Two reviewers independently screened titles/abstracts and then full texts for relevance to biomarker use in hemophilia A/B. Disagreements were resolved by discussion and, when needed, adjudication by a third reviewer. Reference lists of included primary studies and key reviews/guidelines were checked to identify additional eligible primary studies.

We included primary human studies (original research articles, clinical trials, systematic reviews, registry analyses, and assay-validation studies) that evaluated diagnostic, prognostic, predictive, or monitoring biomarkers in hemophilia A or B. Guidelines were used for background only and were not treated as primary evidence. We excluded case reports, editorials, animal-only studies, and reports without biomarker-related outcomes. For each included primary study, we extracted biomarker type, specimen matrix, assay/platform (when reported), diagnostic or prognostic performance, and study design and cohort context. The synthesis employed a narrative (qualitative) approach, emphasizing key trends, biomarker validation efforts, and clinical implications.

Given the narrative design and heterogeneity of included study designs and outcomes, we did not perform a formal risk-of-bias assessment using standardized tools. Instead, studies were descriptively appraised based on study design, sample size, outcome definitions, and clarity of biomarker measurement and reporting.

Significance of biomarkers in different studies Biomarkers are broadly defined by the United States Food and Drug Administration (FDA) as molecular, histologic, radiographic, or physiologic characteristics that can be assessed for diagnostic, monitoring, prognostic, predictive, pharmacodynamic, and safety purposes. Not only can diagnostic markers be used to identify a disease, but some can also identify a particular subtype, thereby guiding future management. Other monitoring biomarkers can be used to follow up on disease progression or treatment response. Prognostic biomarkers can identify high-risk individuals, determine resource allocation, and guide clinical decision-making. Certain biomarkers can stratify patients to predict the risk of certain outcomes, proving to be particularly useful in clinical trials [⁹,¹⁰,⁶].

In real life, biomarkers are not just numbers from lab reports. They provide important information about how hemophilia manifests in each patient and how they might respond to a particular treatment. Based on the BEST framework, biomarkers can be divided into four main groups: diagnostic, prognostic, predictive, and monitoring. This system helps make the scientific data more practical for daily clinical work [⁹,¹⁰].

Diagnostic markers, such as FVIII or FIX activity, thrombin generation, and inhibitor testing, are used to define the type and severity of hemophilia. In addition, genetic analysis for F8, F9, or F11 mutations can confirm the disease and detect carrier status. Prognostic biomarkers include baseline factor levels, high-risk genotypes (like intron 22 inversion), and inflammatory molecules such as CRP or VEGF. These can help predict the frequency of bleeding or the extent of joint involvement. Predictive markers are useful in determining which therapy will be most effective. For instance, a patient’s baseline thrombin generation may influence their response to emicizumab or gene therapy. Finally, monitoring biomarkers, including D-dimer and viscoelastic tests (TEG, ROTEM), is used to assess hemostatic stability and to follow the effects of therapy over time. Thinking in this structured way helps physicians integrate laboratory results, genetic data, and imaging findings into a single picture. This approach improves individual care and supports early recognition of complications [⁹,¹⁰,⁷].

Motion analysis, including gait analysis and surface electromyography (sEMG), is being studied as an indicator of early joint changes and muscle affection, which are often missed by radiography and clinical examination. The non-invasive nature of this tool makes it especially suitable for pediatric patients. Magnetic resonance imaging (MRI) is also very sensitive to early joint changes, such as synovial hypertrophy. In addition, it can detect the presence of hemosiderin, indicating joint bleeding. However, it is not ideal in the pediatric population, as they often require sedation to tolerate the procedure. This makes ultrasound a better option for children with hemophilia despite its relatively lower sensitivity. Ultrasound can assess effusions and synovial changes, but cannot detect hemosiderin deposition. Although X-rays are sometimes used, they can only detect late joint changes [¹⁰].

The role of biochemical markers in hemophilic arthropathy is also being explored. For instance, a study by Xu et al (2020; n=144 severe hemophilia A patients and 90 healthy controls; cross-sectional study, China) found levels of leukocytes, C-reactive protein (CRP), macrophage migration inhibitory factor (MIF), and vascular endothelial growth factor (VEGF) to be elevated in patients with hemophilia, and even more so in those with active bleeding. Though this could indicate their potential use as biomarkers, the study is limited by a relatively small sample size and inadequate long-term follow-up (55). A case-control study by Knowles et al (2023; n=59 hemophilia A/B and 54 healthy controls; cross-sectional with longitudinal sub-analysis, Germany) found serum levels of interleukin-6 (IL-6), CRP, LPS-binding protein (LBP), and soluble IL-6 receptor \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α (sIL-6R\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α) to be elevated in hemophilia patients with acute bleeding or recent bleeding within the last month, suggesting a possible diagnostic role [⁷].

C2M, COMP, and CTX-II are biomarkers of cartilage degradation that are elevated in patients with hemophilia. Compared with controls, CTX-1, a marker of bone resorption, was elevated, and PINP, a marker of bone formation, was increased in patients with hemophilia. In early joint bleeding, serum levels of D-dimer, fibrin degradation products (FDPs), and plasminogen were significantly decreased. On the other hand, plasma levels of epidermal growth factor (EGF), colony-stimulating factor 2 (CSF2), interleukin 4/13 (IL4/13), fibroblast growth factor 2 (FGF2), and MIP-1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α were significantly lower during acute bleeding [¹¹,⁷].

One study, López-Jiménez et al. (2019; n=50 pediatric patients with hemophilia; cross-sectional genetic association study, Mexico), examined the association between genetic biomarkers and the severity of hemophilic arthropathy in pediatric patients. After analyzing data from 50 patients, those expressing the MTHFR 677TT genotype were found to have more affected joints. On the other hand, those expressing the MTHFR 1298AC genotype had more severe effusions, as assessed by MRI. Subchondral cysts were significantly more common among participants with the TNF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α‐308GA genotype (58). However, further studies are needed to identify the mechanisms underlying the relation between genetic polymorphisms and the clinical and radiographic manifestations in pediatric patients with hemophilic arthropathy [⁹,¹⁰].

Interestingly, a cross-sectional study by Susanah et al. (2021; n=216 patients with severe hemophilia A; cross-sectional observational study, Indonesia) found a significant correlation between high serum tumor necrosis factor \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α (TNF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α) levels and high factor VIII inhibitor titers, suggesting a potential role in treatment selection. However, further studies are needed to further explore this correlation [⁹,¹⁰].

Beyond treatment selection, biomarkers are being explored to monitor therapeutic efficacy. For instance, rurioctocog alfa pegol is a PEGylated recombinant factor VIII used to prevent bleeding in patients with hemophilia A. Manon-Jensen et al (2023; n=98; secondary biomarker analysis of a phase 3 randomized trial (PROPEL), multinational) evaluated the use of collagen turnover markers to assess the efficacy of rurioctocog alfa pegol. PRO-C4 is a biomarker that reflects type 4 collagen formation, while C4M represents type 4 collagen degradation. Both together can assess basement membrane remodeling. PRO-C5 reflects type 5 collagen formation, and C3M reflects type 3 collagen degradation; they assess interstitial matrix remodeling. PRO-C2 reflects type 2 collagen formation, and C2M reflects type 2 collagen degradation. Thus, they can assess cartilage remodeling [⁹,¹⁰].

Serum levels of these biomarkers were measured at baseline and at 3-month intervals until 1 year post-treatment. PRO-C4, C4M, PRO-C5, and C3M levels decreased after treatment with rurioctocog alfa pegol [¹²].

On the other hand, PRO-C2 had increased at 6 months. However, this increase was not observed in C2M levels. In addition to their potential use in monitoring treatment response, these biomarkers have allowed us to better understand the mechanisms through which the medication works. Thus, they can be valuable in research settings and clinical trials. Though promising, the small number of participants in this study limits the generalizability of findings [¹²].

For biomarkers to be truly useful, they must be validated and standardized. Many are promising, but only a few are well tested for routine practice. According to FDA BEST and ISTH guidance, validation should include analytical accuracy, clinical significance, and reproducibility. Analytical validation focuses on the consistency and precision of measurements. For example, FVIII and FIX assays should yield identical results regardless of the reagents or instruments used. Thrombin generation and viscoelastic tests also require careful calibration to minimize inter-laboratory variability. Clinical validation means linking biomarker levels to real outcomes like bleeding frequency or joint health. However, most studies remain small, and differences in methods complicate comparisons. Reproducibility across laboratories remains a major challenge. Global efforts led by WFH and WHO reference centers aim to unify testing methods, use shared reference materials, and define acceptable ranges of variation. Standardization of how and when samples are collected will help produce comparable results between studies. When this is achieved, biomarkers will become reliable tools not only for research trials but also for personalizing therapy in everyday hemophilia care [¹,⁵,²,⁶,⁴].

Historically, the life expectancy for individuals with hemophilia was less than 11 years. Still, the introduction of clotting factor concentrates has significantly improved the quality of life and life expectancy, now extending to 60-70 years. In the Asia-Pacific region, prophylactic treatment for young children with hemophilia has been adopted based on successful randomized controlled trials in the US. However, many healthcare systems still struggle with advanced hemophilic arthropathy (HA) due to various challenges, including inhibitor development. To better understand HA, researchers are using advanced imaging, molecular biology, and genetic assessment [⁵,⁷,⁴].

Biomarkers can aid in diagnosing challenging cases, predicting disease progression, and developing targeted therapies. Biochemical markers in serum, urine, or synovial fluid can reflect joint tissue changes, enabling early detection and potentially preventing joint damage. Further research on biomarkers is essential to improve decision-making and management of HA [⁴].

Validating biomarkers involves evaluating their efficacy in documenting disease progression and activity, thereby enabling their widespread use in therapeutic interventions. While imaging techniques such as MRI are preferred for detecting arthropathy in patients with hemophilia, biomarker analysis has also shown promise. Studies have identified biomarkers with high sensitivity and specificity for diagnosing acute joint bleeding, such as CRP and VEGF. Combinations of biomarkers, such as cartilage degradation markers, have been used to monitor joint damage and assess treatment efficacy [¹,⁷,⁴].

However, the heterogeneity of biomarkers and inter-individual differences pose challenges in decision-making. Further research is needed to identify novel biomarkers specific to hemophilic arthropathy, leveraging omics approaches like proteomics. Personalized treatment strategies, including clotting factor replacement and physiotherapy, may benefit from biomarker-driven monitoring of joint health. Potential biomarkers, such as cartilage iron and inflammatory factors, hold promise for early diagnosis and primary prophylaxis [¹³].

Research has identified several biomarker combinations that can accurately distinguish between patients with joint disease and healthy individuals. For instance, serum levels of bone turnover markers, such as sRANKL and OPG, are lower in patients with hemophilia, and these levels correlate with joint outcomes. Conversely, elevated sclerostin levels, a regulator of bone formation, have been associated with joint damage [¹³].

The proinflammatory cytokine TNF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α is elevated in patients with hemophilic arthropathy and correlates with joint deterioration. However, its lack of specificity limits its use as a biomarker. Other potential biomarkers, such as iron accumulation in cartilage and vascularization factors like VEGF, have been associated with joint damage and disease progression. Additionally, low bone mineral density and vitamin D deficiency have been associated with poor joint health and reduced quality of life in patients with hemophilia. Despite these findings, biomarker research in hemophilia faces challenges, including heterogeneous patient populations, small sample sizes, and the lack of a reference standard for assessing arthropathy [¹⁴,⁴].

Several limitations constrain biomarker research in hemophilic arthropathy (HA). One major challenge is the lack of a standardized reference for assessing HA severity. Physical function scores and imaging techniques have limitations in reflecting joint damage, and discrepancies between clinical function and imaging findings are common. Radiographic assessments, such as X-rays, are useful for visualizing bone structures but may not capture changes in cartilage and synovial tissue. Alternative imaging modalities, such as ultrasonography and MRI, can provide more representative assessments, but they also have limitations [⁸,⁷,⁴].

Moreover, biomarkers can be influenced by various factors, including co-infections like HIV or HCV, which can affect inflammatory marker levels. Bone markers may not accurately reflect bone status due to age-dependent variations and differences between children and adults. Tissue specificity and assay variability can also contribute to heterogeneous assessments and contradictory results. To overcome these challenges, future studies should prioritize homogeneous study designs, minimize inter-individual variability, and consider combined biomarker indices to improve correlations [¹⁵,⁴].

Biomarkers can be categorized by utility, and numerous biomarkers have been reported in the literature, including those related to cartilage, synovium, bone, and inflammation. Examples include miRNAs, soluble vascular cell adhesion molecule-1 (sVCAM-1), and markers of collagen turnover, bone formation, and resorption [¹,¹⁰,⁷,⁴].

MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been identified as potential biomarkers for hemophilic arthropathy. Research has shown that specific miRNAs, such as miR-208a-3p and miR-524-3p, are associated with severe hemophilic arthropathy and may serve as early diagnostic or prognostic tools. These miRNAs target genes involved in bone and cartilage degradation, including PTEN, which regulates the PI3K/AKT pathway and endothelial growth [¹⁶].

Studies have also found changes in miRNA expression patterns in response to joint bleeding and arthropathy development. While some miRNAs, such as miRNA-15b, have been linked to hemophilia in animal models, others, such as miRNA-155 and miRNA-9, have yielded inconsistent results in human studies. Further research is needed to validate the potential pathogenic role of miRNAs in hemophilic arthropathy and explore their utility as biomarkers [¹⁶].

Research on microRNAs (miRNAs) as biomarkers for hemophilia arthropathy (HA) is limited. One major concern is the uncertainty about the tissue source of miRNAs and whether plasma levels accurately reflect changes in osteochondral tissues. Without analyzing osteochondral tissue, it’s challenging to draw conclusive correlations between tissue activity and circulating miRNA levels. Studies have focused on patients with advanced arthropathies, but future research should investigate miRNA signatures indicative of early arthropathy. Overlapping miRNA levels between controls and patients can complicate the determination of thresholds for early diagnosis. To overcome these challenges, homogeneous study designs, minimized inter-individual variability, and combined biomarker indices are essential [⁴].

Although microRNA (miRNA) has been proposed as a potential biomarker for rheumatoid arthritis and hemorrhagic arthritis, as demonstrated in this study (Pasta et al.), the evidence remains limited due to several factors that affect the quality of results, such as small sample sizes and inconsistencies in analytical methods. There are several uncertainties regarding the accuracy of pathological tissue analysis. Consequently, miRNA-based biomarkers should currently be considered research tools rather than clinically validated indicators. Therefore, more clinical trials are needed to clarify the accuracy of the results [⁴]. Similarly, genetic polymorphisms beyond causative F8 and F9 mutations have been associated with disease severity and joint outcomes; however, these findings lack direct therapeutic implications and therefore have a very limited role in clinical decision-making. Further research and verification are needed before their routine and fundamental use in clinical practice can be established to ensure optimal patient outcomes [⁴].

Prognostic biomarkers help us understand how hemophilia evolves, who is likely to bleed more and who might develop complications such as arthropathy or inhibitor formation later on. One of the most important indicators remains the baseline factor level. Patients with FVIII or FIX activity < 1% usually have spontaneous bleeding and more rapid joint damage, whereas those with moderate levels tend to have milder symptoms and fewer spontaneous episodes [¹,⁵,⁴].

Genetic changes also carry strong prognostic value. For example, large deletions, nonsense mutations, and intron 22 inversions in the F8 gene have consistently been associated with a substantially higher risk of inhibitor development than smaller or missense mutations. There are also immune-related genes, such as IL10, TNFA, and CTLA4, that may modify this risk, although results across populations remain somewhat inconsistent. In recent years, inflammatory markers have become another useful group of prognostic tools. Higher levels of C-reactive protein (CRP), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP-1, MMP-9) have been associated with persistent joint inflammation and early cartilage degradation. Studies that follow patients over time have shown that when these markers remain elevated, joint damage and functional decline typically progress more rapidly – even in patients receiving regular prophylaxis. A newer approach is to analyze multiple markers simultaneously, combining genetic, coagulation, and inflammatory data to develop predictive models for bleeding severity and inhibitor risk. Still, these tools need validation in larger, more diverse groups [¹³,¹⁴,⁶,⁷].

Unfortunately, there is no specific prognostic biomarker to predict the risk of inhibitor development during the treatment of patients with hemophilia. However, many studies investigate potential biomarkers, but they are not yet fully validated. The study by Işık et al. (2021; n=3,248 hemophilia A cases with missense F8 variants; retrospective database study) reported that elevated levels of G-CSF and IL-6, reduced levels of IL-10, higher core-fucosylation and galactosylation, and lower sialylation were observed in patients with inhibitors [¹⁴,¹⁷,⁴].

Large registry studies and systematic reviews have consistently shown that specific F8 mutation types, particularly large deletions, nonsense mutations, and missense variants affecting the A2, A3, and C2 domains, are associated with an increased risk of inhibitor development in hemophilia A [¹³].

Exploratory transcriptomic analyses reported in conference abstracts have suggested up-regulation of B-cell – related pathways in patients with inhibitors; however, these findings remain preliminary and require validation in peer-reviewed prospective studies [¹⁶].

Numerous prognostic biomarkers have been studied for their ability to predict hemophilic arthropathy; however, MRI remains the gold standard for evaluation. López‐Jiménez et al.’s study revealed that certain genetic variants, such as MTHFR 677TT and MTHFR 1298AC, were associated with more affected joints and increased joint effusion. In contrast, the TNF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α‐308GA genotype was associated with subchondral cysts [¹⁸,⁸].

Additionally, increased levels of inflammatory biomarkers such as TNF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α and cartilage and bone remodeling biomarkers, including CTX-II, COMP, CS-846, and collagen turnover markers such as C4M, PRO-C4, C3M, PRO-C5, and PRO-C2, were associated with joint bleeding and joint tissue bleeding. Future studies should aim to standardize cutoff values and laboratory techniques to improve the reliability of these biomarkers for clinical decision-making [¹,¹²,¹⁹,²⁰].

Diagnostic biomarkers are still the foundation for identifying hemophilia type and severity. Routine tests include prothrombin time (PT), activated partial thromboplastin time (aPTT), and specific factor assays. A pattern of prolonged aPTT with normal PT almost always suggests FVIII or FIX deficiency. One-stage or chromogenic assays confirm the diagnosis and help distinguish hemophilia A from B. It’s also essential that reagents be standardized and assays calibrated using WHO reference plasma to avoid interlaboratory variability [¹,⁵,⁴].

Beyond these basic tests, newer diagnostic markers are making the process more detailed. Global coagulation tests, such as thrombin generation (TGT) and viscoelastic methods (ROTEM, TEG), provide a broader view of the hemostatic system. They are especially helpful when monitoring patients receiving non-factor therapies, such as emicizumab, for which routine FVIII assays are unreliable [²¹].

Genetic biomarkers now play a central role as well. Sequencing the F8 and F9 genes not only confirms the diagnosis but also supports genetic counseling, prenatal diagnosis, and gene therapy planning. Next-generation sequencing (NGS) panels can test multiple coagulation genes at once, saving time and increasing diagnostic yield [²¹,⁶,⁷,⁴].

Other experimental markers, such as circulating microRNAs (miR-15b, miR-30e, miR-150) and inflammatory mediators (IL-6, TNF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\alpha$\end{document}α), are being studied to detect subclinical inflammation or early joint changes. While early results are promising, they still need to be validated in large, standardized studies before being used in clinical practice [²¹].

In short, combining traditional factor assays with molecular and genetic testing provides a clearer diagnostic picture, enabling physicians to tailor management and move toward precision medicine in hemophilia [²¹,¹,⁵,⁷,⁴].

Monitoring biomarkers is critical for tracking therapeutic response and identifying potential problems such as inhibitor formation or insufficient hemostatic protection. Routine monitoring includes FVIII or FIX activity, aPTT, and inhibitor titers measured by the Bethesda or Nijmegen-modified Bethesda assay. Checking trough levels helps ensure patients, especially those on extended half-life or gene therapy products, are adequately protected from bleeding [²¹,¹,⁵,¹²,⁷,⁴].

However, standard factor assays don’t always reflect the true clinical picture, particularly for patients on non-factor therapies such as emicizumab, fitusiran, or concizumab. In such cases, global assays such as thrombin generation (TGT), ROTEM, and TEG are more informative. Thrombin generation parameters – such as lag time, peak thrombin, and endogenous thrombin potential – have been shown to correlate with bleeding tendency and to guide dose adjustment or the timing of prophylaxis [²¹].

Newer molecular and inflammatory markers (for example, D-dimer, VEGF, MMPs, and CRP) can reveal ongoing inflammation or vascular stress. If these markers remain elevated even when factor levels appear adequate, this may indicate silent joint bleeding or early arthropathy, prompting imaging or review of treatment. For patients receiving gene therapy, markers such as vector DNA copy number, transgene expression, and immune response signals (ALT levels, anti-AAV antibodies) are essential for assessing the durability and safety of the therapy. Long-term monitoring helps detect any early loss of vector activity or immune-mediated reduction in gene expression. Ultimately, integrating laboratory data, molecular markers, and imaging into a single monitoring plan enables physicians to personalize prophylaxis, detect complications early, and improve long-term outcomes for people living with hemophilia [²¹,¹⁴].

Hemophilia treatment has transitioned from standard factor replacement therapy to extended half-life (EHL) concentrates, non-factor therapies, and gene therapy. While these classes of therapies and biomarkers have pioneered the monitoring of biomarkers uses for (i) dose tailoring and safety monitoring and (ii) surrogate endpoints in clinical trials and longitudinal studies [¹²].

For EHL factor products and PK-guided prophylaxis, factor activity (FVIII/FIX), PK metrics (e.g., trough and time below target), and global hemostatic function assays (thrombin generation; viscoelastic assays) are often used as pharmacodynamic outcome monitoring biomarkers to bleed outcomes [¹²].

Lastly, because the clinical burden is predominantly attributable to hemophilic arthropathy, imaging and tissue-turnover biomarkers are used more frequently as outcome measures alongside the annualized bleeding rate. Musculoskeletal ultrasounds (e.g., HEAD‑US) and MRIs are paired with synovitis, osteochondral damage, and hemosiderin scores. They are often used in conjunction with inflammatory and cartilage/bone turnover markers to assess and document disease activity and progression at the joint level. Consequently, the following subsections are organized by biomarkers, biological family, and intended use within the BEST framework (diagnostic/prognostic/monitoring/predictive), with an emphasis on the level of validation and the pragmatic issues that limit the routine use of biomarkers‬‬‬‬‬‬‬‬‬‬‬‬ [²¹,¹,⁸,¹²].

The majority of the above studies are small, single-center, and often cross-sectional. Inflammatory markers are more consistently raised in participants with severe hemophilic arthropathy than in well-controlled patients on effective prophylaxis. However, the studies differ substantially in age (pediatric vs. adult cohorts), definitions of joint disease (clinical scores vs. ultrasound/MRI), timing of sampling relative to bleeding episodes, and the assays used. The reported effects are often similar (with greater inflammation associated with increased joint damage), yet they lack effect sizes and clinical relevance. Additionally, neutral and negative results are more common in smaller cohorts, in studies with low-grade inflammation, and in studies with diverse target outcomes. In summary, inflammation-related biomarkers remain of interest but lack the necessary standardization to enable routine joint health assessments without cross-sectional imaging and detailed clinical information‬‬‬‬‬‬‬‬‬ [¹,⁸,¹²].

The lack of consistency in assay methodologies is a key limitation in hemophilia biomarker studies, reducing comparability across studies. The same biomarker may yield different results, and, depending on the analytical platforms used, many reports do not specify assay details [¹,²²,⁴].

Inflammatory and Angiogenic biomarkers (e.g., CRP, VEGF, cytokines) are commonly measured using ELISA or multiple assays; both are affected by interassay and interlaboratory variability. On the other hand, factor VIII and IX activity assays are supported and standardized by World Health Organization reference standards. However, their reliability is decreased in patients taking non-factor therapies. Cartilage turnover and molecular biomarkers, particularly circulating microRNAs, are still poorly standardized and lack validated reference ranges. Only factor activity assays are well harmonized; therefore, most other biomarker classes require further methodological standardization to confirm reproducibility and clinical utility [²¹,¹⁴,²²,⁵,¹²].

Biomarker validation in hemophilia often disregards the validation of different analytical assays. Avoiding technology selection that entails considerable variability is advisable to mitigate issues in data interpretation. The Enzyme-Linked Immunosorbent Assay (ELISA) is still the ’go-to’ approach for assessing quantifiable biomarkers because of cost and sensitivity. The single-analyte detection inherent to ELISA approaches, along with the higher sample volumes required, are constraints of the approach. There are also multiplex bead-based arrays (e.g., Luminex) and plate electrochemiluminescence sensors (e.g., Meso Scale Discovery), which are more widely used because they enable simultaneous assessment of multiple markers of tissue turnover and cytokines from small sample volumes. The more common multiplex approach, particularly with respect to cost, still does not provide a defined marker for some ELISA assays and, in fact, yields a broader range for some less abundant markers. There is also the option of more sophisticated research mass spectrometry (LC-MS/MS), which is even less common due to the high cost and specialized expertise required. The primary challenge when comparing findings across studies is inter-assay variability. A substantial portion of this issue is attributable to the lack of a consistent standard for antibody selection‬‬ [²²].

Each commercial kit will ultimately use antibodies that bind to different epitopes on the same model protein. Therefore, when using Kit A, one study may show a biomarker level as ‘high,’ while another study using Kit B may show the same biomarker level as ‘normal.’ This is purely a function of the different analytical measurement systems used, not of any biological differences. This phenomenon is even more pronounced when assessing bone and cartilage turnover biomarkers (COMP, CTX-II, and C2M), as they are not standardized, unlike coagulation factors [²²].

Although assays for FVIII, FIX, and CRP are standardized against World Health Organization (WHO) International Standards, ensuring globally comparable results, most biomarkers for the musculoskeletal system are still governed by “research-use-only” (RUO) manufacturing standards. These RUO assays use arbitrary internal calibrators that vary from lot to lot. Data unification is impossible until international reference materials are created for these specific markers of joint tissue. Consequently, researchers are left with no choice but to provide specific details about the assay manufacturer, catalog number, and whether lot-to-lot control samples are available. The most pressing need for future validation is the creation of Certified Reference Materials (CRMs) to close the gap between discovery and clinical use [²²,⁵,¹²].

Some previous analytical considerations have a major role in influencing the biomarkers of hemophilia, as the choice of specific biological matrices, such as using plasma or synovial fluid, has a clear and significant role in the concentration of chemical markers. The latest studies (Van Bergen et al.) have shown differences between plasma and serum C-reactive protein (CRP) levels, underscoring the need to select the appropriate matrix, as an incorrect choice can lead to inconsistent results [²²,²³].

Furthermore, circulating microRNA (miRNA) levels may vary between serum and plasma. This necessitates expanding the number of clear protocols for collecting standardized data to ensure highly reliable data. Although synovial fluid plays a significant and clear role in the joint environment, many recent studies have focused primarily on urinary and hematological markers because they are noninvasive [²⁴,²³,²⁵]. Recent studies (Pasta et al.) have highlighted the importance of timing in sample collection for clinical events, as it is a crucial variable that significantly influences the results. Several joint tissue indices exhibit transient elevations following joint hemorrhage. Specifically, urinary carboxylate peptide-1 (uCTX-II) and serum chondroitin sulfate 846 (CS846) levels show a substantial and noticeable increase lasting from two to seven days after joint bleeding. Similarly, inflammatory markers like macrophage migration inhibitory factor (MIF) and CRP show marked intra-individual changes when measured during an acute bleed compared to baseline levels one month later [²⁴,²³]. Methodological differences in sample handling and storage conditions significantly affect biomarker stability. As this study (Leuci et al.) demonstrated, plasma samples for microRNA (miRNA) analysis must be processed immediately after collection to ensure measurement accuracy. The use of different laboratory assays, varying reagent batches, and materials from multiple suppliers contributes to the observed lack of standardization across the literature. Ideally, biomarkers should demonstrate high stability during both the collection phase and long-term storage to be clinically feasible [²²,²³].

Several individual patient factors influence the results, including the timing of sample collection, which clearly affects the interpretation of vital signs data. It is also known that common co-infections in hemophilia patients, such as hepatitis C virus or HIV, have a significant impact on inflammatory marker levels. However, several studies do not account for these variables, which significantly affect the interpretation of the results [²⁴,²³].

Moreover, bone turnover markers are highly age-dependent, showing significant variations between pediatric and adult populations, which complicates the establishment of universal reference ranges. Recent studies (van Bergen et al.) (2025; in press; narrative review of hemophilia biomarker methodology) have shown that levels of physical activity and the presence of systemic inflammation in conditions such as rheumatoid arthritis play a significant role in the metabolism of indicators traditionally used to assess joint damage. This necessitates verifying all results and considering all factors that influence their interpretation. These diverse pre-analytical factors collectively contribute to substantial inter-study heterogeneity and limited reproducibility in hemophilic arthropathy research [²⁴,²³].

Several obstacles still significantly hinder the success of clinical applications of biochemical markers. Among the most important are the lack of a global reference standard for assessing joint diseases, the small sample size, which contributes significantly to trial failures, and the variation in laboratory techniques between different laboratories. All these obstacles must be overcome to achieve the best clinical outcomes for patients. Establishment of more homogeneous study designs and longitudinal measurements within the same patient cohort is required to overcome these challenges [²³].

From a clinical perspective, understanding the fundamentals of kinetic and temporal dynamics has numerous crucial applications to biomarkers of rare diseases. The complexity of hemophilia patients’ situations necessitates a broad and appropriate interpretation, compounded by missing and underexplored data on clinical events and therapeutic interventions [²⁴,²³,²⁶].

Classifying the biomarkers mentioned in this review will clarify their responses and place them within a systematic behavioral timeline. (CRP, IL-6) They are protein-based inflammatory markers and act as acute-phase reactants. Their function rises after hemarthrosis within the early hours and peaks at 1-2 days post-bleed. Reaching 7-14 days following the resolution of bleeding can be considered as the point of returning to baseline. We can infer that they can detect acute episodes and treatment responses [²⁴,²³,²⁶,²⁵].

On the other hand, cartilage turnover markers (CT-II, COMP, PRO-C4), joint damage markers such as VEGF, and other microRNA species act as chronic-phase reactants through the slow pathway over weeks to months. Cumulative tissue damage and continuous pathological remodeling are widely covered in the chronic pathway, with less suitability for individual bleeding events. Cartilage turnover markers require sustained use over the next 6-12 months to prevent bleeding and ensure measurable improvement. A possible delay in detectable response may overlap with a lack of experience, leading to an inaccurate suggestion of treatment failure. Prolonged observation can provide us with the real benefits of prophylactic action [²⁴,²³,²⁶,²⁵].

(D-dimer and thrombin generation) reach the normalization point within 3-7 days in the effective hemostasis period. On the contrary, Factor VIII/IX levels respond immediately to replacement therapy within hours. All those variable changes alarm us to focus on measuring the time of every event with clear necessity [¹⁰,²⁴,⁸,²³,²⁶,²⁵].

We can highlight the variation in therapeutic response times by providing examples of the aforementioned classes under the initiative’s prophylaxis. Factor VIII/IX levels reach the normalization point immediately post-infusion, paving the way for acute pharmacokinetic follow-up. The inflammatory biomarker class requires 2-4 weeks of continuous prophylaxis before reaching the point of decline, after which it reaches maximal suppression at 2-3 months. We can’t depend on cartilage degradation markers because of the minimal early change during the first 3-6 months. Improved values can be measured only after more than 6 months of effective therapy. Emicizumab, a novel therapy, can alter thrombin generation within weeks, whereas changes in inflammatory and joint damage markers persist for longer, requiring follow-up beyond 1 year [⁹,¹⁰,²⁷,²⁴,⁸,²⁸,²³,²⁶,²⁵].

Few studies have specified the timing of biomarker sampling relative to clinical events, limiting interpretation. Optimal strategies likely include: daily to every-other-day monitoring of inflammatory markers during acute bleeding; monthly to quarterly assessments during prophylaxis optimization; and 6-12 month intervals for long-term prognostic markers. Standardization of sampling protocols – including time since last bleed, time since factor infusion, and time of day – is essential for reproducibility [¹⁰,²⁷,²⁴,⁸,²⁸,²³,²⁶].

Although we have gathered all relevant data on hemophilia biomarkers, there remains a knowledge gap regarding sampling frequency and timing. We still need standards for sampling protocols to record bleeding episodes, time since last bleeding, time since factor infusion and treatment administration, and diurnal insights relevant to assessing reproducibility. Optimal checkpoints can be matched with a factor class, such as: 2-3 days follow-up during acute bleeding using inflammatory markers; 1-4 months follow-up during prophylaxis using factor VIII/IX; and 6-12 months follow-up intervals for prognostic assessment over a long time by using cartilage markers and microRNA forms [²⁸,²³,²⁶].

The included studies shared a common methodological limitation: reliance on single-checkpoint biomarker measurements, without accounting for dynamic variation. Serial measurement provides us with more and superior privileges, such as: controlling for high biological variability and temporary events (trauma grade, recent activity, illness presence), trajectory analysis allowance instead of absolute numbers and values in decreasing or increasing forms to guarantee more prognostic data, setting better individualized treatment response data, and perfect capability of detecting subclinical disease activity before progression and clinical case deterioration. It is difficult to establish a correlation between single-measurement risk misclassification and accurate findings at a large scale [²⁸,²³].

We recommend, in the near future, focusing on longitudinal study designs that use predefined protocols with clear specifications for hemophilia biomarkers. Prepared protocols with specific serial sampling at well-known intervals can establish evidence-based medicine. Our best options are: bleeding, timing, factor handling, physical activity, and time of day. There are necessary steps for reproducing and translating clinical findings; serial measurements are the preferred method for longitudinal studies [²³].

Interpretations within the same category of our review must be critically evaluated, particularly when they concern publication bias in the scientific committee. Most reviewers and researchers prefer hearing good news about a disease such as hemophilia, so we observe a tendency to publish only studies with promising positive statistical findings rather than studies with negative or null outcomes. The diversity of reasons has shaped the current situation, including rare epidemiology, with studies reporting fewer than 50 patients, the absence of a pre-registered protocol with specific hypotheses, leading to more selective and statistical reporting of biomarker efficacy, and overreliance on exploratory biomarkers. (miR-15b, miR-208a-3p, miR-524-3p), As novel microRNAs, added to (IL-4, IL-13), as specific inflammatory mediators, and cartilage markers, have more attention to follow. The disproportionate emphasis on initial studies with positive reports has led to unfair validation, with neglected studies receiving less scrutiny and potentially yielding poorer results.

As noted earlier, the class of biomarkers can have significant consequences, leading to the standardization of use among clinicians across multiple medical centers. Factor VIII/IX and F8/F9 genetic variants have been widely validated and used for many years without being affected. However, Novel markers require fairer interpretation through involvement in large, prospective, multicenter validation studies. Another suitable solution for all relevant limitations is to receive all submitted studies in the future, subject to prior registration. Documenting the prognostic value with equal numbers of positive and negative outcomes is also highly recommended, followed by a follow-up on clinical effects, while reducing the number of current limitations [¹⁰,⁸,¹²,²³].

Previous reviews have largely focused on hemophilic arthropathy or imaging-based assessment of joint disease. In contrast, the present review integrates biochemical, molecular, genetic, and imaging biomarkers within a unified framework, highlighting both potential clinical applications and persistent gaps in validation and standardization. While our findings are generally consistent with prior reviews, this synthesis emphasizes the limited readiness of most non-traditional biomarkers for routine clinical use [¹,⁸,¹²,²³,⁴,²⁵].

This narrative review summarizes current evidence on biomarkers in hemophilia across protein-based, genetic, molecular, inflammatory, and imaging domains. Established biomarkers such as factor VIII/IX activity levels, inhibitor assays, and imaging modalities remain central to diagnosis and disease monitoring. In contrast, emerging biomarkers, including inflammatory markers, cartilage turnover products, and molecular signatures, are supported primarily by small and heterogeneous studies. Overall, preliminary evidence suggests potential clinical utility for selected biomarkers, although their roles vary substantially across biomarker classes and clinical contexts.

The most important pathways to be addressed to translate evidence into clinical practice concern biomarkers. Data on the association of C-reactive protein (CRP) and vascular endothelial growth factor (VEGF) with inflammatory flares suggest that these biomarkers can be prospectively validated as adjuncts to the identification and monitoring of acute joint bleeding. For chronic joint damage and hemophilic arthropathy, cartilage turnover markers CTX-II and COMP demonstrate the most robust and reproducible associations with joint structural changes. They should be prioritized for longitudinal studies to define prognostic thresholds. The microRNA and genetic polymorphism biomarker families have, for the most part, remained largely unexplored. They offer promising mechanistic insights and are potential candidates for precision medicine, but they lack standardized assays and validated cutoffs. They also need robust multicenter studies, which are lacking and remain in the exploratory stage; these should be pursued as such rather than implemented.

This review can be interpreted as a step toward hemophilia biomarker evaluation and characterization, especially since most studies didn’t focus on biomarkers only as a diagnostic or prognostic tool. However, the results in this review should be interpreted with caution due to the small sample size and limited data on specific biomarkers, given that hemophilia is a rare X-linked recessive disorder. Most studies included small sample sizes and also examined other potential confounders, such as BMI, treatment adherence, and prophylactic use.

In conclusion, the identification and validation of biomarkers specifically with patient-reported quality-of-life and joint-health outcomes hold great promise for improving various aspects of patient care, including diagnosis, prognosis, and treatment selection. The field of biomarker research in hemophilia is still evolving, and further validation and standardization are necessary before widespread clinical implementation. However, this process is hindered by significant financial and logistical barriers, underscoring the need for WHO-referenced standards and cost-effectiveness frameworks. Large-scale multicenter studies, aligned with established ISTH and WFH biomarker adoption frameworks and involving diverse patient populations, are needed to confirm the reliability and generalizability of the identified biomarkers. Specific biomarker panels, such as inhibitor-risk genotypes combined with baseline Bethesda inhibitor assays and thrombin generation profiles for patients on non-factor therapies, warrant prospective multicenter validation to establish their diagnostic and monitoring accuracy. Additionally, future studies should also standardize biomarker reporting, including specimen type, timing relative to bleeding episodes, and analytic platform, to improve reproducibility. To move these findings toward clinical application, there is a need for a clear research pathway that includes:

Integrating these efforts with patient-reported quality-of-life and joint-health outcomes may further strengthen the clinical relevance and overall value of biomarker use in hemophilia.