Diagnosing primary immunodeficiencies using protein-based screening assays

Abstract

Primary immunodeficiencies, now more commonly referred to as inborn errors of immunity (IEIs), are a diverse group of inherited conditions characterized by immune system defects, leading to recurrent infections, immune dysregulation, and autoimmune symptoms. Early diagnosis is crucial for effective management and treatment. This document explores the role of protein-based assays in diagnosing and characterizing IEIs. Protein-based assays, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting, have significantly advanced the identification and understanding of IEI. These assays enable rapid screening and functional analysis of immune proteins, providing critical insights into the phenotypic manifestations of genetic mutations. ELISA technology allows for the detection of specific antigens or antibodies, while flow cytometry facilitates the analysis of multiple cell populations and their functional states. Immunoblotting remains valuable for detecting protein expression and identifying truncated or non-functional proteins. This review highlights case studies demonstrating the application of these assays in diagnosing conditions such as X-linked lymphoproliferative syndrome type 2, activated phosphoinositide 3-kinase delta syndrome, severe combined immunodeficiency, and Wiskott–Aldrich syndrome. It also discusses the importance of functional assays in confirming the pathogenicity of genetic variants of unknown significance and guiding targeted genetic analysis. Overall, protein-based assays are essential tools in the diagnostic workflow for IEI, enabling timely and accurate diagnosis, guiding therapeutic decisions, and monitoring patient outcomes post-treatment, including haematopoietic stem cell transplantation and gene therapy.

The review sets out how protein-based assays: flow cytometry and ELISA, allow rapid diagnosis of inborn errors of immunity, ahead of or alongside genetic testing. It distinguishes expression assays from functional ones, illustrating both with cases including XLP2, APDS, X-linked SCID, hyper-IgM, WAS, DOCK8 deficiency and IPEX. It concludes that these assays are particularly valuable for resolving variants of unknown significance, directing genetic analysis and monitoring patients after transplant or gene therapy.

Graphical Abstract

Graphical Abstract.

Introduction

Primary immunodeficiencies (PIDs), now more commonly referred to as errors of immunity (IEIs), are a group of inherited conditions resulting from defects in the immune system. These defects cause patients to suffer from recurrent infections, immune dysregulation, and autoimmune symptoms [1–3]. Given the nature of these conditions, early diagnosis is essential to ensure expert care and prompt treatment. The spectrum of IEI is continuously expanding, with increasingly diverse clinical manifestations, making diagnosis based solely on clinical presentation and basic laboratory tests challenging [4]. Over the past decades, protein-based assays have been developed and refined, contributing significantly to the discovery of new IEI and the diagnosis and treatment of more patients. As the number of genes associated with IEI continues to grow, so does the need to categorize these defects both genetically and phenotypically [5]. Since genetic testing can be time-consuming and expensive, rapid protein-based screening assays offer a valuable diagnostic tool. The short turnaround time of these assays is of particular clinical value in suspected haemophagocytic lymphohistiocytosis (HLH), where results can be available within a few hours of sample receipt. In this setting, protein expression testing can support or exclude a genetic diagnosis early enough to influence immediate management decisions, including the avoidance of invasive investigations and the timely initiation of HLH-directed therapy. Additionally, protein screening can help direct genetic investigations and justify further analysis. For example, abnormal gamma chain expression and impaired STAT5 phosphorylation supported the need for extended genetic testing, ultimately identifying a mutation in the E twenty-six (ETS) binding domain of the gamma chain [6–8].

Genetic analysis can reveal mutations in IEI genes; however, in some IEI cases, identical gene mutations can present with very different phenotypes. This heterogeneity is best assessed through immunological laboratory testing, which enables not only diagnosis but also, more importantly, targeted treatment. Genetic analysis may identify variants of unknown significance (VUS), and protein or functional testing can confirm or exclude the pathogenicity of these variants. A frequently cited example is the A91V variant of PRF1 (p.Ala91Val), which is best regarded as a hypomorphic or disease-modifying allele. A91V is common in the general population and most carriers remain healthy, but it impairs perforin maturation and reduced perforin expression on flow cytometry has been documented in a proportion of individuals carrying a single A91V allele. An abnormal perforin screen therefore does not by itself indicate a biallelic disease-causing PRF1 defect. HLH has been reported both in patients homozygous for A91V and, more frequently, in patients carrying A91V in trans with a predicted loss-of-function (LOF) PRF1 variant, often with a later onset than classical FHL2. Penetrance is nevertheless clearly incomplete, and disease risk cannot be predicted from genotype alone, so functional assessment (perforin expression and cytotoxic activity) interpreted together with the clinical picture remains necessary [9–12]. First-line screening is performed after the initial consultation with a suspected IEI patient. Investigations are guided by the suspected area of immune dysfunction, inferred from the clinical presentation, including the types of infectious organisms involved. A general immunological work-up may include full blood count, immunoglobulin levels, complement (C3 and C4), and assessment of antibody responses. Depending on the initial findings, immunophenotyping for T and B cells and neutrophil function tests may also be performed. The initial assessment is followed by more specialized tests that may confirm the final diagnosis in the appropriate clinical context. This is where protein-based assays are particularly useful, as they provide more detailed insight into IEI phenotypes.

The 2024 International Union of Immunological Society (IUIS) classification includes 508 disease-associated genes and 17 phenocopies. Against this denominator, the number of genes for which a protein expression or functional assay is available for routine clinical use remains small, on our estimate fewer than 10%. Assays in routine diagnostic practice include perforin expression and granule release assay (CD107a) for familial HLH, SLAM-associated protein (SAP) and as X-linked inhibitor of apoptosis (XIAP) expression for X-linked lymphoproliferative disease type-1 (XLP-1) and type-2 (XLP-2), the Dehydrorhodamine 123 and Nitro blue tetrazolium (DHR/NBT) assays and NADPH oxidase component expression for chronic granulomatous disease (CGD), induced CD40L expression for X-linked hyper-IgM syndrome, CD132 (common gamma chain) and CD127 expression for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome protein (WASP) expression for Wiskott–Aldrich syndrome (WAS), Dedicator of cytokinesis 8 (DOCK8) expression for autosomal recessive (AR) hyper IgE syndrome, Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression for Immune-dysregulation, Major Histocompatibility Complex (MHC) Class I and Class II expression for Severe combined immunodeficiency (SCID-like, adhesion molecule expression for leukocyte adhesion deficiency (LAD), FAS-induced apoptosis and double-negative T cells for autoimmune lymphoproliferation syndrome (ALPS), phospho-STAT1 for chronic mucocutaneous candidiasis (CMC), phospho-STAT3 for autosomal dominant (AD) hyper IgE syndrome, and phospho-STAT5 for SCID. The assays presented in this review reflect part of the diagnostic repertoire of our laboratory, and the XIAP functional assay, phospho-AKT, and ADA2 activity are the most recently introduced. Several of the more established tests referred to above, including the HLH panel and CGD testing, are also performed in other routine diagnostic laboratories.

Protein-based screening assays may involve various techniques that either detect a protein or assess its function. Expression-based assays have limitations, as they may detect stable, non-functional, or truncated proteins. Functional studies, on the other hand, can identify protein dysfunction and are thus more informative; examples include the phosphoprotein assays (phospho-STAT1, phospho-STAT3, phospho-STAT5, and phospho-AKT), cytotoxicity and degranulation assays [Natural Killer (NK) killing and CD107a], the XIAP functional assay, and C1 inhibitor functional testing. The value of this approach is illustrated by X-linked SCID, where surface expression of the common gamma chain may be preserved despite a pathogenic IL2RG variant, while STAT5 phosphorylation in response to cytokine stimulation is clearly abnormal, and by hereditary angioedema type II, in which the antigenic level of C1 inhibitor is normal and only the functional assay is diagnostic

Common protein-based assays include enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. ELISA technology uses the catalytic properties of enzymes to detect specific antigens or antibodies. The most common ELISA format involves adding a known concentration of antigen to a microtiter plate coated with a specific antibody, allowing antigen–antibody binding [13]. After washing the plate, an enzyme-labelled conjugate is added, forming a sandwich complex (antibody–antigen–antibody). Following thorough washing to remove unbound material, a substrate is added, producing a colourimetric change that is measured spectrophotometrically. Depending on the ELISA type, the signal may be directly or inversely proportional to the antigen concentration. Flow cytometry is an extremely valuable tool for IEI screening (Table 1). It uses lasers to generate scattered and fluorescent light signals, which are detected by photomultiplier tubes and converted into electronic signals for analysis via computer software [14]. Multiparametric analysis is possible, as multiple fluorescently conjugated antibodies can be used to detect various cell populations, depending on the flow cytometer's configuration. Flow cytometry can assess both protein expression and function using functional assays. One such example is phosphoflow technology. Since the immune system depends on numerous signalling pathways for the development, differentiation, and activation of immune cells, many of these can be measured through phosphorylation events. Cells are stimulated with specific antigens or cytokines relevant to the pathway under study, then fixed to preserve phosphorylation status, and permeabilized to allow intracellular and surface staining [15, 16]. A challenge with fixed cells is that some epitopes may be altered by the fixation process, so protocols must ensure antibodies can still bind effectively and be detected accurately.

Table 1.

Examples of immunological defect and laboratory tests to detect abnormalities.

Defect	Assay type	Expected immunological assay results
Phagocytic
CGD)	Flow	DHR: Low percentage and mean fluorescent intensity to stimulants (FMLP - Formyl-Methionyl-Leucyl-Phenylalanine, Escherichia coli, and PMA) NBT: Absent/reduced number of burst neutrophils on film after stimulation with PMA CGD proteins expression via flow (gp91phox, p22phox, p47phox, p67phox, p40phox)
LAD	Flow cytometry	Reduced/absent expression of CD18 on neutrophils
Combined
SCID	Flow cytometry	Low/absent T cells with +/− B cells and NK cells on a lymphocyte subset panel Depending on SCID further protein testing is targeted to the immunophenotype
Gamma chain expression	Flow cytometry	Gamma chain absent, or present (non-functional somantical reversion)
pSTAT5	Flow cytometry	Hypophosphorylation to IL-2/IL-7/IL-15 in gamma chain deficient patients or to single cytokines in other SCID conditions (e.g. IL7R alpha deficiency)
CD40L HIGM	Flow cytometry	Reduced/absent CD40L expression on lymphocytes when stimulated with PMA and PHA. CD25 expression is normal
MHC I deficiency	Flow cytometry	Reduced/absent HLA ABC expression
MHC II deficiency	Flow cytometry	Reduced/absent HLA DR expression
DOCK8	Flow cytometry	Reduced/absent DOCK8 expression in T cells and B cells
APDS	Flow cytometry: phosphoflow	Hyperphosphorylation of AKT after stimulation with CD3/CD28.
Humoral defects
Common variable immunodeficiency	Flow cytometry	Extended B-cell immunophenotyping
X-linked hypogammaglobulinemia	Flow cytometry and	Absent BTK (Bruton's tyrosine kinase) expression Absent phosphorylation of BTK if present but non-functional
Combined with Syndromic features
WAS	Flow cytometry	Reduced/absent WAS protein expression in lymphocytes
DiGeorge syndrome	Flow cytometry	Reduced/absent T cells
Immune dysregulation
HLH	Flow cytometry, ELISA	Reduced/absent perforin: perforin deficiency Reduced/absent SAP: XLP1 Reduced absent XIAP: XLP2 Reduced/absent degranulation on granule release assay: FHL3, FHL4, and FHL5 Raised soluble CD25 (IL2Rα) for all conditions
ALPS	Flow cytometry and ELISA	Raised double-negative αβ T cells Abnormal functional apoptosis assay Raised soluble Fas ligand
Innate
STATs	Flow cytometry	Reduced or increase phosphorylation of STAT protein to various stimulants depending on STAT pathway
CARD9	Flow cytometry	Absent protein expression
TLR signalling	Flow cytometry	Reduced/absent CD62L shedding and CD11b upregulation to various stimulants depending on Toll-Like Receotor (TLR) pathway

Validation of these assays presents particular difficulties in rare inborn errors of immunity, and a brief description of our approach may be useful. Each new assay is first established on samples from healthy adult volunteers collected in lithium heparin or EDTA tubes. We generally perform it on fresh whole blood and on peripheral blood mononuclear cells (PBMCs) that have been cryopreserved and thawed, in order to determine whether the assay remains reliable in stored material, an important consideration when samples are referred from other centres. We typically test between 10 and 20 healthy controls to define the normal range, followed by up to 5 patients with a genetically confirmed diagnosis to establish the range of abnormal results and the separation between the two. This last step is the principal limitation: for many of these conditions only a small number of patients exist nationally, so the numbers available for validation are far below those used for assays in common diseases, and the boundary between normal and abnormal is therefore defined on limited data and refined as further cases accrue. Standard operating procedures, risk assessment, health and safety, staff competency record,s and quality control documentation are prepared alongside the analytical work. In our experience, the process from initial development to clinical release takes between 6 months and 1 year. Assays released before formal accreditation are done on R&D basis with an explicit statement released with the report that the test is not UKAS (United Kingdom Accreditation Service) accredited, and results are interpreted in conjunction with the clinical picture and, where available, genetic findings [17].

Proteomics is increasingly being explored as a diagnostic tool for PIDs, with recent work highlighting its ability to detect disease-specific molecular signatures. A study recently published showed that proteomic profiling of dried blood spots (DBSs) can identify subclinical type I interferon signatures in neonates with type I interferonopathy [18]. This is particularly relevant as DBS sampling is minimally invasive and suitable for early-life screening. The ability to detect interferon-driven pathology prior to symptom onset highlights a shift towards predictive diagnostics and individualized care, offering the potential for earlier, targeted intervention during a critical developmental window.

Translation from research and development

Phospho-Akt by phosphoflow cytometry

To increase diagnostics by protein expression/function, it is essential to translate assays from research laboratories into the clinical laboratory. An example of a translated assay from research is the assay to help diagnose activated phosphoinositide 3-kinase delta syndrome (APDS) [19–21]. This assay uses PBMCs that are starved overnight with and without a phosphoinositide 3-kinase delta inhibitor. The following morning, cells are stimulated with anti-CD3 and CD28, then fixed and permeabilized, and finally stained with CD3 to detect the T-cell population and phosphorylated Akt (at the Ser473 position). The level of phosphorylated Akt (pAkt) is measured for unstimulated, stimulated, and inhibited cells. As seen in Fig. 1, APDS patients have higher levels of pAkt when their cells are stimulated, which is expected, as they have hyperactive PI3K signalling causing hyperphosphorylation of Akt.

Figure 1.

Phosphorylated Akt levels in healthy control and APDS patient. Left hand plot is the control, and right side is the patient. The stimulated pAkt MFI is much higher in the patient compared to the control, with both being inhibited using the PI3K delta inhibitor. MFI, mean fluorescent intensity.

APDS is a complex condition that is difficult to diagnose in a laboratory without genetics and is often misdiagnosed as other IEIs. As genetic analysis can take a long time, it was necessary to develop an assay to assist in the diagnosis of this condition. However, as it is a functional phosphorylation assay, the assay is open to interpretation and requires experience to report; however, it can be useful when hyperphosphorylation of Akt is seen, as it confirms defects in this pathway. This assay is beneficial to patients who have a mutation in PIK3CD or PIK3R1 that is classified as a variant of unknown significance. This is due to patients with VUS's not being able to access targeted therapies unless the mutation is found to be pathogenic or disease-causing. Moreover, this assay is considered valuable as it can assess the PI3K signalling pathway, giving indication of Akt hyperphosphorylation, which is often associated with the clinical manifestation of the syndrome. Finally, it is important to highlight that one limitation to using this assay for diagnostic use is the absence of standardization. Many laboratories only perform Akt phosphorylation assays as research due to the lack of published methods and/or reference ranges. It is difficult to create a reference range for phosphorylation events such as in this assay. This is due to the variability seen in healthy donors, a result of heterogeneity seen in a healthy population when assessing cellular function.

XIAP functional assay

X-linked lymphoproliferative syndrome type 2 (XLP2) is a complex immune disorder caused by mutations in BIRC4, also known XIAP. XIAP regulates the body's immune response by coordinating inflammatory signalling events and controlling cell survival in cell death pathways. XIAP also mediates NOD2 pro-inflammatory signalling by promoting RIPK2 ubiquitination, which facilitates the recruitment of downstream molecules that activate NF-κB (Nuclear Factor Kappa B Subunit-2) pathway, leading to the transcription of genes encoding pro-inflammatory cytokines, chemokines, anti-microbial peptides, and pro-survival proteins.

A timely diagnosis is essential for XLP2 patients when symptoms present in a severe and progressive way from early infancy and the chance of a successful haematopoietic stem cell transplant (HSCT), which is the only definitive treatment for the condition, increases if the patient has not developed a clinical picture of severe inflammation. Moreover, in more recent years, the growing adoption of reduced-intensity and reduced-toxicity conditioning regimens has contributed to better survival rates in patients with genetic HLH and XLP2-related disorders [22–24].

At Great Ormond Street Hospital (GOSH), a functional diagnostic assay was validated to assess XIAP function by measuring p38 mitogen-activated protein kinases (MAPKs) phosphorylation in monocytes. P38 MAPK phosphorylation indicates successful NOD2 stimulation followed by XIAP E3 ligase activity, and it occurs when cells from whole blood are stimulated with L18 muramyl dipeptide (MDP), a synthetic derivative of MDP, which is the minimal bioactive component of bacterial peptidoglycan. LPS (Lipopolysaccharide) is used as a positive control to induce p38 phosphorylation via Toll-like receptor 4 [25].

This method allows XIAP function to be assessed in whole blood, providing a functional readout of the immune response.

Patients with XIAP deficiency had a significant decrease in p38 phosphorylation, which correlated with BIRC4 mutations and XIAP impaired expression assessed by flow cytometry (Fig. 2).

Figure 2.

XIAP functional data. Figure showed percentages of p38 phosphorylation in healthy controls (n = 18) and patients (n = 5) after LPS or L18-MDP stimulation. ***P < 0.001.

One case was particularly insightful, whereby a patient with a frameshift mutation in BIRC4 showed normal XIAP expression but had an impaired function, indicating that protein expression and genetic tests alone may not fully capture the extent of XIAP dysfunction. In such cases, a functional assay is crucial for accurately diagnosing XLP2 and guiding treatment. This ability to detect functional impairment early is particularly important when deciding whether to proceed with HSCT before the onset of an uncontrolled inflammatory phenotype [26], improving the odds of a successful transplant considering that the complications associated with HSCT in XIAP patients are very challenging, with a higher rate of mortality than some other IEI. During the validation process, it was established that the assay can diagnose other NOD2-related conditions such as Blau Syndrome and inflammatory bowel disease (IBD), where the immune regulation is similarly disrupted [27].

In summary, this assay offers a rapid and reliable method to assess XIAP function in patients with XLP2, IBD, and Blau syndrome. By providing a functional assessment when genetic results are inconclusive or highlight VUS, this assay helps guide early intervention decision-making, ensuring that patients can receive early HSCT before significant inflammation occurs, maximizing the likelihood of success.

ADA2 enzyme assay

The most severe cases of ADA2 (Adenosine deaminase 2) deficiency usually manifest within the first decade of life with recurrent strokes or cerebral haemorrhage; some patients have been described as presenting with joint pains, hepatomegaly, splenomegaly and gastrointestinal problems. It is thought that, over time, upregulation of pro-inflammatory cytokines in these patients causes organ damage. The reduced ADA2 enzyme activity is the result of an AR mutation in the CECR1 (also referred to as ADA2) [28].

We can measure patients’ total ADA activity in the presence of a known ADA1 inhibitor [EHNA: erythro-9-(2-hydroxy-3-nonyl)adenine] using enzymatic deamination in a kinetic manner (Fig. 3), allowing the determination of ADA2 activity. Continued development is currently ongoing, and sensitivity and specificity have yet to be fully evaluated.

Figure 3.

ADA2 preliminary data. Box plot comparing all healthy controls with patients and carrier with genetically confirmed mutations.

Flow cytometry–based assays in a routine diagnostic laboratory

Common gamma chain (or CD132) expression and STAT5 phosphorylation

X-linked SCID is a rare and life-threatening condition caused by mutation in IL2RG, which encodes an essential and shared subunit (gamma chain) for interleukin (IL) 2, IL-4, IL-7, IL-9, IL-15, and IL-21. The advanced newborn screening programme for SCID allows for the fast and prompt diagnosis of SCID in affected individuals. Any newborn with low T-cell receptor excision circle (TREC) values is immunophenotyped, and if T cells, and usually NK cells, are low or absent, further testing includes assessing γ chain expression and STAT5 phosphorylation [29, 30]. The utility of this assay is illustrated by the following case, assessed at GOSH. Born to non-consanguineous parents, the infant's newborn screening showed TREC = 0. Immunophenotyping showed absent T cells with preserved B and NK cells (T⁻ B⁺ NK⁺), although the NK cell proportion could have been overestimated due to non-specific binding caused by sample age due to transport issues. This contrasts with the canonical X-linked SCID, where the immunophenotype is typically T− B+ NK−. When gamma chain expression was assessed, B and NK cells showed 2 populations with high expression of gamma chain seen in only 43.5% of CD19 (B cells) and 73.2% of CD56 (NK cells) (Fig. 4). The patient was genetically confirmed to carry a mutation: IL2RG ChrX (GR37): g70330826C>T NM_000206.3: c.190G>A p.(Val64Met), hemizygous.

Figure 4.

Gamma chain expression. Figure showed gamma chain expression in a healthy control and in gamma chain mutated patient.

In gamma chain–deficient patients, a mutation in the IL2RG prevents T and NK cell development. In rare instances, a non-functional somatic reversion can occur. However, despite the reversion and recovery of the gamma chain with different levels of protein expression, the essential STAT5 signalling pathway remains inactive (Fig. 5a), abrogating full immune function restoration [31].

Figure 5.

STAT5 phosphorylation before and after normal gamma chain expression. Figure showed impaired STAT5 phosphorylation in response to IL-2, IL-7, and IL-15 in whole blood derived from the patient compared with a healthy control (a). (b) Shows gamma chain in different patient lineages 2 months post-gene therapy treatment compared with a healthy control. (c) Showed restored STAT5 phosphorylation in response to IL-2, IL-7, and IL-15 in whole blood derived from the Patient 3 months post-gene therapy compared with a healthy control.

The patient underwent lentiviral gene therapy because no HLA (Human Leukocyte Antigen)-identical family donor or unrelated donors were identified. Protein expression may be used to monitor patients post-HSCT or gene therapy [32].

A few months after the procedure, gamma chain expression (Fig. 5b) and STAT5 phosphorylation (Fig. 5c) were re-assessed showing gamma chain expression comparable to a healthy control and productive STAT5 phosphorylation.

In our laboratory, gamma chain (CD132) expression is always assessed in conjunction with the pSTAT5 assay, due to the non-functional somatic reversion; in these patients, pSTAT5 responses are abnormal within both the total lymphocyte gate (Fig. 5a) and gated T cells (not shown). Results should nevertheless be interpreted with caution where maternally engrafted is present, as T-cell-gated responses may then appear normal.

CD40 ligand expression

Hyper immunoglobulin M syndrome (HIGM) is a rare primary immunodeficiency, first described as a mutation in CD40LG. It is characterized by low or absent levels of serum IgG, IgA, IgE, and normal or elevated levels of serum IgM. To date, other mutations in several other genes involved in B-cell signalling have been described with an AR inheritance. However, the X-linked HIGM is the most common and accounts for about 65–70% of the cases. These patients have a humoral defect due to low switched immunoglobulin production and susceptibility to respiratory bacterial infections. They also have a cellular defect; CD40L, a member of the tumour necrosis factor family, is expressed on activated CD4+ T cells and interacts with CD40 expressed on antigen-presenting cells, acting as an important co-stimulatory signal. For this reason, patients are also susceptible to opportunistic infections such as Pneumocystis jirovecii, Cryptosporidium, Toxoplasma, and others [33, 34].

To determine whether CD40 Ligand is expressed on activated lymphocytes, whole blood is incubated with PMA (Phorbol 12-myristate 13-acetate) and PHA (Phytohaemagglutinin) to stimulate the lymphocytes. Cells are stained with directly conjugated monoclonal antibody recognizing CD45, CD4, CD40L, and CD25 as an activation marker. Based on data collected over the years at GOSH, this test shows a sensitivity of 96% and a specificity of 86% [35]. We illustrate the application of this test with the following patients: two brothers with a family history of X-HIGM syndrome presented with a mild phenotype consistent with some CD40L expression in stimulated cells and were evaluated and diagnosed at GOSH (Fig. 6) [36]. The family carries the C.31C>T, p.(Arg11*) and should have full CD40 ligand deficiency. However, immunological investigations showed some ambiguity, where the expression of CD40L was n't fully abrogated in either brother. Despite the test being highly specific and sensitive for the condition, cases like these require genetic testing to provide a conclusive answer. The test can enable rapid diagnostics, helping support patient management and treatment.

Figure 6.

CD40 L expression in whole blood. The three plots represent CD40L expression upon PHA-PMA whole blood stimulation from two brothers. Healthy control first, Patient 1 (older brother) and Patient 2 (younger brother).

STAT1 phosphorylation

Patients with inborn errors of immunity in STAT1 exhibit different functional abnormalities in STAT1, leading to a wide variety of infections. Patients with AR STAT1 LOF mutations typically present with recurrent viral infections [influenza A/B, parainfluenza, Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), etc.], while patients with AD gain-of-function STAT1 mutations are highly susceptible to CMC and show impaired Th17 differentiation and function. STAT1 plays a crucial role downstream of interferon signalling, and STAT1 phosphorylation in response to interferon-α (IFN-α) is instrumental in assessing STAT1 function in patients with genetic variants in this gene [37–39]. The following case demonstrates how this assay can contribute to the diagnosis and monitoring of patients. A young patient was assessed at GOSH and clinically presented with idiopathic thrombocytopenic purpura, recurrent oral and vaginal ulcers, autoimmune neutropenia, recurrent tonsillitis, severe chickenpox requiring anti-viral therapy, and occasional headaches. Genetic analysis revealed a heterozygous mutation in the JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signalling suppressor pathway gene SOCS1 [c.391C>T, p.(Gln131Ter)], which can be associated with lymphoma. The patient exhibited hyper-phosphorylated STAT1 in response to IFN-α when compared with controls.

The patient has been clinically managed with anti-viral prophylaxis, and more recently, the therapy has been co-administered with the JAK inhibitor ruxolitinib. STAT1 phosphorylation is being monitored over time (Fig. 7). Interestingly, a month after starting treatment, the patient showed a significant decrease in STAT1 phosphorylation, bringing it closer to the control sample [40].

Figure 7.

STAT1 phosphorylation in response to IFN-α in whole blood. The first two set of plots represent STAT1 phosphorylation in response to interferon-α (IFN-α) in a healthy control and a patient highlighted a SOCS1 mutation. The second set of plots showed the same patient following treatment with ruxolitinib.

Wiskott–Aldritch protein expression

WAS is an X-linked primary immunodeficiency that frequently presents early in infancy with the typical triad of severe immunodeficiency, thrombocytopenia, and eczema. The gene responsible for this condition is located on the short arm of Chromosome X at Xp11.22–p11.23 and encodes a ∼50-kDa protein involved in transducing signals from cell surface receptors to the actin cytoskeleton, regulating its reorganization, specifically in lymphocytes and megakaryocytes.

Diagnosing and managing WAS requires a comprehensive approach, including clinical evaluation, laboratory testing, and long-term management strategies. Flow cytometry analysis of WAS protein expression in blood cells provides strong evidence for the condition, particularly when the protein is absent in the context of a clinical presentation. Also, X-linked thrombocytopenia (XLT) is caused by mutation in WAS and presents with a milder phenotype; XLT patients usually have abnormal but not absent WAS protein expression. Genetic testing should confirm the mutation; however, protein expression can be used to differentiate patients with WAS from those with XLT. WAS is an X-linked recessive disorder, meaning it primarily affects males, who have only one X chromosome. Females, having two X chromosomes, are typically carriers of the disorder but usually do not exhibit symptoms due to X-inactivation (lyonization) and genetic compensation [41, 42].

In female carriers of WAS variants, skewed lyonisation normally favours the non-mutated X chromosome, so most cells express normal WAS protein and carriers remain asymptomatic. Rarely, skewing in the opposite direction preferentially inactivates the X chromosome carrying the normal allele, and such carriers may become symptomatic [43].

At GOSH, 99% of WAS cases are observed in male patients, consistent with the X-linked recessive inheritance pattern. Only one female case has been identified in which X-inactivation favoured the mutated X, resulting in clinical manifestations. That case was highly unusual, both due to the female gender of the patient and the severe phenotype arising from unfavourable X-inactivation.

The value of this approach in routine practice is exemplified below; a typical male WAS patient seen at GOSH presented with thrombocytopenia, eczema, recurrent infections, and failure to thrive, which aligns with the classical triad of WAS (Fig. 8). A HSCT was performed after genetic mutation was confirmed [44].

Figure 8.

WASP expression in a patient compared to a healthy control.

DOCK8 expression and Th17 differentiation

AR hyper IgE syndrome (AR-HIES) is a combined immunodeficiency marked by recurrent viral and bacterial infections, CMC, eczema, food allergies, autoimmunity, and virus-associated cancers. Most cases result from LOF mutations in DOCK8, which encodes a guanine nucleotide exchange factor involved in actin cytoskeleton regulation via small GTPases like Cdc42.

While AR-HIES shares many features with AD-HIES caused by STAT3 mutations, such as high IgE, eczema, staphylococcal abscesses, and hypereosinophilia, only AD-HIES is consistently linked to impaired Th17 differentiation due to STAT3 dysfunction. Although Th17 defects have also been observed in DOCK8 deficiency, these findings are based on limited and heterogeneous patient data, leaving the role of DOCK8 in STAT3 signalling unclear [45, 46]. A representative case in which this assay informed management is described below. At GOSH, we diagnosed a patient with DOCK8 deficiency but with normal STAT3 activation that would have supported normal Th17 cell differentiation. The child presented with severe eczema, skin infections (Molluscus contagiosus and Herpes Zoster), recurrent respiratory tract infections, chronic asthma, and multiple food allergies. At that point, DOCK8 deficiency was suspected and confirmed by a genetic test, which highlighted a compound heterozygous mutation. This patient showed an abnormal DOCK8 expression by flow cytometry analysis (Fig. 9a). The patient's lymphocyte subsets revealed abnormal expression of the DOCK8 protein. B cells displayed minimal to no detectable expression, whereas T cells showed a bimodal pattern: the majority lacked DOCK8 expression, while a smaller fraction (23.8%) retained partial expression. Although reversion mosaicism, a phenomenon previously reported in DOCK8 deficiency, could account for this partial expression in T cells, it has not yet been confirmed through genetic testing of the individual cell populations. The patient also showed an equivocal Th17 differentiation following Staphylococcus aureus enterotoxin stimulation (Fig. 9b). The genetic confirmation of DOCK8 mutation made repeat Th17 expression analysis unnecessary. Additionally, a recent retrospective study from the laboratory evaluated 52 patients with low Th17%. Of these, 52% had pathogenic or likely pathogenic variants in STAT1, STAT3, or DOCK8. In contrast, no disease-causing variants in these genes were found in patients with normal Th17 levels. These findings support the value of Th17 analysis as a screening tool, particularly for excluding hyper IgE syndrome and CMC, while emphasizing that molecular genetic testing remains essential for definitive diagnosis [47].

Figure 9.

DOCK8 expression and Th17 enumeration. D8 expression by flow cytometry is shown in a healthy control and a DOCK8 deficient patient (a). Th17 differentiation following S. aureus enterotoxin (SEB) stimulation is shown in a control and the DOCK8 deficient patient (b).

Tregopaties—CTLA4 assay

The concept of Tregopathies refers to monogenic immune disorders that impair the function or number of regulatory T cells (Tregs), leading to immune dysregulation. Among the most well-characterized are CTLA4 deficiency and LRBA (LPS-responsive beige-like anchor protein) deficiency, both of which disrupt the critical immune checkpoint molecule CTLA4. In CTLA4 deficiency, heterozygous mutations in CTLA4 result in reduced expression or function of CTLA4 on Tregs, leading to unchecked T-cell activation and autoimmunity. Clinically, patients may present with lymphoproliferation, autoimmunity, hypogammaglobulinemia, and recurrent infections. LRBA deficiency, caused by biallelic mutations in LRBA, leads to impaired recycling and degradation of CTLA-4, causing its intracellular retention and functional deficiency. While the genetic mechanisms differ, both disorders share overlapping clinical features and pathophysiology due to impaired CTLA-4-mediated immune regulation. Importantly, therapies aimed at restoring CTLA-4 function, such as abatacept (a CTLA4-Ig fusion protein), have shown promise in treating these conditions [48]. Although the Tregopathies described above arise from defects in the CTLA-4 pathway, they share with IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked) a common final consequence and loss of functional Treg control and are therefore interrogated by the same flow cytometric approach. In our laboratory, CTLA-4 testing with FOXP3 upregulation allows a more accurate enumeration and functional assessment of Tregs than is otherwise achievable by the standard immunophenotyping (CD4+, CD25+, CD127+). We illustrate this with a case in which both parameters were abnormal: a 21-month-old boy diagnosed with IPEX syndrome, presenting with the classical triad of dermatitis or eczema, enteropathy, and autoimmune cytopenia, specifically thrombocytopenia and anaemia. He also developed alopecia, further supporting the clinical suspicion of a severe autoimmune dysregulation syndrome. Genetic analysis confirmed a pathogenic mutation in the FOXP3 (c.1108A>G; p.Met370Val), which is known to impair Treg development and function [49]. Of note, he experienced multiple thrombotic episodes, primarily in relation to central venous catheter placements, raising additional concerns around immune activation and vascular inflammation. Parental genetic testing was performed, and the mother was found to be a carrier. Flow cytometry–based functional assays demonstrated a markedly reduced proportion of Tregs, accounting for <4% of the parental memory CD4+ T-cell population. This reduction was associated with diminished intracellular FOXP3 expression and poor CTLA-4 upregulation, consistent with impaired Treg differentiation and function, supporting the diagnosis of IPEX syndrome (Fig. 10). Given the severity of his presentation and limited response to supportive therapies, haematopoietic stem cell transplantation (HSCT) remains the main curative approach. HSCT has shown success in reconstituting normal immune regulation in IPEX, particularly when performed early in the disease course. Emerging treatments such as gene therapy are also under consideration, particularly in light of the identified monogenic defect [50]

Figure 10.

CTLA4 and Tregs expression. CTLA4 (top) and Tregs expression (bottom) following CD3/CD28 PBMCs stimulation in a healthy control and a patient with IPEX syndrome. CD4⁺CD45RA⁻ (RA^-) memory T cells has been gated and evaluated for the concomitant expression of CTLA4 and Foxp3 at high mean fluorescent intensity (MFI) (RA-Foxp3++CTLA4++). CD4⁺CD45RA⁻ memory T cells have also been gated for Tregs differentiation by the CD3/CD28 mediated CD25/Foxp3 expression.

Conclusion

As illustrated above, protein-based testing, flow cytometry or enzyme assays, provides a rapid diagnosis that can be confirmed by targeted genetic testing. In the era of whole-genome sequencing, several VUSs are identified that require protein expression and ideally functional confirmation to determine their pathogenicity. In addition, protein assays can be used to monitor patients post-HSCT or gene therapy. A recent study showed that combining proteomic profiling with RNA sequencing enhanced the diagnostic yield in IEI by identifying protein-level abnormalities that were not detected through genomic analysis alone [17].

Author contributions

Micol Ferro (Data curation, Formal analysis, Investigation, Validation, Writing—original draft, Writing—review & editing), Mercedes Selby (Data curation, Formal analysis, Investigation, Validation, Writing—original draft, Writing—review & editing), Mark Davis (Data curation, Formal analysis, Investigation, Validation, Writing—original draft), and Kimberly Gilmour (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Supervision, Validation, Writing—original draft)

Funding

This work was supported by National Institute for Health and Care Research (GOSH NIHR BRC).

Data availability

Standard operating procedures for each assay are available on request from the corresponding authors.

Ethical approval

Test development was approved by National Research Ethics (NRES) Committee London—Bloomsbury. 06/Q0508/16 Development of Diagnostic Tests for Primary Immunodeficiency.