Combined primary immune deficiency: diagnosis by clinical flow cytometry

A mutation/defect affecting any of the processes that aid in the natural ability of the human body to protect itself from infection and disease may compromise the immune system of an individual. Susceptibility to acute or chronic disorders may be biologically explained due to failure or dysfunction of several factors, both internal and external; thus the scientific community constantly engages in advancing technologies and methodologies to delineate the pathophysiological processes involved in causing the damage. 

Some of these disorders are inherited congenitally, leading to the growing list of primary immune deficiency diseases (PIDDs), a set of diseases distinguished from acquired syndromes or those occurring secondary to known sequelae. The International Union of Immunological Societies (IUIS) Expert Committee on Primary Immunodeficiency revises the classification of these challenging disorders and compiles an update on a biennial basis. Based on typical immunologic manifestations supported by clinical presentation and genetic abnormalities, all reported and known PIDDs are grouped into eight major categories. For purposes of this article, not all 150+ disorders will be listed, and the reader is referred to the current published report.¹ An illustration of flow cytometry application by immune-phenotyping patient’s blood supplemented by functional testing in the first category of combined immunodeficiencies (CID) will be described here.

Severe combined immunodeficiency (SCID), as well as deficiencies in DNA ligase IV, ZAP-70, Ca2+ channel, MHC I & II, STAT5b and DOCK8, are among the 22 different classes of CIDs described so far. SCID is characterized by profound impairment of both cell- and antibody-mediated immunity due to marked reduction in T cells. A decrease in B cells or natural killer (NK) cells may also present in some forms of SCID due to specific genetic defect. Infants typically present with recurrent bacterial and viral infections, failure to thrive, and reactions to live viral vaccines. Therefore, if undetected, SCID leads to life-threatening complications requiring treatment with hematopoietic stem cell transplantation (HSCT).

Wisconsin was the first state in the United States to initiate a statewide newborn screening program in 2008 for identifying babies born with SCID or other T cell lymphopenias. Several states followed suit, including California, Colorado, Connecticut, Delaware, Florida, Massachusetts, etc. The initial screening consists of quantitating the number of T-cell excision circles (TRECs) using real-time quantitative polymerase chain reaction on DNA extracted from dried blood spots on newborn screen (Guthrie) cards.^2-4 All full-term and pre-term infants who have reached 37 weeks of gestation that do not meet the criteria of passing the NBS-SCID screen for TREC (specified by each state’s public health laboratory) are followed up by flow cytometry confirmation and consult by a clinical immunologist. 

Fluorochrome conjugated antibodies are used to enumerate the percent and absolute cell counts of T helper cells (CD3+ CD4+), T cytotoxic cells (CD3+ CD8+), B cells (CD19+), and NK cells (CD56+) in the peripheral blood of a patient. This limited panel would suffice to classify the SCID into T-, B+, NK+/-, or T- B- NK+/- types. γ chain deficiency is the only T- B+ NK- SCID type [Figure 1] that is inherited in an X-linked manner, while others such as JAK3, IL7Rα, CD45, and Coronin-1A follow the autosomal recessive trait. Several cytokines including IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, etc., produced by a variety of immune and non-immune cells (fibroblasts, stromal cells, epithelial cells) signal through the JAK (Janus associated kinase) – STAT (signal transducer and transcription) pathway. The cytokine receptors are composed of heterotrimeric chains one of which is shared amongst these family members—the common γ chain as the signaling component. Cases have been reported of γc receptor signaling deficiencies with subtle manipulations of the in vitro systems to determine the specific cytokine receptor defect.⁵ 

Peripheral blood mononuclear cells (PBMCs) obtained from patients with CIDs usually do not respond to T cell mitogens, such as lectin from Phaseolus vulgaris, phytohemagglutinin (PHA), and Concanavalin A from Canvalia ensiformis (Con A). Flow cytometry provides the advantage to determine T cell proliferation to these stimulants without resorting to the traditional [³H]-thymidine incorporation studies, thus avoiding radiation. Briefly, 5(6)-carbosyfluorescein diacetate N-succinimidyl ester (CFSE) labeled PBMCs are incubated with stimulants for four to five days at 37oC/5% CO2 and determine the number of fluorescent peaks corresponding to cell divisions of the parent cell population (Figure 2).

PIDDs can also present clinically with aberrations of defense, homeostasis, or impaired surveillance—the three basic functions of the immune system. In addition to PIDDs, which have been observed mainly in children, there exist a more frequently encountered group of secondary immune deficiencies that occur both in children and adults. These are the consequences of  another disorder or underlying condition (autoimmune disease, sickle cell disease); infectious process (HIV, bacterial infections); genetic disorder  or chromosome abnormality (cystic fibrosis, Down syndrome); age (premature birth, elderly population); surgery or trauma (splenectomy, burns): lymphoproliferative malignancies (multiple myeloma, lymphomas) or treatment with immunosuppressive agents (corticosteroids, anti-thymocyte globulin) that result in increased susceptibility to infections.

Dr. Ogden Bruton’s observation of agammaglobulinemia in a male child suffering from recurrent otitis media, lacking tonsillar tissue, led to the ground-breaking diagnosis of XLA in 1952.⁶  Immunoglobulin replacement is the treatment of choice with intravenous infusion or sub-cutaneous administration of highly purified and concentrated IgG antibodies from pooled healthy donors.^7-8 

Flow cytometry applications are not limited to the two examples described here. Detection of intracellular cytokines, phosphorylated proteins, and apoptotic events is also possible with the newer instruments with lasers capable of multi-color analyses.

References

1. Al-Herz W, Bousfiha A, Casanova JL, et al. Primary immunodeficiency diseases: an update on the classification from the international union of immunological societies expert committee for primary immunodeficiency. Front Immunol. 2011;2:54.
2. Baker MW, Grossman WJ, Laessig RH, et al. Development of a routine newborn screening protocol for severe combined immunodeficiency. J Allergy Clin Immunol. 2009;124(3):522-527.
3. Puck JM. Population-based newborn screening for severe combined immunodeficiency. Biol Blood Marrow Transplant. 2008;14:78-80.
4. Routes JM, Grossman WJ, Verbsky J, et al. Statewide newborn screening for severe T-cell lymphopenia. JAMA. 2009;302(22):2465-2470.
5. Verbsky JW, Baker MW, Grossman WJ, et al. Newborn screening for severe combined immunodeficiency; the Wisconsin experience (2008-2011). J Clin Immunol. 2012;32(1):82-88.
6. Bruton OC. Agammaglobulinemia. Pediatrics. 1952;9(6):722-728.
7. Berger M. Choices in IgG replacement therapy for primary immune deficiency diseases: subcutaneous IgG vs. intravenous IgG and selecting an optimal dose. Curr Opin Allergy Clin Immunol. 2011;11(6):532-538.
8. Bonagura VR. Using intravenous immunoglobulin (IVIG) to treat patients with primary immune deficiency disease. J Clin Immunol .2013;33:Suppl 2:S90-S94.