Flow cytometry for the diagnosis of inborn errors of immunity

INTRODUCTION — Flow cytometry is a powerful technique for the measurement of multiple characteristics of individual cells within heterogeneous populations. This topic review gives an overview of the technical aspects of flow cytometry and highlights some of its uses in the diagnosis of inborn errors of immunity (IEI; formerly "primary immunodeficiencies" [PIDs]). Each of these immunodeficiencies is discussed in greater detail separately in specific topic reviews.

TECHNICAL ASPECTS

Flow cytometer components and function — A basic flow cytometer consists of five main components: a flow cell (through which cells flow), a laser, optical components, detectors to amplify signals, and an electronics/computer system. With these five components, the flow cytometer is capable of performing instantaneous measurements by passing thousands of cells per second through a laser beam and capturing the emerging light from each cell as it passes through the interrogation point. Any suspended cell or particle ranging from 0.2 to 150 micrometers in size is suitable for analysis. Analyses to determine cellular characteristics, such as size, granularity, viability, and immunophenotyping, are the most common types of studies done.

Sample types — Theoretically, any biologic sample can be analyzed by flow cytometry, although peripheral blood is the most common sample analyzed. Depending upon the specific assay, whole blood may be used, or peripheral blood mononuclear cells (PBMCs) may be isolated and used for analysis. Bone marrow samples are used during the workup of suspected leukemia. Other biologic sources have been used for flow cytometry, such as cerebral spinal fluid or bronchoalveolar lavage, although the lack of published normals from these biologic sites can make interpretation difficult.

Immunophenotyping — Immunophenotyping is a technique used to characterize the makeup of cell populations by detecting cellular protein expression. Immunophenotyping uses an antibody specific for the antigen of interest that is conjugated to a fluorescent compound known as a fluorophore or fluorochrome (figure 1). These fluorescent compounds absorb energy from the laser source, causing an electron to be raised to a higher energy level. The excited electron quickly returns to its ground state, emitting the excess energy as a photon of light of a characteristic wavelength that is detected by the flow cytometer.

Different fluorochromes are excited by different wavelengths of light and emit light at different wavelengths. Thus, it is possible to simultaneously detect several different fluorescently labeled antigens on a cell by using lasers of different wavelengths and filters of specific wavelengths to detect the fluorescent emission. As an example, flow cytometers may have as many as five lasers of different wavelengths. Each laser can be paired with three to four different filters, allowing for an analysis of up to 20 different proteins simultaneously. However, there is considerable overlap of excitation and emission spectra of individual fluorochrome, which can limit certain combinations of fluorochromes. Most immunophenotyping panels in clinical use consist of 10 to 12 different markers.

Spectral flow cytometry is similar to conventional flow cytometry but with multiple detectors for each fluorochrome rather than one detector. This allows for better differentiation of fluorochromes based upon the fluorescent spectral signature of each fluorochrome, enabling the use of more fluorochromes in a single sample. Some spectral flow cytometers may be able to resolve 40 different fluorochromes in one sample.

Data collection and analysis — Each cell is analyzed by the following parameters as the cell suspension passes through the flow cytometer:

●The intensity of the forward scatter (FSC) of light, which is a reflection of cell size

●The intensity of the side scatter (SSC) of light, which is a reflection of cell granularity

●The intensity of light emitted by each fluorochrome, which reflects the level of expression of the antigen of interest

The results are typically presented by two-dimensional dot plots or one-dimensional histograms. As an example, flow cytometric analysis of peripheral blood by FSC (ie, size) and SSC (ie, granularity) yields a characteristic two-dimensional plot that distinguishes different cell populations (figure 2):

●Neutrophils are large and highly granular and therefore exhibit high SSC and high FSC signal.

●Monocytes are large but less granular. Thus, they exhibit lower SSC signal but similar FSC signal compared with neutrophils.

●Lymphocytes are the smallest and least granular of these cells and therefore exhibit the lowest SSC and FSC signals.

The level of expression of specific antigens can be examined in each of the populations defined by FSC and SSC profiles when flow cytometry is combined with fluorescent staining using antibodies against these antigens. As an example, when peripheral blood is stained with antibodies to CD15 (a neutrophil antigen) and CD14 (a monocyte antigen), three populations of cells are detected: a population of cells that only express CD15 (neutrophils), a population of cells that only express CD14 (monocytes), and a population of cells that express neither CD15 nor CD14 (figure 3). The percentage of each of these populations is used together with a complete blood count (CBC) with differential to determine the absolute numbers of the different cell populations.

Mass cytometry — A technology has been developed that combines flow cytometry with mass spectrometry, known as mass cytometry or cytometry by time-of-flight (CyTOF) [1]. This technology uses labeled antibodies to evaluate cell populations similar to fluorescence-based readouts. However, in CyTOF, the antibodies are labeled with heavy-metal ions and interrogated by mass spectrometry. The advantage of this technique is that the readout is based on the atomic number of the heavy metal, and there is little overlap between the isotopes. Thus, a greater number of antibodies can be used simultaneously, allowing for the analysis of over 40 targets per cell. CyTOF is not widely available clinically but will probably become more prominent in the future [2].

USE FOR DIAGNOSIS OF INBORN ERRORS OF IMMUNITY

Indications for use — IEI are a group of disorders, which are usually inherited, that can affect cells of the innate or adaptive immune systems [3,4]. Flow cytometry is an essential tool in the diagnosis of immunodeficiencies. Its use is not limited to IEI, since this methodology may be used to evaluate the immune system in secondary or acquired immunodeficiencies, such as those that may occur during viral infections (eg, human immunodeficiency virus [HIV] infection), post-bone marrow transplantation, during chemotherapy, and during immunosuppression for systemic autoimmunity.

There has been a rapid increase in discovery of novel IEI, mainly due to next-generation deoxyribonucleic acid (DNA) sequencing technologies. In addition, there are now commercially available DNA sequencing panels that rapidly and economically screen genes responsible for a large number of IEI. The advancement in DNA sequencing technologies has led to a shift in our approach to the workup of suspected IEI, and we and others in the field use sequencing panels earlier during the assessment. However, flow cytometry remains a crucial technique, particularly when there are variants of uncertain significance detected in DNA sequencing panels. Immune phenotyping and functional testing by flow cytometry can help determine if the variants are functionally significant in order to ascertain the genetic cause of an IEI. (See "Genetic testing in patients with a suspected primary immunodeficiency or autoinflammatory syndrome".)

Defects in lymphocyte numbers — A complete blood count (CBC) with differential is an essential first step in the workup of suspected IEI, although this test does not determine the different lymphocyte subsets. Flow cytometry enables qualitative and quantitative enumeration of lymphocyte subsets, including CD4- and CD8-positive T cells, B cells, and natural killer (NK) cells (figure 4).

T cells — Markedly diminished numbers of naïve T cells, with or without diminished numbers of B and NK cells, are a hallmark of severe combined immunodeficiency (SCID) (figure 5). SCID can be divided into several subtypes based upon the cell types that are deficient. As examples, pathogenic variants in CD3 subunits, CD45, or interleukin (IL) 7 receptor alpha (IL-7-R-alpha) result in T-B+NK+ SCID. Pathogenic variants in the recombinase-activating genes (RAG) or Artemis result in T-B-NK+ SCID. Pathogenic variants in adenosine deaminase (ADA) result in T-B-NK- SCID. Pathogenic variants in the genes for Janus kinase 3 (JAK3) or the gamma chain of the IL-2 receptor (IL-2-R-gamma) result in T-B+NK- SCID. (See "Severe combined immunodeficiency (SCID): An overview" and "Severe combined immunodeficiency (SCID): Specific defects".)

Flow cytometry using markers specific for T cells (CD3), B cells (CD19), or NK cells (CD56) is the first step in evaluating children with suspected SCID due to clinical manifestations or a failed newborn screen for SCID. It is also important to determine the percentage of naïve and memory T cells in infants with suspected SCID. In normal newborns, nearly all of the T cells are naïve. However, in some situations such as maternal engraftment of T cells or hypomorphic variants in SCID-causing genes, the numbers of T cells may be normal in the affected infant, but nearly all are memory T cells. CD45RA and CD45RO are the two antigens that distinguish naïve from memory T cells, respectively. Staining with CD62L, CD45RA, and CD31 can enumerate the number of recent thymic emigrants, which is also low in SCID.

Enumeration of naïve T cells is also used to determine T cell counts in children with defects affecting thymic development, such as DiGeorge syndrome, also known as 22q11 deletion syndrome, and CHARGE syndrome. Analysis of T cell numbers is important because T cell counts correlate with mortality in these individuals [5]. Affected individuals exhibit varying degrees of defects in the development of the thymus. Most patients with DiGeorge have mild defects in T cell numbers and are not clinically immunodeficient (incomplete DiGeorge syndrome). Rarely, infants may have a complete absence of thymic function (complete DiGeorge syndrome) with a profound reduction in the number of T cells similar to that found in SCID. (See "DiGeorge (22q11.2 deletion) syndrome: Management and prognosis".)

Regulatory T cells — Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a disorder causing widespread autoimmune manifestations, usually beginning soon after birth. Pathogenic variants in the forkhead box P3 gene (FOXP3) are the cause of IPEX. Foxp3 is a transcription factor that is crucial to the development, survival, and effector function of regulatory T (Treg) cells, which are essential in maintaining tolerance and preventing autoimmunity [6]. Treg cells account for approximately 5 to 10 percent of peripheral blood CD4+ T cells. Treg cells express high levels of CD25, the high-affinity binding subunit of the IL-2 receptor, and low levels of CD127, the alpha subunit of the IL-7 receptor [7]. Use of antibodies to CD4, CD25, CD127, and Foxp3 allows for the quantitation of Treg cells in peripheral blood that are usually reduced in patients with IPEX (figure 6). The presence of Foxp3-positive cells does not rule out IPEX, since some pathogenic variants decrease or abolish Foxp3 function but not protein stability. Sequence analysis of the FOXP3 gene should be performed if the clinical history suggests IPEX but Foxp3 positive cells are present on flow cytometry. IPEX-like diseases can also be caused by deficiencies in CD25 or signal transducer and activator of transcription 5b (STAT5b), key signal transduction subunits of the IL-2 receptor [8,9]. (See "IPEX: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked".)

Follicular helper T cells — Germinal center (GC) formation in secondary lymphoid organs indicates B cell clonal expansion, somatic hypermutation (SHM), and class-switch recombination (CSR) after proper activation of B cells by helper T cells. Three cell types comprise a GC: B cells, follicular dendritic cells (FDC), and follicular helper T (Tfh) cells. Tfh cells migrate to the follicular region and provide costimulatory signals and cytokines to GC B cells, which then proliferate and undergo SHM and CSR. Tfh are characterized by the expression of the C-X-C motif chemokine receptor 5 (CXCR5, where C = cysteine and X = any amino acid; also called CD185 (figure 7)), which enables these cells to migrate towards a chemokine cytosine-X-cytosine motif chemokine ligand 13 (CXCL13) gradient secreted by the FDC [10,11]. Tfh are detected in elevated numbers in patients with systemic autoimmunity [12] and in patients with common variable immunodeficiency. Tfh cells are reduced in hyperimmunoglobulin E syndrome (HIES) due to signal transducer and activator of transcription 3 (STAT3) pathogenic variants [13]. (See "Autosomal dominant hyperimmunoglobulin E syndrome", section on 'T cell defects'.)

B cells — Severe reduction in all serum immunoglobulin isotypes with profoundly diminished or absent B cells are hallmarks of congenital forms of agammaglobulinemia. X-linked agammaglobulinemia (XLA) is the most common form of congenital agammaglobulinemia, accounting for approximately 85 percent of patients. Patients with XLA present in early childhood with recurrent infections, particularly with encapsulated bacteria and chronic enteroviral infections. XLA is caused by pathogenic variants in the Bruton tyrosine kinase (BTK) gene, leading to a developmental block at the pre-B cell stage of B cell development in the bone marrow that results in near complete deficiency of all mature B cells. B cell enumeration is performed using the pan B cell markers CD19 or CD20. The numbers of B cells are usually profoundly reduced in XLA. Intracellular expression of the BTK protein can be analyzed in monocytes to confirm loss of expression of the BTK protein since patients with XLA typically lack B cells (figure 8). Females who carry the mutated allele express normal numbers of B cells and normal levels of BTK expression in B cells due to nonrandom X-chromosome inactivation, but only approximately 50 percent of their monocytes express BTK. Thus, the percent of monocytes expressing BTK can be used to determine the carrier status in relatives of affected children [14,15]. BTK is also expressed in platelets, and platelet expression of BTK can be used to determine the presence of this protein when B cells are absent [16]. (See "Agammaglobulinemia".)

Autosomal recessive (AR) forms of agammaglobulinemia present with a clinical phenotype similar to XLA [17]. AR agammaglobulinemias are caused by pathogenic variants in genes essential for the development of B cells (eg, immunoglobulin heavy constant mu [IGHM], immunoglobulin lambda-like polypeptide 1 [IGLL1], CD79A, CD79B, B cell linker [BLNK], phosphoinositide-3-kinase regulatory subunit 1 [PIK3R1], and transcription factor 3 [TCF3]). Similar to XLA, the numbers of B cells are usually markedly decreased or absent in AR forms of agammaglobulinemia, but BTK expression is normal. (See "Agammaglobulinemia".)

Natural killer cells — NK cells are an innate lymphoid population that exhibit cytotoxicity toward virally infected cells and tumor cells. Unlike CD8 cells, which also exhibit cytotoxicity toward tumors and viruses, NK cells exhibit spontaneous cytotoxicity without a requirement for antigen activation. NK cells express unique cell lineage markers such as CD56, as well as CD16, an Fc receptor that mediates antibody-dependent cellular cytotoxicity. Most NK cells do not express CD3, although there is a small population of cells, called NKT cells, that are CD3+CD56+. NK cell deficiencies can occur due to a lack of NK cells (eg, minichromosome maintenance complex component 4 [MCM4] or GATA binding protein 2 [GATA2] deficiency) or a lack of NK function (eg, defects in CD16) [18-21]. These patients are susceptible to herpetic viral infections. Defects in perforin and other genes involved in the cytotoxic machinery also lead to defective NK killing, as well as defective CD8 killing. These patients are at risk for hemophagocytic lymphohistiocytosis (HLH). (See 'Cytotoxic lymphocyte defects' below and "NK cell deficiency syndromes: Clinical manifestations and diagnosis".)

Defects in B cell function — The functional B cell defects seen in common variable immunodeficiency (CVID) are reviewed below.

Common variable immunodeficiency — CVID is a relatively common IEI that typically presents with upper and lower respiratory tract and gastrointestinal tract infections. Frequently, patients with CVID also have disorders of immune regulation (eg, autoimmune cytopenias, inflammatory bowel disease, interstitial lung disease). The diagnostic criteria for CVID include a low age-adjusted immunoglobulin G (IgG) and a low immunoglobulin A (IgA) or immunoglobulin M (IgM), impaired specific antibody responses following immunization, and exclusion of other causes of hypogammaglobulinemia in a patient greater than two years of age [22]. (See "Clinical manifestations, epidemiology, and diagnosis of common variable immunodeficiency in adults" and "Common variable immunodeficiency in children".)

In general, the diagnosis of CVID is reserved to those cases in which no genetic cause is identified. Monoallelic mutations in the transmembrane activator and calcium modulator and cyclophilin ligand protein interactor (TACI) gene (TNFRSF13B) are a risk factor to develop CVID in a small percentage of cases [23]. With the increased use of high-throughput DNA sequencing, an increasing number of monogenic causes of "CVID-like" diseases have been identified [4,22,23].

Patients with CVID have been grouped based upon their respective B cell phenotypes as determined by flow cytometry [24,25]. These classification schemes involve analysis of the maturational state of B cells and other antigens (eg, CD21, CD38), which may help to predict noninfectious complications of CVID, such as splenomegaly, lymphadenopathy, or granulomatous disease. B cells go through a maturational program from naïve B cells (characterized by the expression of immunoglobulin D [IgD] but lacking the memory marker CD27) to memory B cells (IgD+CD27+) and switched memory B cells (IgD-CD27+) upon antigen exposure (figure 9).

The EUROclass criteria for CVID distinguished patients based upon the extent of B cell lymphopenia (<1 percent B cells), the reduction of switched memory B cells, the expansion of CD21 low B cells, and the expansion of transitional B cells (CD38++IgM++) (figure 10) [24]. Granulomatous disease was associated with a reduction in switched memory B cells, splenomegaly with low switched memory B cells and increased CD21 low B cells, and lymphadenopathy with increased transitional B cells. (See "Pathogenesis of common variable immunodeficiency".)

Defects in T cell function — T cell function can be assessed by a variety of assays using flow cytometry. Upon activation, T cells proliferate and differentiate into cytokine secreting (CD4) or cytotoxic (CD8) cells. CD4 cells can be further differentiated into different subsets based upon the type of cytokines that they express, such as T helper type 1 (Th1) cells expressing interferon (IFN) gamma, Th2 cells expressing IL-4, and Th17 cells expressing IL-17. CD4 Tfh cells are also critical for B cell stimulation, which they accomplish through cell surface molecules (eg, CD40L) and cytokines.

SCID or SCID-like illnesses — Complete loss of T cells results in severe combined immunodeficiency (SCID) (see 'T cells' above), but, in some instances of SCID or SCID-like illnesses, there are a significant number of T cells that do not proliferate or function (eg, stromal interaction molecule 1 [STIM1] and Orai1 deficiencies). The workup of these patients requires assessment of the proliferative ability of T cells. Historically, tritiated thymidine incorporation was used to measure the proliferation of T cells, but this can also be accomplished by flow cytometry. One manner to test this is to label cells with a fluorescent molecule, such as carboxyfluorescein succinimidyl ester (CSFE), and then activate the cells with T cell mitogens [26]. As cells divide, the amount of fluorescence is reduced in half, which can be measured by flow cytometry. An alternative to this is to use fluorescently labelled thymidine analogs, such as BrdU or EdU, to measure proliferation. (See "Combined immunodeficiencies: An overview".)

Hyperimmunoglobulin M syndrome — Hyperimmunoglobulin M syndrome (HIGM) is characterized by recurrent infections and very low levels of serum IgG, immunoglobulin E (IgE), and IgA, with normal or elevated IgM. The most common form of HIGM is X-linked HIGM (XHIM) syndrome caused by pathogenic variants in the CD40 ligand (CD40L, also called CD154) gene. CD40L is transiently expressed upon CD4+ T cell activation, which then interacts with CD40 on B cells, resulting in B cell activation, proliferation, immunoglobulin class switch recombination, and affinity maturation. Surface expression of CD40L on CD4+ T cells can be measured by in vitro activation of peripheral blood by phorbol esters (eg, phorbol 12-myristate 12-acetate [PMA]) and calcium ionophore that induce the expression of CD40L (figure 11). The failure to detect CD40L on activated cells is used as a screening flow cytometric assay to diagnose XHIM [27]. In addition, the expression of CD40 on B cells can also be analyzed since defective CD40 expression is known to be one of the causes of autosomal recessive HIGM (AR-HIGM) syndrome (figure 11). In a small percentage of cases of XHIM, CD40L is expressed on activated T cells but is unable to bind to CD40 due to missense mutations. In these instances, functional CD40L-CD40 binding assays can be performed by flow cytometry [28].

Autoimmune lymphoproliferative syndrome — Autoimmune lymphoproliferative syndrome (ALPS), also known as Canale-Smith syndrome, is caused by genetic defects in proteins involved in the Fas-mediated apoptotic pathway. ALPS patients present with lymphadenopathy, hepatosplenomegaly, and autoimmune cytopenias and are at high risk of developing lymphomas [29]. ALPS is categorized into Ia, Ib, IIa, IIb subtypes due to gene pathogenic variants in Fas/CD95, Fas ligand (FasL), caspase10, and caspase8, respectively. Normally, <1 percent of T cells that express the T cell receptor (TCR) alpha and beta chains do not also express the CD4 or CD8 coreceptors. These cells are called double-negative T cells (DNTs). In ALPS, the number of TCR alpha beta DNTs is increased (figure 12). Additionally, these TCR alpha beta DNT cells coexpress the B cell antigen B220. Evans syndrome has features of ALPS (eg, autoimmune cytopenias); therefore, it is important to rule out ALPS in this disorder [30]. (See "Autoimmune lymphoproliferative syndrome (ALPS): Epidemiology and pathogenesis" and "Autoimmune lymphoproliferative syndrome (ALPS): Clinical features and diagnosis" and "Apoptosis and autoimmune disease", section on 'Autoimmune lymphoproliferative syndrome' and "Autoimmune hemolytic anemia (AIHA) in children: Classification, clinical features, and diagnosis", section on 'Evans syndrome'.)

Hyperimmunoglobulin E syndrome (HIES) — Autosomal dominant (AD) HIES is characterized by pulmonary infections, staphylococcal abscesses, eczema, bone and connective tissue abnormalities, and elevated serum IgE levels. The defects in HIES are caused by missense pathogenic variants in the transcription factor STAT3, which is required to induce CD4+ T cells to produce IL-17a, a cytokine important in the generation of effective immune responses to bacteria and fungi [31]. These pathogenic variants are inherited in an AD manner due to a dominant-negative effect of the mutated protein. A flow cytometry-based functional assay that measures the ability of T cells to synthesize IL-17a can be used to screen for this disorder (figure 13). In this assay, peripheral blood mononuclear cells (PBMCs) are stimulated with phorbol ester (PMA) and calcium ionophore in the presence of molecules (eg, brefeldin A) that prevent exocytosis of IL-17a. After several hours, the cells are stained with an IL-17a specific antibody in the presence of detergents that allow for intracellular staining of IL-17a. The production of IL-17a in this assay is impaired in patients with AD HIES syndrome [32]. A low percentage of IL-17 producing T cells is not specific to AD HIES, as this can be seen in atopic disorders [32]. Other testing abnormalities in HIES include a reduced percentage of Tfh cells as described above. (See "Autosomal dominant hyperimmunoglobulin E syndrome" and 'Follicular helper T cells' above.)

X-linked lymphoproliferative syndrome, types 1 and 2 — X-linked lymphoproliferative syndrome type 1 (XLP-1) and X-linked lymphoproliferative syndrome type 2 (XLP-2) are rare, X-linked disorders that may present with HLH, often following Epstein-Barr virus (EBV) infection. XLP-1 is caused by pathogenic variants in the Src homology 2 domain protein 1A (SH2D1A) gene that encodes the signaling lymphocyte activation molecule (SLAM) associated protein (SAP), whereas XLP-2 is caused by pathogenic variants in the baculoviral inhibitor of apoptosis protein repeat-containing protein 4 (BIRC4) gene that encodes the X-linked inhibitor of apoptosis protein (XIAP). Both XLP-1 and XLP-2 may present with hypogammaglobulinemia. XLP1 is also associated with EBV-associated lymphomas. XLP-2 and carriers of the mutant BIRC4 gene are at risk to develop inflammatory bowel disease. These life-threatening IEI can be cured with hematopoietic cell transplantation. Decreased or absent expression of SAP or XIAP proteins in lymphocytes can be used to screen for XLP1 and XLP2, respectively (figure 14) [33,34]. In addition, functional testing of the XIAP protein can be performed by stimulating monocytes with muramyl dipeptide and measuring intracellular tumor necrosis factor (TNF) alpha, which requires functional XIAP [35]. (See "X-linked lymphoproliferative disease" and "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis", section on 'Specialized testing'.)

Wiskott-Aldrich syndrome — Wiskott-Aldrich syndrome (WAS) is a congenital, X-linked disorder characterized by the triad of bleeding due to thrombocytopenia, eczema, and frequent infections. There is an overlap of symptoms in WAS patients with a closely related disease, X-linked thrombocytopenia and congenital X-linked neutropenia [36]. The WAS protein (WASp) is important for actin polymerization of immune cells upon activation and can affect both T and B cell responses. Deficient expression of WASp in lymphocytes is indicative of WAS (figure 15) [37]. Flow cytometry can also be used to evaluate heterozygosity carrier status in mothers of affected male patients [38]. (See "Wiskott-Aldrich syndrome".)

Cytotoxic lymphocyte defects — NK cells and CD8+ T cells are cytotoxic cells that possess the ability to recognize and kill virally infected cells and tumor cells. Upon activation of NK cells or CD8+ T cells, cytotoxic granules translocate toward the point of contact with the target cell and fuse with the plasma membrane, releasing the cytotoxic molecules perforin and granzymes into the intercellular space. This process is facilitated by several proteins including Rab27a, Munc13-4, Syntaxin 11, and Lyst1. Perforin then facilitates the delivery of granzymes into the target cell and the initiation of apoptosis. Genetic pathogenic variants in proteins involved in this process may result in HLH in response to viral infections, particularly cytomegalovirus and EBV [39]. Flow cytometry can be used to detect deficiencies in these molecules, as well as to test for defects in killing of target cells. (See "NK cell deficiency syndromes: Clinical manifestations and diagnosis" and "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis" and "Treatment and prognosis of hemophagocytic lymphohistiocytosis".)

Cytotoxicity assays — The cytotoxic pathway can be evaluated using a flow cytometry-based NK cell cytotoxicity assay since NK cells exhibit the ability to kill tumor cells without preactivation or other manipulation. Briefly, a target tumor cell line (K562) is labeled with a fluorescent dye (carboxyfluorescein succinimidyl ester [CFSE]) so that these target cells can be identified from the subject's effector cells. These target cells are incubated with increasing ratios of PBMCs containing NK cells. Upon apoptosis, target cells shrink (reflected by a reduction in cell size) and incorporate the fluorescent dye (7-aminoactinomycin D [7-AAD]), which intercalates into the DNA of apoptotic cells (figure 16). Defective killing of effector cells indicates cytotoxic lymphocyte dysfunction and possible variants in gene(s) in the cytotoxic pathway described above. Flow cytometry can then be used to analyze the expression of cytotoxic molecules (eg, perforin), as well as determine if there are defects in the degranulation of these cells. (See "NK cell deficiency syndromes: Clinical manifestations and diagnosis".)

Expression of cytotoxic molecules and testing for degranulation — Defective cytotoxicity is the hallmark of HLH. The expression of perforin as detected by intracellular flow cytometry is an important analysis in evaluating patients with defective cytotoxicity since 20 to 30 percent of HLH patients present with defects in perforin expression (figure 17) [40,41]. Genetic mutational analysis should be performed if perforin expression is normal to examine other pathogenic variants associated with HLH (Rab27a, Munc13-4, and Lyst1) [39,42]. These proteins are critical to the translocation and fusion of cytotoxic granules to the plasma membrane. Measurement of surface expression of CD107a (lysosomal-associated membrane protein 1 [LAMP-1]) by flow cytometry can be used for assessment of the cytotoxic pathway since this protein is displayed on the cell surface upon cytotoxic granule fusion with the plasma membrane. Normally, approximately 5 to 15 percent of NK cells express CD107a on the cell surface when in contact with the target cells (figure 18). (See "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis".)

Innate immune system defects — Neutrophil defects are associated with skin infections, abscesses, poor pus formation, and defects in wound healing. Neutrophils must adhere to the endothelium at sites of infection, migrate toward the infectious organisms, and then phagocytose and kill the organisms. Defects in any step along this pathway can result in disease. (See "Primary disorders of phagocyte number and/or function: An overview".)

Leukocyte adhesion deficiencies — Movement of leukocytes from the vascular space to the active site of inflammation involves extravasation of neutrophils by rolling and adhering to the endothelial cell surface. Leukocyte adhesion deficiency type 1 (LAD I) is an AR disorder due to pathogenic variants in the beta-2 integrin CD18. LAD II, also an AR disorder, occurs due to pathogenic variants in a fucose transporter that causes sialyl Lewis X/CD15a deficiency. Both CD15 and CD18 are involved in the rolling and firm adhesion of neutrophils to endothelium. Flow cytometry can assess the expression of CD18 and CD15 and should be tested in any infant with poor wound healing, abscesses, or skin infections (figure 19). LAD III is caused by defects in the intracellular protein Kindlin 3, and patients have infections as well as a bleeding disorder since this protein is involved in signaling to activate the integrin proteins. LAD III is diagnosed through genetic testing and platelet studies. (See "Leukocyte-adhesion deficiency".)

Chronic granulomatous disease — Chronic granulomatous disease (CGD) is a rare immunodeficiency but one of the most common phagocyte defects. CGD is characterized by the absence or severe reduction of the neutrophil oxidative burst that is required to produce molecules, such as superoxide, that are critical to the killing of microorganisms. CGD patients suffer from a wide variety of recurrent infections typically caused by catalase-positive bacteria and fungi. In CGD, microbial killing is defective due to pathogenic variants in one of four known components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. These diseases are X linked (gp91phox/CYBB) or AR (p22phox/CYBA, p47phox/NCF1, p67phox/NCF2). (See "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis", section on 'Genetic defects'.)

Flow cytometric diagnosis of CGD is made based upon reduced oxidation of the fluorescent dye dihydrorhodamine 123 (DHR123). In this assay, neutrophils are loaded with DHR123, then stimulated with phorbol esters or soluble bacterial peptides (eg, fMLP) (figure 20) [43]. A strong shift in fluorescence is typically seen when the neutrophils are activated. This fluorescence is either absent or severely reduced in patients with CGD. This assay can also be used to detect carrier status of female relatives of patients with X-linked CGD since CGD carriers will exhibit a bimodal peak due to random X-inactivation. However, skewed X-inactivation may alter this finding, especially if the carrier has some CGD features. Antibodies to each of the NADPH oxidase subunits are available and can be used to detect expression of these proteins in neutrophils. This is particularly helpful for p47phox since sequencing confirmation is not possible due to a p47phox pseudogenes. (See "Chronic granulomatous disease: Pathogenesis, clinical manifestations, and diagnosis", section on 'Diagnosis'.)

Activation of the oxidative burst pathway in neutrophils by N-formyl-methionyl-leucyl-phenylalanine (fMLP) is G-protein dependent. Thus, this assay is also helpful in evaluating children with a suspected immunodeficiency due to activating pathogenic variants in the gene RAC2, which may present in a similar manner to CGD. In this case, the DHR123 response will be normal when stimulated with PMA but will be absent when stimulated with fMLP [44].

Mendelian susceptibility to mycobacterial diseases — Individuals with defects in the generation of or responsiveness to IFN-gamma are susceptible to infections with mycobacteria and Salmonella since IFN-gamma plays a major role in the activation of macrophage and the clearance of intracellular pathogens. (See "Mendelian susceptibility to mycobacterial diseases: Specific defects".)

Macrophages and dendritic cells secrete IL-12 upon intracellular bacterial infection. IL-12 then binds to the IL-12 receptor on CD4+ T cells, thereby inducing production of IFN-gamma. IFN-gamma produced by CD4+ T cells binds to the IFN-gamma receptor (IFNGR) on macrophages, leading to activation of signal transducer and activator of transcription 1 (STAT1). This results in activated macrophages capable of eliminating intracellular microbes. Defects in any of these proteins can lead to increased susceptibility to mycobacteria and Salmonella.

Monogenic IEI in this pathway include mutations in subunits of the interferon gamma receptor (IFNGR1) or the IL-12 receptor (IL12RB1), STAT1, and IL-12B. Flow cytometry is useful in the evaluation of patients with increased susceptibility to mycobacterial infection (figure 21). Determination of IFNGR1 (CD119) and IL12RB1 (CD212) expression by flow cytometry can be used to look for loss of protein expression. Other assays such as phosphorylation of STAT1 and STAT4 in response to IFN-gamma and IL-12 stimulation, respectively, can be used to test whether the receptors are functional [45,46].

Toll-like receptor defects — The ability to recognize infectious microorganisms is a critical first step in the generation of an immune response. Toll-like receptors (TLRs) are a group of receptors that recognize pathogen-associated molecular patterns (PAMPs) of bacteria, viruses, and fungi and mediate this effect. Defects in the TLR signaling pathway proteins IL-1 receptor associated kinase 4 (IRAK-4) and myeloid differentiation primary response gene 88 (MyD88) are implicated in invasive pneumococcal and staphylococcal disease, and pathogenic variants in genes involved in TLR3 signaling are associated with herpetic encephalitis [47,48]. Flow cytometry can be used to assess the response of immune cells to TLR ligands by measuring the intracellular production of cytokines (figure 22) [49,50]. Similarly, defects in the nuclear factor (NF) kappa-B essential modifier (NEMO) or NF-kappa-B inhibitor protein alpha (also called inhibitor of kappa B alpha [IKBA]) can result in defective TLR signaling, which can be detected by this assay. Flow cytometry can also be used to measure the degradation of IKBA [51]. There are no known flow cytometric studies to evaluate the function of the TLR3 pathway. (See "Toll-like receptors: Roles in disease and therapy" and "Combined immunodeficiencies: An overview".)

Gain-of-function pathogenic variants in immune deficiency/immune dysregulation syndromes — Point pathogenic variants in important immune response genes, such as STAT1 or STAT3, that result in enhanced signaling (gain-of-function variants) can lead to a variety of disorders with features of immunodeficiency and autoimmunity/immune dysregulation [52,53]. Phosphorylation of STAT1 or STAT3 in response to cytokines that activate these pathways, such as IFN-gamma and IL-6, respectively, can be measured by flow cytometry [54]. Cells are typically stimulated with cytokines for variable amounts of time, then fixed and stained with antibodies that bind specific phosphorylated residues on these proteins (ie, phosph-STAT1 or phosphor-STAT3) (figure 21). Any reduction of phosphorylation following cytokine stimulation or failure of these proteins to normally dephosphorylate over time can aid in the diagnosis of these syndromes. Similar assays can be used to assess loss-of-function or dominant-negative pathogenic variants in these genes. Theoretically, these assays can be used for any signaling pathways that result in phosphorylation of proteins as long as there are specific antibodies for the phosphorylated protein. STAT4, STAT5, and STAT6 gain-of-function disorders have been described. (See 'Mendelian susceptibility to mycobacterial diseases' above.)

SUMMARY

●Technical aspects – Flow cytometry is most commonly used in cellular analysis to determine characteristics, such as size, granularity, viability, and cellular protein expression (immunophenotyping). (See 'Technical aspects' above.)

●Use as a diagnostic tool for inborn errors of immunity – Flow cytometry is an essential tool in the diagnosis of inborn errors of immunity (IEI). Lymphocyte subset enumeration can be determined by measuring expression of certain cell surface molecules. Flow cytometry-based functional assays are also available to test for defects in immune pathways. (See 'Use for diagnosis of inborn errors of immunity' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Trivikram Dasu, PhD, who contributed to earlier versions of this topic review.

The UpToDate editorial staff acknowledges E Richard Stiehm, MD, who contributed as a Section Editor to an earlier version of this topic review.