Cytometry-based blood immune cell markers associated with clinical outcomes in systemic sclerosis: protocol for a systematic review

Abstract

Introduction

Systemic sclerosis (SSc) is a rare autoimmune disease characterised by skin and organ fibrosis, vasculopathy and immune dysregulation. Given the disease heterogeneity and severity, accurate prognostic and predictive markers are needed. Blood immunophenotyping by flow or mass cytometry offers a promising non-invasive approach to identify immune signatures associated with disease subtypes, complications (eg, interstitial lung disease, scleroderma renal crisis, digital ulcers) and treatment responses. However, findings remain inconsistent and lack clinical standardisation. This systematic review aims to identify cytometry-based blood immune markers associated with clinical outcomes in SSc.

Methods and analysis

A comprehensive search will be conducted on three databases: PubMed, Web of Science and Cochrane Library, from their inception to the date of the final search (21 December 2025). Data will be extracted and analysed using a predefined charting form. Studies published in English or French reporting the use of flow or mass cytometry for peripheral blood cells phenotyping in adults with SSc will be included. Comparators will include healthy controls, other autoimmune diseases, disease severity groups and treatment response subgroups. The primary outcome will be the association with the diffuse cutaneous subtype, while secondary outcomes will include overall survival and disease-specific mortality, organ involvement, progression of the disease and treatment response.

Ethics and dissemination

Ethical approval is not required as this review is an analysis of published scientific literature and does not involve patients. The results of this systematic literature review will be submitted for publication in a peer-reviewed journal and presented at relevant conferences.

PROSPERO registration

CRD420250644594.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Protocol registration in PROSPERO, rigorous reporting of the review protocol following PRISMA-P (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols) guidelines, as well as protocol publication before conducting the review analysis, ensure transparency and reproducibility.

• The broad inclusion of study designs increases comprehensiveness but may also introduce heterogeneity.

• Restriction to English and French language studies and to three databases may limit the exhaustiveness of the evidence base.

• Variability in cytometry techniques, phenotypic definitions and clinical endpoints across studies is expected to complicate comparisons and may restrict the generalisability of findings.

• Exclusion of grey literature may introduce a potential publication bias.

Introduction

Background and rationale

Systemic sclerosis (SSc), also known as scleroderma, is a heterogeneous and complex autoimmune connective tissue disease characterised by a triad of pathogenic features: (1) fibrosis of the skin and internal organs, (2) chronic vasculopathy and (3) immune system dysregulation.¹ SSc is the rheumatic disease associated with the highest disease-specific mortality rate,¹ demonstrating the challenges associated with the management of patients with SSc. The disease course is highly variable and often unpredictable, with significant heterogeneity in clinical presentation and related trajectories. SSc is subclassified into three major subtypes based on the extent of skin involvement: (1) limited cutaneous SSc (lcSSc), formerly called CREST syndrome (calcinosis, Raynaud’s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia); (2) diffuse cutaneous SSc (dcSSc), characterised by an increased risk of renal crisis and cardiac involvement and a high frequency of interstitial lung disease; (3) sine scleroderma SSc, with chronic vasculopathy and internal organ involvement but without skin fibrosis.² This classification is grounded in the criteria jointly developed by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR) in 2013.³ Over the past two decades, efforts have increasingly focused on early recognition and diagnosis. This has led to the development of the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) criteria in 2011, aiming to identify patients at an early stage of the disease, potentially more accessible to disease modifying therapies.^{4 5} The severity and prognosis of SSc are largely determined by the extent and progression of fibrotic changes in both cutaneous and visceral tissues, with dcSSc subtype more likely to be associated with early and severe organ dysfunction.⁶ However, despite advances in classification and early diagnostic criteria, assessing disease extent and activity in SSc remains difficult, limiting timely therapeutic intervention.¹ Serological markers such as anti-nuclear antibodies, which are present in over 90% of patients, are accessible and help diagnosis and immunological stratification, but they have limited utility in predicting disease trajectory or response to therapy.⁷ From a pathophysiological perspective, SSc begins by immune cell infiltration (primarily monocytes/macrophages and T cells) into affected tissues, a hallmark that reflects the inflammatory and autoimmune nature of the disease. Dysregulation involves both innate and adaptive immunity, including imbalances in T and B lymphocyte and monocyte subsets, which contribute to the breakdown of immune tolerance.⁸ Assessing immune dysregulation in tissues is challenging and sometimes requires invasive sampling procedures, but circulating immune cells could reflect these immune defects. Immunological phenotyping of peripheral blood therefore appears to be a particularly promising approach to better understand SSc heterogeneity and underlying pathogenesis, and to improve stratification. Several studies have highlighted specific phenotypic alterations in circulating lymphocytes and myeloid cells, sometimes associated with clinical features, organ involvement or disease progression.9,14 For example, decreased levels of CD8+cells, activated memory CD8+cells and CD56+T/NK cells seem to be correlated with lung disease severity and capillaroscopic abnormalities.⁹ The CD16+monocytes subpopulation appears to be correlated with the severity of skin fibrosis and the occurrence of pulmonary fibrosis.¹⁰ Furthermore, multiparametric cytometry approaches allow the distinction of specific SSc immune profiles differing from other autoimmune diseases such as systemic lupus or rheumatoid arthritis, even at early stages and in untreated patients.¹¹ Certain blood signatures seem to reflect key immunopathological mechanisms, such as type I interferon activation or innate recognition pathways, as revealed by blood transcriptomic profiles.¹² However, investigations of peripheral blood immune cells profiles have given inconsistent findings. The CD4+/CD8+ ratio has been described as elevated^{9 13}; but this increase has been variably attributed to either elevated CD4+ T cell counts¹³ or decreased CD8+ T cell counts.⁹ This reflects the heterogeneity and complexity of immune dysregulation in SSc.¹⁴ Overall, these findings support the idea that peripheral blood is an accessible and informative indicator of systemic immune dysregulation in SSc, yet it remains underexplored. Thus, blood immunophenotyping could refine disease characterisation, identify subgroups and serve as a tool for prognostic assessment or treatment stratification. Nevertheless, despite these potential benefits, there is currently no consensus regarding which immunophenotypic markers are clinically relevant, and only a few studies have comprehensively or systematically evaluated the utility of flow or mass cytometry in this population. Consequently, there is a need for further research to identify, validate and standardise relevant immune parameters that could be incorporated into clinical practice, whether to predict the progression of the disease (prognosis biomarkers) or to predict the response to treatment (predictive biomarkers).

This systematic review aims to synthesise current evidence on the application of cytometry-based techniques for the characterisation of circulating immune cell populations in patients with SSc. Specifically, it aims to explore associations between immune cell profiles and clinical outcomes, including disease severity, progression and response to therapy, with the goal of identifying robust phenotypic biomarkers that could guide personalised care strategies.

Objectives

The aim of this systematic review is to identify blood immune cells and related phenotypic markers of prognostic or predictive interest in SSc, as well as their association with disease characteristics.

Methods and analysis

Design and registration

This systematic review was prospectively registered in the PROSPERO registry in March 2025 (CRD420250644594).

Main research question

In patients with SSc, which potential prognostic and/or predictive markers based on blood immune cell phenotyping by cytometry can be identified, and how are they associated with clinical outcomes?

Search strategy

MEDLINE via PubMed, Web of Science and Cochrane Library will be used to identify eligible articles. Searches in Embase and Scopus could not be conducted due to the unavailability of institutional access to these databases. Additionally, we will perform backward and forward citation tracking of all included studies. The search strategy will include the identification of articles written in French or English, published from the inception of the databases to the date of the final search (21 December 2025) and mentioning cell phenotyping in patients with SSc. Search strategies are designed as mentioned in table 1.

Table 1. Search strategies.

Database	Search strategy
PubMed	(((cytometry(All Fields)) OR (immunophenotyping(All Fields)) OR (cell sorting(All Fields)) OR (FACS(All Fields))) AND ((scleroderma(All Fields)) OR (CREST(All Fields)) OR (CRST(All Fields)) OR (systemic sclerosis(All Fields)) OR (lSSc(All Fields)) OR (dSSc(All Fields)) OR (VEDOSS(All Fields))))
Web of Science	TS=(“cytometry” OR “immunophenotyping” OR “cell sorting” OR “FACS”) AND TS=(“scleroderma” OR “systemic sclerosis” OR “lSSc” OR “dSSc” OR “CRST” OR “CREST” OR “VEDOSS”)
Cochrane Library	(cytometr* OR immunophenotyp* OR “cell sort*” OR FACS) AND (scleroderma OR CREST OR CRST OR “systemic sclerosis” OR lSSc OR dSSc OR VEDOSS) in All Text

Inclusion criteria

Studies meeting the criteria outlined below, reported in English or in French, and published from database inception to the date of the final search (21 December 2025) will be eligible for inclusion.

Population

Articles mentioning adults with systemic sclerosis, lcSSc, dcSSC, VEDOSS, CREST syndrome, SSc sine scleroderma or limited SSc will be included.

Intervention/exploration

Use of cytometry (flow or mass) for blood immune cells phenotyping in patients with SSc.

Comparator

Comparisons may include patients with SSc versus healthy controls, patients with SSc versus patients with another autoimmune disease, presence versus absence of severity criteria and treatment responders versus non-responders.

Outcomes

The primary outcome of this systematic review will be the association with the diffuse cutaneous subtype (dcSSc, which is considered a proxy for disease severity and survival. Secondary outcomes will include overall survival and disease-specific mortality, association to lcSSc and SSc sine scleroderma, as well as key clinical and biological indicators of disease severity in SSc, such as the presence of interstitial lung disease (ILD) or pulmonary fibrosis and relevant autoantibody subtypes. Additional secondary outcomes will encompass the occurrence of specific complications, such as scleroderma renal crisis, myocardial involvement and digital ulcers, as well as the evaluation of early disease states, notably VEDOSS (Very Early Diagnosis of Systemic Sclerosis) syndrome and progression to definite SSc according to ACR/EULAR criteria. Identification of treatment responders and non-responders will also be analysed as a secondary outcome, in order to assess the predictive value of biomarkers for therapeutic efficacy and management optimisation.

Studies will be included regardless of which of these outcomes they report. All reported outcomes from each selected study will be extracted and analysed if available.

Study design

All study designs, including both randomised and non-randomised studies, will be considered for inclusion. Eligible studies comprise observational analytical designs (cross-sectional or longitudinal), case–control studies, prospective and retrospective cohort studies, randomised controlled trials (RCTs) and non-RCTs. Uncontrolled or non-randomised interventional and observational studies (such as before-and-after studies conducted in routine clinical practice) will also be included. Furthermore, clinical trials assessing a treatment or therapeutic strategy, as well as translational science studies, will be considered.

Exclusion criteria

Population

Articles mentioning patients under the age of 18, patients with localised scleroderma or morphoea or animal subjects only will be excluded.

Intervention/exploration

Studies using other technological approaches than mass or flow cytometry and studies exclusively reporting cytometry data of tissues other than blood will be excluded.

Comparator

Articles mentioning groups of patients with autoimmune diseases without separate data on SSc patients will be excluded. Single-arm descriptive immune profiling studies will be excluded.

Outcomes

Identification of markers not relevant to SSc or outcomes demonstrated by other technological approaches than cytometry will be excluded.

Study design

Narrative and non-systematic reviews, conference abstracts, biography, case report, case series of less than 10 cases, comment, editorial, directory, interviews, lectures, legal cases, legislation, letter, news, newspaper article, patient education handout, popular works, congresses, consensus development conference, meta-analyses and systematic reviews and practice guideline will be excluded. Observational studies specifically evaluating the prevalence of the disease, as well as basic science studies, including genetic and epigenetic research, will also be excluded. Articles with no abstract available will be excluded.

Selection process

Articles will be reviewed independently by two referees via Rayyan (https://www.rayyan.ai/): JF and MS. There will be two screening stages for the selection of articles. The first consists of screening titles and abstracts to exclude papers that do not meet the eligibility criteria (table 2). The full text of the eligible articles will be screened by JF and MS. A third reviewer, ED, will resolve disagreements between the reviewers to make the final selection of articles.

Table 2. Inclusion and exclusion criteria for the systematic review.

Inclusion criteria	Exclusion criteria
Language: English, French Publication date: from the inception of the databases to the date of the final search (21 December 2025)	Other languages
Population	Population
Articles that only mention systemic sclerosis/scleroderma without specifying dcSSc or lcSSc will be kept, and articles mentioning both lcSSc and dcSSc will be kept, articles mentioning lcSSc or dcSSc only, SSc sine scleroderma, CREST/CRST, limited SSc or VEDOSS will be kept as well.	Articles only focusing on localised scleroderma/morphoea without including systemic sclerosis/systemic scleroderma patients will be excluded. Studies not involving adult human subjects will be excluded.
Intervention/exploration	Intervention/exploration
Use of cytometry (flow or mass) for blood immune cells phenotyping in patients with systemic sclerosis	Studies using other technological approaches than mass or flow cytometry will be excluded. Studies exclusively reporting cytometry data of tissues other than blood will be excluded.
Comparator	Comparator
Comparator groups include patients with SSc versus healthy patients to assess disease-specific differences, and patients with SSc versus patients with another autoimmune disease to assess differences between conditions.	Groups of patients with autoimmune diseases, without separate data on patients with SSc, will be excluded. Single-arm descriptive immune profiling studies will be excluded.
Outcomes	Outcomes
The primary outcome of this systematic review will be the association with the diffuse cutaneous subtype, which is considered a proxy for disease severity and survival. Secondary outcomes will include overall survival and disease-specific mortality, association to lcSSc and SSc sine scleroderma, as well as key clinical and biological indicators of disease severity in SSc, such as the presence of interstitial lung disease or pulmonary fibrosis, and relevant autoantibody subtypes. Additional secondary outcomes will encompass the occurrence of specific complications, such as scleroderma renal crisis, myocardial involvement and digital ulcers, as well as the evaluation of early disease states, notably VEDOSS (Very Early Diagnosis of Systemic Sclerosis) syndrome and progression to definite SSc according to ACR/EULAR criteria. Identification of treatment responders and non-responders will also be analysed as a secondary outcome, in order to assess the predictive value of biomarkers for therapeutic efficacy and management optimisation. Studies will be included regardless of which of these outcomes they report. All reported outcomes from each selected study will be extracted and analysed if available.	Identification of markers not relevant to SSc. Outcomes demonstrated by other technological approaches than cytometry.
Study design	Study design
All study designs, including both randomised and non-randomised studies, will be considered for inclusion. Eligible studies comprise observational analytical designs (cross-sectional or longitudinal), case–control studies, prospective and retrospective cohort studies, randomised controlled trials and non-randomised controlled trials. Uncontrolled or non-randomised interventional and observational studies (such as before-and-after studies conducted in routine clinical practice) will also be included. Furthermore, clinical trials assessing a treatment or therapeutic strategy, as well as translational science studies, will be considered.	Narrative and non-systematic reviews, conference abstracts, biography, case report, case series of less than 10 cases, comment, editorial, directory, interviews, lectures, legal cases, legislation, letter, news, newspaper article, patient education handout, popular works, congresses, consensus development conference, meta-analyses and systematic reviews and practice guideline will be excluded. Observational studies specifically evaluating the prevalence of the disease, as well as basic science studies, including genetic and epigenetic research, will also be excluded.

ACR, American College of Rheumatology; CREST/CRST, calcinosis, Raynaud’s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia; dcSSc, diffuse cutaneous systemic sclerosis; EULAR, European League Against Rheumatism; lcSSc, limited cutaneous systemic sclerosis; SSc, systemic sclerosis; VEDOSS, Very Early Diagnosis of Systemic Sclerosis.

Assessment of risk of bias

For RCTs, risk of bias will be assessed at the outcome level using the RoB 2 (Cochrane Risk of Bias 2) tool.¹⁵ Each outcome of interest will be evaluated across the five RoB 2 domains (randomisation process, deviations from intended interventions, missing outcome data, measurement of the outcome and selection of the reported result), leading to a domain-based judgement for each outcome. For observational studies, the Newcastle-Ottawa Scale¹⁶ will be used to assess methodological quality and allow standardised and reproducible evaluation across a large and heterogenous set of studies. All assessments will be conducted independently by two reviewers (MS and JF), with detailed justifications recorded. Potential publication bias or selective outcome reporting will be addressed through a critical appraisal of the included studies and a contextual discussion of how such biases may have influenced the reported findings.

Data extraction

Data extraction will be performed independently by two reviewers using a predefined form that recapitulates relevant information including: (1) study characteristics, (2) population details (systemic sclerosis subtype, demographics), (3) cytometry methodology (flow or mass cytometry, markers assessed), (4) comparator groups and (5) outcomes of interest (survival, organ involvement, disease progression, treatment response). Disagreements between reviewers will be resolved by consensus or, if necessary, by a third reviewer. When data are incomplete or not directly extractable, efforts will be made to derive the information from available text, tables or figures. Missing or unclear data will be explicitly reported in the extraction form and discussed in the synthesis to avoid bias. If key information remains unavailable, corresponding authors will be contacted by email to request clarification or provision of missing data. If no response is obtained, the study will remain eligible but will be flagged as having missing data in the review.

Planned data synthesis

For each included study, we will extract detailed information on study design, population characteristics, cytometry parameters (including the markers assessed, gating/phenotyping strategy and cellular subsets), definitions of clinical outcomes, follow-up duration and treatments. Considering the expected substantial clinical and methodological heterogeneity, studies will be grouped based on study design, populations included and cytometry methodology. Main results will be collected and presented in summary tables to allow transparent comparison of study characteristics and findings across groups. Data will be synthesised descriptively and qualitatively. A narrative analysis will compare key outcomes, including survival, clinical and biological factors associated with SSc severity and specific complications and responses to treatment. A comparative analysis will identify trends and variations across studies, taking methodological differences and inclusion criteria into account. A qualitative analysis will further explore the heterogeneity of the studies and identify factors that may influence results (eg, subtypes of SSc, presence of comorbidities, outcome measurement methods). Subgroup analysis will examine variations in key endpoints according to specific clinical and biological characteristics. Patients will be stratified according to several parameters, including SSc cutaneous subpopulation (lcSSc, dcSSc, SSc sine scleroderma), presence of ILD, autoantibody subtypes and precursor status (VEDOSS vs definite SSc). In addition, treatment responders and non-responders will be distinguished. The results will be compared descriptively to identify any clinically significant trends.

Validity and reliability

This study will follow reporting standards set by PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines,¹⁷ and PRISMA guidelines for the publication of systematic review Protocols (PRISMA-P)¹⁸ were applied for this protocol redaction. The overall certainty of the evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation¹⁹ approach to generate a summary of findings.

Patient and public involvement

None.

Ethics and dissemination

Ethical approval is not required as this review is an analysis of published scientific literature and does not involve patients. The results of this systematic literature review will be submitted for publication in a peer-reviewed journal and presented at relevant conferences.