Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

Abstract

Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements.

FIG 1

Assessment of CD40L expression and function for diagnosis of X-linked hyper-IgM syndrome (XL-HIGM). The top left panel (panel 1) shows expression of CD40L (CD154) on activated CD4⁺ T cells after stimulation with PMA. A control for T cell activation, CD69, is included in the assay (not shown). Functional activity of the ligand is measured by binding to a soluble form of the receptor—CD40muIg (panel 2). The bottom panels show flow cytometric data for a male patient with XL-HIGM with lack of expression of CD40L on activation of CD4⁺ T cells (panel 3) and therefore, no binding to the soluble receptor (panel 4). The grey peak represents the unstimulated sample. The purple peak represents the specific antibody.

FIG 2

LRBA protein expression in lymphocytes from a healthy individual and a patient. LRBA protein is expressed intracellularly, and data are shown for T cells (panel 1), B cells (panel 2), and NK cells (panel 3) from a healthy donor (A) and a patient (B). Peripheral blood mononuclear cells (PBMCs) are isolated from blood samples collected in sodium heparin or EDTA and assessed for LRBA expression without stimulation, using an isotype control and a specific primary antibody. Intracellular protein expression is assessed by cell fixation and permeabilization prior to simultaneous staining with cell lineage markers and primary antibody. The protein is visualized using a fluorescently labeled secondary antibody. Both percent-positive lymphocyte subsets (T, B, or NK cells) along with mean fluorescence intensity (MFI) information are captured. LRBA protein is robustly expressed in the majority of lymphocyte subsets without stimulation. In the B panels, data from three healthy donors are represented as donor 1, donor 2, and donor 3. The patient shows a normal proportion of lymphocyte subsets expressing LRBA; however, the mean fluorescence intensity, which correlates with the amount of protein expression, is significantly reduced, which is consistent with LRBA deficiency. The patient had a clinical phenotype of very early onset inflammatory bowel disease, failure to thrive, and multiple autoimmune manifestations. A homozygous 2-bp deletion was identified (c.3958_3986del; p.D1329Yfs*18) in the LRBA gene.

FIG 3

DOCK8 protein expression in lymphocytes from a healthy individual. DOCK8 protein is expressed intracellularly, and data are shown for T cells (A), B cells (B), and NK cells (C). Peripheral blood mononuclear cells (PBMCs) are isolated from blood samples treated with sodium heparin or EDTA and assessed for DOCK8 expression without stimulation, using an isotype control and a specific primary antibody. Intracellular protein expression is assessed by cell fixation and permeabilization prior to simultaneous staining with cell lineage markers and primary antibody. The protein is visualized using a fluorescently labeled secondary antibody. Both percent-positive lymphocyte subsets (T, B, or NK cells) along with mean fluorescence intensity (MFI) information are captured. DOCK8 protein is robustly expressed in the majority of lymphocyte subsets without stimulation.

FIG 4

Assessment of neutrophil oxidative burst using DHR flow cytometry. NADPH oxidase, which produces the respiratory burst in neutrophils, can be assessed following in vitro stimulation with PMA. The oxidation of DHR to fluorescent rhodamine is measured by flow cytometry. The unstimulated fluorescence is represented by the green histogram, while the PMA-stimulated oxidative burst in neutrophils is represented by the red histogram. Both percent-positive neutrophils and mean fluorescence intensity (MFI) are captured for diagnostic interpretation. (A) A normal neutrophil oxidative burst is demonstrated by a complete shift in the stimulated signal. (B) Data on the assessment of neutrophil oxidative burst using DHR flow cytometry in a patient with X-linked CGD. In a patient with a mutation in the CYBB gene (encoding gp91phox protein), there is no evidence of a normal neutrophil oxidative burst, and the unstimulated (green) and stimulated (red) histograms overlap completely. (C and D) Assessment of neutrophil oxidative burst using DHR flow cytometry in a patient with autosomal recessive CGD due to NCF1 and NCF2 mutations, respectively. Patients with autosomal recessive CGD, due to defects in p47phox, have a different pattern of neutrophil oxidative burst, with a proportion of neutrophils negative (stimulated histogram overlapping with unstimulated) and the remaining neutrophils showing significantly reduced fluorescence. Patients with defects in p67phox have a similar pattern of neutrophil oxidative burst to those with NCF1 mutations, with a proportion of neutrophils negative (stimulated histogram overlapping with unstimulated) and the remaining neutrophils showing significantly reduced fluorescence. (E) Assessment of neutrophil oxidative burst using DHR flow cytometry in a patient with complete myeloperoxidase deficiency (cMPO). Patients with cMPO having mutations in the MPO gene may demonstrate a variable pattern of DHR fluorescence, ranging from partially positive to completely absent. In this example, there is complete shift of the neutrophils, indicative of the majority of neutrophils showing DHR fluorescence, but there is an overall significant reduction in the mean fluorescence intensity. This would suggest partially reduced but not completely absent neutrophil oxidative burst in this patient example. (F) Assessment of neutrophil oxidative burst using DHR flow cytometry in a male patient with atypical X-linked CGD. Neutrophil oxidative burst may be preserved in some patients with mutations in the CYBB gene, leading to an atypical clinical phenotype as well as flow cytometric pattern of DHR fluorescence. The pattern is similar to that seen with NCF4 gene mutations (p40phox) and the form of complete MPO deficiency seen in panel E. This is a young male patient with one episode of Burkholderia pneumonia with no other manifestations of CGD who had a missense mutation (c.1061A>G; p.H354R) in the CYBB gene, which has been previously reported to be associated with decreased but not absent NADPH oxidase activity in neutrophils. (G) Assessment of neutrophil oxidative burst using DHR flow cytometry in a female patient with extreme skewing of lyonization, resulting in a phenotype of X-linked CGD. Neutrophil oxidative burst assessment by DHR fluorescence in female carriers of X-linked CGD characteristically shows two populations consistent with the presence of mutant and normal alleles. However, if there is skewing of lyonization, the proportion of neutrophils that are negative for DHR fluorescence can increase. This is an example of an elderly female patient with a history of being a carrier for XL-CGD and who has an affected male offspring demonstrating age-related extreme skewing of lyonization with a DHR flow pattern similar to that seen in a male patient with XL-CGD. (H and I) Side scatter (SSC) and forward scatter (FSC) separation of neutrophils, monocytes, and lymphocytes in whole blood. The DHR flow analysis is performed on neutrophils. (H) A transported sample received within validated stability under optimal conditions with abundant viable neutrophils. INT, integral. (I) DHR fluorescence for PMA-stimulated neutrophils in this sample, indicating a normal and robust result. (J and K) Another transported sample, also received within validated stability, but under suboptimal conditions. (J) There is significant neutrophil cell death observed with reduced viable neutrophils. (K) DHR fluorescence from PMA-stimulated neutrophils of the same sample with high background in unstimulated control (green histogram) and two peaks (red histogram), indicating poor sample quality.

FIG 5

Immunophenotyping of lymphocyte subsets. (A) Lymphocyte subset quantitation. T, B, and NK cells can be quantitated by flow cytometry. The top panels (panels 1 to 3) show identification of lymphocytes using CD45 and side scatter (SSC). The use of CD14 allows discrimination between monocytes that may inadvertently be included in the lymphocyte population. CD45⁺ lymphocytes are further subdivided into CD3⁻ lymphocytes and CD3⁺ T cells. The CD3⁺ T cells are further analyzed for CD4⁺ and CD8⁺ T cells (panel 4), and the CD3⁻ lymphocytes are further assessed for B and NK cells (panel 5). Absolute quantitation (number of cells per microliter) can be performed with the use of fluorescent beads. (B) Identification of naive and memory T cells by flow cytometry. CD3⁺ T cells can be further divided based on specific cell markers (typically CD45RA and CD45RO though additional markers can be used) into naive and memory subsets. A healthy newborn infant shows predominantly naive CD45RA⁺ T cells in the CD4⁺ T cell compartment with only a few memory CD45RO⁺ T cells. (C) Identification of naive recent thymic emigrants by flow cytometry. CD4⁺ T cells are newly derived from thymic output and have typically not undergone antigen-induced cell expansion in the periphery and express CD31 (recent thymic emigrant marker). A large proportion of the naive CD45RA⁺ CD4⁺ T cells in a healthy newborn infant express CD31. (D) Identification of naive and memory T cells by flow cytometry in a patient with Omenn syndrome. In infants with leaky SCID or Omenn syndrome, there are usually no naive CD45RA⁺ CD4⁺ T cells, and the majority of T cells that are present are oligoclonally expanded and express the memory marker, CD45RO. They also have no recent thymic emigrants due to absent thymic output. This patient shows that more than 99% of CD4⁺ T cells present in blood express CD45RO with no CD45RA⁺ expression. Further, this patient demonstrated severely skewed T cell receptor repertoire diversity and absent thymic function, in addition to the phenotypic features associated with Omenn syndrome. This female patient had a RAG1 gene mutation associated with a hypomorphic form of SCID.

FIG 6

Assessment of lymphocyte telomere length in a patient with dyskeratosis congenita. (A) Leukocyte telomere length assessment is performed by measuring the median fluorescence intensity (MFI) of distinct cell subpopulations while controlling for DNA content (not shown) and controlling for the intrinsic fluorescent properties of the cell type analyzed (green [performed in a separate tube and overlaid on the graph]). The MFI of the gated lymphocyte cell population is shown on the graph (red), along with the internal positive hybridization control cells (fixed cow thymocytes [orange]). FITC, fluorescein isothiocyanate. (B) An example of a young patient with dyskeratosis congenita showing in the selected lymphocyte cell population example very short lymphocyte telomere length (red circle) compared to the reference curve summarizing data from healthy individuals – or below the first percentile of distribution for age (blue curve); green curves represent the 10th, 50th, and 90th percentiles, and the red curve represents the 99th percentile of distribution.