 8 weeks were excluded from study. In addition, patients having any known psychiatric condition or major cognitive impairment hampering a self-reported HRQoL evaluation were excluded. The study was approved by the institute ethics committee (IEC-INT-2023/DM-1207) and all patients were provided with written informed consent or assent form from patients less than 18 years of age. The study was conducted in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy Procedures\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThe Functional Assessment of Chronic Illness Therapy (FACIT) questionnaire was administered by study investigators at the hospital in each participant’s preferred language (Hindi or English), with prior approval from FACIT.org, to assess fatigue burden and HRQoL. The FACIT scale is divided into five subscales: Physical Wellbeing (PWB), Social Wellbeing (SWB), Emotional Wellbeing (EWB), Functional Wellbeing (FWB), and Fatigue Subscale (FS) and each item is scored on a scale of 0–4. The Fatigue Subscale, consisting of 13 items, measures the impact of fatigue on cognitive, physical, and psychosocial functioning. The FS score ranges from 0–52 with higher values indicating less fatigue. A total of 100 consecutive patients of ITP meeting the eligibility criteria were recruited as cases. Information regarding the baseline demographic variables, clinical factors related to ITP and WHO bleeding score (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) at the time of study enrolment was gathered. In addition, 50 age-matched healthy voluntary blood donors coming to the hospital were selected as controls and underwent FACIT scoring prior to their blood donation. All controls were screened based on self-declarations of not having any major comorbidities and not being on any medication. As the presence of co-existing anxiety and depression can impact HRQoL, all study participants also underwent Generalised Anxiety Disorder 7 (GAD-7) scoring to assess the burden of anxiety and Patient Health Questionnaire 9 (PHQ-9) scoring for the assessment of depression (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytokine profiling\u003c/b\u003e:\u003c/p\u003e\u003cp\u003e For cytokine analysis, 10 ml of blood was collected from study participants (100 patients with persistent and chronic ITP and 50 age matched controls) in EDTA vials at the time of HRQoL assessment. The samples were centrifuged immediately at 2500 rpm for 10 minutes, after which the plasma was aliquoted and stored at -20°C. After thawing, the plasma samples were analysed for cytokine profiles (IL-2, IL-12, interferon gamma, IL-4, IL-10, IL-6, and IL-17) using a sandwich enzyme-linked immunosorbent assay (ELISA) (Diaclone, France). The absolute plasma concentrations of these cytokines were used for statistical analysis.\u003c/p\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003cp\u003eData was expressed as percentages (%), mean ± SD, or median, as appropriate. The normality of quantitative variables was assessed using the Kolmogorov-Smirnov test. Continuous variables with a normal distribution were compared using the t-test, while non-normally distributed continuous variables were compared using the Mann-Whitney U test. Categorical variables were compared using the chi-square test. Exploratory correlation analysis using the Spearman coefficient was performed to assess the relationship between two continuous variables. Variables with a bivariate p-value ≤ 0.2 in the univariable analysis, along with those with possible clinical relevance, were included in the multivariable Cox regression model. A p value of ≤ 0.05 was taken to indicate statistical significance. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS®), version 20.0 (SPSS Inc., IBM Corp.).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 100 eligible patients were included in the study. The baseline characteristics are summarized in Table 1. Among the 100 patients, 53 were females and 47 were males. The median age of the participants was 33 years (range:13-61 years). Thirty-one (%) patients had persistent ITP, while the remaining 69 (%) had chronic ITP. The median duration of diagnosis was 25 months (range: 3-300 months). Of the 100 patients, 52 patients had history of bleeding manifestations. Among these, 13 (25%) had a WHO grade 3 bleed, and none had a WHO grade 4 bleed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Baseline demographic and disease characteristics of study patients with ITP:\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"538\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003eValue (n=100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eAge, years, median (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e33 (13-61)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eGender:\u003c/p\u003e\n \u003cp\u003eMale, n (%)\u003c/p\u003e\n \u003cp\u003eFemale, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e47 (47)\u003c/p\u003e\n \u003cp\u003e53 (53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003ePhase of ITP\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePersistent, n (%)\u003c/p\u003e\n \u003cp\u003eChronic, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31 (31)\u003c/p\u003e\n \u003cp\u003e69 (69)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eDuration of diagnosis (months), median (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e25 (3-300)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eReceipt of Red blood cell transfusion in past, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e23 (23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eHaemoglobin, gm/dl, median (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e12.4 (10.2-15.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eTotal Leucocyte count (x 10\u003csup\u003e9\u003c/sup\u003e/L), median (range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e6.8 (4-14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003ePlatelet counts (x10\u003csup\u003e9\u003c/sup\u003e/L), median (range)*\u003c/p\u003e\n \u003cp\u003eAt the time of study enrolment\u003c/p\u003e\n \u003cp\u003eNadir platelet count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e45 (10-94)\u003c/p\u003e\n \u003cp\u003e15 (1-55)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eMean platelet volume (MPV) (fl), mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e12.1 (7.8-14.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eAntinuclear antibody positivity, n (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e7 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003ePatients experiencing history of bleeding event, n (%)\u003c/p\u003e\n \u003cp\u003eGrade of bleed among bleeders, n (%)\u003c/p\u003e\n \u003cp\u003eWHO grade 1\u003c/p\u003e\n \u003cp\u003eWHO grade 2\u003c/p\u003e\n \u003cp\u003eWHO grade 3\u003c/p\u003e\n \u003cp\u003eWHO grade 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e52 (52)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26 (50)\u003c/p\u003e\n \u003cp\u003e13 (25)\u003c/p\u003e\n \u003cp\u003e13 (25)\u003c/p\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003ePatients receiving ITP-directed therapy prior to enrolment, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e53 (53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eMedian no of previous ITP directed therapies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e1 (0-3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003ePrevious splenectomy\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 74.7681%;\"\u003e\n \u003cp\u003eDuration from last therapy, median, months*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.2319%;\"\u003e\n \u003cp\u003e8 (2-98)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eHRQoL parameters in the study population\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eDetails of the FACIT scores within the study population are presented in Supplementary Figures 1\u0026ndash;5. The median total FACIT score among patients was 146 (range: 59\u0026ndash;160), significantly lower than the control group\u0026rsquo;s median of 155 (range: 149\u0026ndash;159) [p-value \u0026lt; 0.001]. Likewise, the median PWB, EWB, and FWB subscale scores were all significantly lower, highlighting the significant impact of ITP on HRQoL (Supplementary table 1). Fifty-two percent of patients reported feeling ill to varying degrees, while 66% reported a lack of energy. Only 30 % of patients did not report any bodily pain (Supplement Figure 1). Notably, 95% of patients reported receiving \u0026quot;very much\u0026quot; support from family, and 93% received \u0026quot;very much\u0026quot; support from friends, indicating strong social support within the community (Supplement Figure 2).\u0026nbsp;Depression (PHQ-9 \u0026ge;5) was identified in 8% of ITP patients, while anxiety (GAD-7 \u0026ge;5) was observed in 7% of patients.\u003c/p\u003e\n\u003cp\u003eFemale patients had a significantly lower median total FACIT score compared to male patients (136 vs. 151.8; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). There was no difference in median total scores between persistent and chronic ITP patients (146 vs. 146; \u003cem\u003ep\u003c/em\u003e = 0.935), bleeders and non-bleeders (141.4 vs. 147.9; \u003cem\u003ep\u003c/em\u003e = 0.42), or between patients with hemoglobin \u0026gt;12 g/dL and \u0026le;12 g/dL (146 vs. 140.9; \u003cem\u003ep\u003c/em\u003e = 0.23). Similarly, no significant difference was noted between those with platelet counts \u0026gt;50 \u0026times;10⁹/\u0026mu;L and \u0026le;50 \u0026times;10⁹/\u0026mu;L (140.9 vs. 146.5; \u003cem\u003ep\u003c/em\u003e = 0.14). However, among patients with chronic ITP, patients with disease duration \u0026gt;2 years had significantly lower median total FACIT scores compared to those with duration \u0026le;2 years (139 vs. 150; \u003cem\u003ep\u003c/em\u003e = 0.039) (Supplementary Figure 6). No significant correlation was observed between the total FACIT score and either age (\u003cem\u003er\u003c/em\u003e = 0.067; \u003cem\u003ep\u003c/em\u003e = 0.51) or nadir platelet count (\u003cem\u003er\u003c/em\u003e = 0.062; \u003cem\u003ep\u003c/em\u003e = 0.54).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFatigue subscale scores and predictors of fatigue\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe median Fatigue Subscale Score (FSS) among patients was 45 (range: 9\u0026ndash;52), significantly lower than the control group\u0026rsquo;s median of 49 (range: 44\u0026ndash;52) [p-value \u0026lt; 0.001]. Seventy-seven percent of patients reported feeling fatigued as compared to 22% in controls, 58% experienced overall body weakness, and 38% described feeling listless and washed out. Nineteen percent had trouble starting tasks due to tiredness, while 69% struggled to complete them. Additionally, 62% reported reduced energy levels, and 37% found it challenging to perform their usual activities (Supplementary Figure 5). Fifty two percent patients had clinically significant fatigue, based on the cutoff of less than 44 (2 SD below the mean of healthy controls). The FSS showed a strong correlation with all domain sub-scores and the total FACIT score (Supplementary table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFemale patients had a significantly higher fatigue burden as reflected by a lower median Fatigue Subscale Score (FSS) compared to males (40 vs. 48; p \u0026lt; 0.001). There was no statistically significant difference in median FSS between patients with persistent and chronic ITP (45 vs. 45; p = 0.84), those with hemoglobin levels \u0026gt;12 g/dL and \u0026le;12 g/dL (46 vs. 43; p = 0.19), bleeders and non-bleeders (43 vs. 45.5; p = 0.39), or between those with nadir platelet counts \u0026gt;50 \u0026times;10⁹/L and \u0026le;50 \u0026times;10⁹/L (43 vs. 46; p = 0.95). However, within the chronic ITP subgroup, patients with a disease duration of \u0026gt;2 years exhibited significantly lower FSS scores compared to those with a duration \u0026le;2 years (47 vs. 43; p = 0.044) (Figure 1). Additionally, no significant correlation was found between FSS scores and either age (r = 0.046; p = 0.65) or nadir platelet count (r = 0.07; p = 0.49).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: Determinants of Fatigue subscale and total FACIT score using linear regression.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"681\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003eFatigue subscale score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003eTotal FACIT score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eUnivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMultivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eUnivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMultivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eBeta coefficient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eBeta coefficient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eBeta coefficient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eBeta coefficient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eFemale sex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eChronic ITP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eDuration of disease \u0026gt;24 months*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.218\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003ePlatelet count at recruitment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eNadir platelet count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eHaemoglobin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eBleeder phenotype\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-2 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-12 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIFN-\u003csub\u003e\u0026iexcl;\u003c/sub\u003e levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-4 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-10 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-6 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e-0.112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eIL-17 levels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e-0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e0.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFatigue and impaired HRQoL are significant concerns for patients with ITP. This study confirms a high burden of fatigue and an impaired HRQoL in patients with persistent and chronic ITP, regardless of platelet count or bleeding symptoms. In addition, female sex, duration of chronic ITP, and IL-12 levels were independent predictors of fatigue. Several studies have suggested an alteration of Th2/Th1 cytokines in patients with ITP. A significant correlation between HRQoL and fatigue scores with IL-12 levels was observed, a novel finding not previously reported and suggesting a role of cytokine in the immune pathogenesis of fatigue in ITP.\u003c/p\u003e\u003cp\u003eThe current study utilised the FACIT score, with the median total FACIT score nearly 16 points lower than that of the control population, with a significant impact on quality of life across all domains: physical, emotional, functional, and social. Previous studies utilising SF-39 questionnaires in patients with ITP had also reported impaired HRQoL in majority of domains including fatigue (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In the recent I-WISH survey, which included 1,507 ITP patients from 13 countries, 73% patients reported fatigue as the most significant symptom at diagnosis while only 30% of the ITP physicians reported hearing of fatigue from the patients. Additionally, fatigue was the primary symptom that patients wished to have resolved (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In line with the I-WISH survey, fatigue was reported by 77% patients of chronic/persistent ITP patients who were currently not on any ITP directed therapy in the previous 8 weeks at the time of survey. These findings highlight that fatigue burden in ITP continues to be frequently under-estimated by the health care providers.\u003c/p\u003e\u003cp\u003eVarious clinical factors have been examined for their association with fatigue and/or HRQoL in ITP. Platelet count is often used as a surrogate marker for disease severity and has been variably correlated with fatigue in several studies. While Newton et al. found an association between fatigue and platelet count, with a cutoff of \u0026lt;\u0026thinsp;100 x 10\u003csup\u003e9\u003c/sup\u003e/L in patients with bleeding and \u0026lt;\u0026thinsp;30 x 10\u003csup\u003e9\u003c/sup\u003e/L in non-bleeding patients, a study by Sarpatwari et. al did not reveal any correlation of fatigue with platelet count (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The I-WISH survey found no correlation between improvements in platelet count and the alleviation of fatigue. Similarly, no correlation of fatigue with platelet count was observed in the current study. The heterogeneity in the findings regarding the correlation of fatigue with platelet counts can partly be explained based on differences in methodology of assessment of fatigue as well as socio-cultural differences in the study populations. Moreover, these findings suggest that pathogenic mechanisms that contribute to the development of fatigue may persist even after improvements in platelet counts, atleast in a subset of patients.\u003c/p\u003e\u003cp\u003eThe immune basis of fatigue in autoimmune disorders is thought to be mediated by cytokines, resulting in neuroinflammation and alterations in neuroendocrine metabolism (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). IL-1β has been shown to positively correlate with fatigue in rheumatoid arthritis, while IL-1Ra has been linked to fatigue in Sj\u0026ouml;gren's syndrome and prostate cancer (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Interferon gamma and TNF-α have also been positively correlated with fatigue in Sj\u0026ouml;gren's syndrome (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). IL-12 was also shown to have a positive correlation with fatigue in Rheumatoid arthritis (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In the current study, out of 7 cytokines tested, IL-12 levels were associated with fatigue in addition to duration of ITP. IL-12 is mainly produced by macrophages and dendritic cells, contributing to the differentiation of na\u0026iuml;ve T cells into Th1 cells (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The correlation of fatigue with IL12 levels suggest possible links of the Th1 pathway in the immune pathogenesis of fatigue in ITP (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Several studies have suggested the role of IL12 in the development of chronic fatigue syndrome, and depression through its effects on serotonin metabolism (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). IL12 mediates increased metabolism of serotonin through the induction of enzyme Indoleamine 2,3-dioxygenase. Increased levels of IL-12 in patients of ITP may lead to decreased levels of serotonin, consequently resulting in an increased fatigue burden in patients with ITP (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe case control approach with a limited sample size in the current study has several inherent drawbacks, particularly the inability to analyze the impact of treatments on the cytokine levels and fatigue and HRQoL scores. The impact of socio-cultural disparities, diet and environment on the measured HRQoL variables and cytokines was not analyzed and therefore the findings of the study need to be confirmed in larger number of patients across diverse ethnicities. Despite these drawbacks, the current study is one of the initial studies attempting to identify the cytokine biomarkers of fatigue in patients with ITP. The findings of the current study suggest the need to explore therapeutic strategies that improvise fatigue and the potential role of targeting inflammatory pathways to ameliorate fatigue in patients with ITP.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the substantial burden of fatigue and impaired HRQoL in patients with persistent and chronic ITP, independent of platelet count or bleeding manifestations. Elevated plasma interleukin-12 levels demonstrated a significant negative correlation with fatigue and overall HRQoL scores. These findings suggest a potential immunological basis for fatigue in ITP, likely mediated via Th1 cytokine pathways, particularly IL-12.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e: Authors thank the patients who participated in this study and their families and caregivers.\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval declaration\u003c/strong\u003e: The study was Approved by the Institutional Ethics Committee, with letter number IEC-INT/23/DM-1207.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Ethics and Consent to Participate declaration\u003c/strong\u003e: All patients provided written informed consent, or assent in the case of those under 18 years of age, and the study was conducted in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e: Funding for the study was provided by Departmental Research Grant, PGIMER Chandigarh.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSW, A Jain, AS and RNS wrote the manuscript. CP, AJ, RH, GP, AK and SG contributed to the study design. RA analysed plasma cytokines. RD and PM reviewed the manuscript and gave final approval.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data will be made available by contacting the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNeunert C, Terrell DR, Arnold DM, Buchanan G, Cines DB, Cooper N, et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. Blood Adv. 2019;3(23):3829-66.\u003c/li\u003e\n\u003cli\u003eBhoria P, Varma N, Malhotra P, Varma S, Luthra-Guptasarma M. Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins. MAbs. 2015;7(6):1212-20.\u003c/li\u003e\n\u003cli\u003eZhang Y, Kolesar JM. Eltrombopag: An Oral Thrombopoietin Receptor Agonist for the Treatment of Idiopathic Thrombocytopenic Purpura. 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Fatigue in primary Sj\u0026ouml;gren\u0026apos;s syndrome is associated with lower levels of proinflammatory cytokines. RMD Open. 2016;2(2):e000282.\u003c/li\u003e\n\u003cli\u003eAlqahtani B, Daghestani M, Omair MA, Alhamad EH, Tashkandy Y, Othman N, et al. Association of Inflammatory Cytokine Levels with Extra Glandular Manifestations, Fatigue, and Disease Activity in Primary Sj\u0026ouml;gren\u0026apos;s Syndrome in Saudi Patients: A Cross-Sectional Study. Diagnostics (Basel). 2023;13(19).\u003c/li\u003e\n\u003cli\u003eLiu J, Cao S, Kim S, Chung EY, Homma Y, Guan X, et al. Interleukin-12: an update on its immunological activities, signaling and regulation of gene expression. Curr Immunol Rev. 2005;1(2):119-37.\u003c/li\u003e\n\u003cli\u003eTrinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Reviews Immunology. 2003;3(2):133-46.\u003c/li\u003e\n\u003cli\u003eSun L, He C, Nair L, Yeung J, Egwuagu CE. Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease. Cytokine. 2015;75(2):249-55.\u003c/li\u003e\n\u003cli\u003eSarmin N, Roknuzzaman ASM, Mouree TZ, Islam MR, Al Mahmud Z. Evaluation of serum interleukin-12 and interleukin-4 as potential biomarkers for the diagnosis of major depressive disorder. Scientific Reports. 2024;14(1):1652.\u003c/li\u003e\n\u003cli\u003eYang T, Yang Y, Wang D, Li C, Qu Y, Guo J, et al. The clinical value of cytokines in chronic fatigue syndrome. J Transl Med. 2019;17(1):213.\u003c/li\u003e\n\u003cli\u003eCatani L, Fagioli ME, Tazzari PL, Ricci F, Curti A, Rovito M, et al. Dendritic cells of immune thrombocytopenic purpura (ITP) show increased capacity to present apoptotic platelets to T lymphocytes. Exp Hematol. 2006;34(7):879-87.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"blood-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"BLOOD RESEARCH","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Interleukin-12, Immune Thrombocytopenia, HRQoL","lastPublishedDoi":"10.21203/rs.3.rs-7030456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7030456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFatigue significantly affects health-related quality of life (HRQoL) in patients with Immune Thrombocytopenia (ITP). The interplay of fatigue with cytokines in ITP remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis study included 100 patients with persistent or chronic ITP who had not received ITP-directed therapy in the previous 8 weeks, along with 50 age-matched healthy controls. Fatigue and HRQoL were assessed using the Functional Assessment of Chronic Illness Therapy (FACIT) scale. Concurrently, plasma levels of IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, and IFN-γ were measured via ELISA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eITP patients reported significantly lower total FACIT scores [median 146 (range 59\u0026ndash;160)] and fatigue subscale scores [median 45 (range 9\u0026ndash;52)] compared to controls [155 (149\u0026ndash;159) and 49 (44\u0026ndash;52), respectively; both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. IL-12 levels were markedly elevated in patients (313.2 vs. 95.5 pg/mL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and showed a significant negative correlation with FACIT scores (Spearman\u0026rsquo;s ρ = \u0026minus;\u0026thinsp;0.272, p\u0026thinsp;=\u0026thinsp;0.007). Multivariate analysis revealed that female sex, longer disease duration among chronic ITP patients (\u0026gt;\u0026thinsp;24 months), and elevated IL-12 levels were independently associated with greater fatigue and poorer HRQoL.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eITP is associated with significant fatigue and HRQoL impairment, with IL-12 emerging as a potential immunologic marker linked to this symptom burden.\u003c/p\u003e","manuscriptTitle":"Plasma Interleukin-12 Levels as a Potential Biomarker of Fatigue and Quality of Life in Primary Immune Thrombocytopenia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 07:16:18","doi":"10.21203/rs.3.rs-7030456/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-21T01:45:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T07:55:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27641418616658503572081848516784457936","date":"2025-08-06T12:01:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-05T10:50:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T01:50:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T01:31:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BLOOD RESEARCH","date":"2025-07-02T14:36:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"blood-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"BLOOD RESEARCH","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d4002045-b70c-430c-a20d-40261e7252ca","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T05:23:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 07:16:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7030456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7030456","identity":"rs-7030456","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}