**6.4 Evaluation of NK function**

The importance of evaluating NK cells in human immunity has been previously underscored, and this is supported by two evidences: significantly increased number of patients with reduced NK cells and/or functions, and over 40 genetically defined congenital immunodeficiencies present with impaired NK cell functions [41]. There are several methods utilized for the examination of NK cell functions including 51chromium release assay, flow cytometry-based perforin/granzyme expression and CD107a degranulation. These assays are particularly valuable for the patients suspected of primary hemophagocytic lymphohistiocytosis [42, 43].

#### **6.5 Evaluation of the complement system**

The complement system can be evaluated by measuring the level or function of complement proteins that are involved in the classical and alternative activation pathways. C3 and C4 are the complements routinely tested. Quantitative and functional assay of C1 esterase inhibitor is essential for the diagnosis of hereditary angioedema. Assays of CH50 and AH50 are, respectively, used to measure the overall complement activity in the classical or alternative pathway. Combining the results of CH50 and AH50 is indicative for further investigation of individual complement proteins that initiate the classical or alternative pathway or common terminal pathway [44].

### **6.6 Flow cytometry**

As our understanding of the defect or dysfunction of immune system increases, immunophenotypic and functional assays based on flow cytometry have been extensively used in identifying the abnormality of various cell types and their functions associated with certain diseases, including PIDs. Furthermore, flow cytometry is also a favorable technique for the measurement of intra- and extracellular cytokine production (e.g., IL12, IFN, TNF, and TH17), cell surface protein expression (e.g., Foxp3, CTLA-4, and BTK), and cellular signaling pathways (e.g., phosphor-STAT) [45]. The information gained from flow cytometry analysis can assist not only in the diagnosis, monitoring, and treatment of the diseases but also in understanding the influence of immune system associated with genetic defects that are newly identified. **Table 5** lists the flow cytometry assays used for common PID disorders. Most of the tests listed are required to be undertaken in a specialized laboratory, with the exception of TBNK cell populations, memory B cells, and some function assays that can be performed in a routine diagnostic laboratory.

Proper instrument setting, standardized operating procedures, and good quality controls must be exercised when performing flow cytometric analysis, as flow cytometry is susceptible to assay variation,. The reported data must include both percentage and absolute number of specific cell population. Moreover, appropriate age-matched reference ranges should also be provided in the final report [47, 48].

Ideally, each laboratory should establish their own normal ranges, but this is often not feasible; alternatively, published reference ranges may be used if a proper validation has been undertaken.

To date, flow cytometry has also been widely used for evaluating cell functions. Traditionally, lymphocyte functions were tested by radioactive methods, such as cytotoxicity of T and NK cells (chromium release) or proliferation of T cells



#### **Table 5.**

*Phenotypic and functional assessment for PIDs by flow cytometry [45, 46].*

(tritiated thymidine uptake). These approaches are still recognized as gold standard by some clinicians. However, radioactive methods have the following intrinsic limitations: involvement of radioactivity, labor intensive, high expertise required, and poor result reproducibility. Additionally, seeking for a consistent healthy

#### *Primary Immunodeficiency DOI: http://dx.doi.org/10.5772/intechopen.89624*


#### **Table 6.**

*The advantages, limitations and recommendations of genetic technologies [50, 53].*

fresh blood as assay normal control and obtaining a proper reference range can be challenging in routine laboratory practice. Therefore, they have been gradually replaced by other methodologies, such as bioluminescence-based assay or flow cytometry–based assay, which use specific dye for the detection of cell proliferation (e.g., CFSE, PKH-2, or PKH-26) or cell death (e.g., 7AAD and Annexing V) [49]. Many assays based on flow cytometry have been increasingly popular as they are easier to perform, have quicker turnaround time, are nonradioactive, are capable of using whole blood, and are more robust compared to the traditional radioactive assays.

#### **6.7 Genetic testing**

Genetic testing plays a critical role in patients with PID in confirming diagnosis, predicting the prognosis, assessing the influences of genotype-phenotype associations, and family planning [50, 51]. Besides, early and accurate molecular diagnosis is vital for guiding the selection of appropriate treatment including genetic therapy. Several molecular tests are available in identifying the genetic defects of PIDs, such as chromosomal analysis, fluorescence in situ hybridization, chromosomal microarray, single gene by Sanger sequencing, gene panels by massively parallel, whole exome, and genome by next-generation sequencing [52]. The selection of these assays should consider their inherent advantages and limitations [50, 53]. The summary of these tests is shown in **Table 6**. Recent emerged simple molecular assays for measuring circular DNA segments namely T-cell receptor excision circles and kappa-deleting recombination excision circles, based on quantitative PCR amplification of DNA extracted from dried blood spots, enable for a quick screening of newborn SCID [54].

The choice of specific gene(s) for examination is suggested by the patient's clinical history and phenotypical and functional results. Clinicians are required to have a basic understanding of the utility, accessibility of different genetic approaches. The selection criteria of molecular methodology should be based on the greatest odds of achieving the diagnosis within an acceptable time frame with the most cost-effective test. There is no specific algorithm for genetic testing in patients with PID as individual's genetic mutation is often unique, the technology, cost, and the assay turnaround time are constantly changing, and each molecular method has inherent advantages and limitations. Practically, two or more approaches are often used together to achieve an optimal diagnosis [50]. For example, single gene Sanger sequencing is considered to be not only a simple and reliable assay for testing patients with known monogenic mutations of PID or their family members, but it can also serve as a tool for confirming the genetic variants detected by whole exome sequencing. When assessing large numbers of mutations, gene panels or whole genome/exome approach may be more cost-effective and faster than single gene analysis. Since genetic testing in primary immunodeficiency is highly personalized, and a specific genetic mutation does not always translate into a disease, test results must be interpreted with caution by genetic consultants and immunologists.

The recent advances of sequencing technologies have facilitated the genomic assays to become the standard of care in some hospitals although these techniques may face the challenges of cost, accessibility, and interpretation issues. The exponential growth of genetic analysis by next-generation sequencing and other novel molecular technologies has enabled quick identification of known and novel mutations, which contributed to a dramatic expansion of the number and types of PIDs [16, 53, 55].

#### **7. Treatment**

Treatments for PIDs involve preventing and controlling recurrent infections, treating symptoms, strengthening the immunity, and treating the underlying cause of the immune defects. Illness associated with PIDs such as autoimmune disorders or malignancies should also be managed [1, 13].

More aggressive and/or longer course of antibiotics than "normal infections" is usually prescribed in patients with PID, in order to control the infections caused by bacteria or fungi. Some patients may require prolonged antibiotic therapy to prevent infections and permanent damage to organs [13]. Routine immunizations can also provide protective immunity to those at risk of infections, but the attenuated

#### *Primary Immunodeficiency DOI: http://dx.doi.org/10.5772/intechopen.89624*

vaccines such as oral polio and measles-mumps-rubella might not be suitable for children with PIDs. For viral infection, interferon-gamma therapy may be of choice besides other antiviral drugs routinely used (e.g., amantadine and acyclovir) [13]. In patients with chronic granulomatous disease, using granulocyte colony–stimulating factor, a glycoprotein that is able to stimulate the proliferation/differentiation and improve the functions of neutrophil, can help increase the levels of immunestrengthening leukocytes to control the infections [56].

Immunoglobulin replacement has been the pillar of therapy for recurrent infections of PIDs, since around 60% of PID cases have impaired antibody production [57]. In fact, most of these patients will require life-time immunoglobulin replacement therapy. Immunoglobulin can be delivered either intravenously (abbreviated IVIG) or subcutaneously (abbreviated SCIG). The choice of which route depends on the circumstance although both of them have been demonstrated to be effective. Because higher IgG levels can be obtained through intravenous administration, IVIG has been routinely used for preventing serious/ recurrent infections [58]; however, SCIG has recently emerged as a popular route for delivery due to its fewer side effects and greater flexibility [57, 59]. Future research direction is focusing on more precise IgG replacement in PIDs, such as the development of IgG subclass-specific enriched preparation and microbespecific IgG [58].


#### **Table 7.**

*Current strategies for the treatment of PIDs [1, 13, 66].*

Apart from controlling infections, the considerable morbidity and mortality caused by noninfectious complications of PIDs can also be troublesome to clinicians. To standardize clinical practices and improve treatment outcome, British Society of Immunology has recently published the first set of recommendations for monitoring and managing the noninfectious complications of CVID [60].

Bone marrow transplantation (BMT) and hematopoietic stem cell transplantation (HSCT) are feasible options for a permanent cure for several types of lifethreatening immunodeficiency, with SCID in particular [61, 62]. Immune system can reconstitute when stem cells harvested from bone marrow or cord blood are transferred to the patients with PID. However, the successful rate of biological match, possibility of life-threatening graft-versus-host-disease, and the risk of uncontrolled infections following the destruction of the patient's own immune system prior to the transplant should be well evaluated.

The technical advances of genetic engineering provide another hope to cure PIDs. Substantial progress has been made in the past decade in treating several types of PIDs (e.g., adenosine deaminase-SCID, SCID-X1, chronic granulomatous disorder, and Wiskott-Aldrich syndrome) with gene therapy [63–65]. Current treatment scenario is mostly based on ex-vivo deliver of therapeutic transgene through viral vectors to autologous stem cells, followed by transplantation back to the same patient. Although the overall outcome from all the clinical trials targeting different PIDs has been extremely promising, however, serious adverse events (e.g., vectormediated oncogenesis) and high cost may be a hindrance to clinical trials and promotion of gene therapy [63, 65]. A summary of current strategies for treatment and management of PIDs is shown in **Table 7**.

#### **8. Prognosis**

The prognosis of patients with PID is extremely variable depending on the type of immune defects. Infants with SCID will die in the first 2 years of life without HSCT/BMT or gene therapy. Individuals who obtained stem cell transplantation in early childhood (before 3.5 months) have better prognosis [67]. Many PID patients who received proper medical care and treatments are able to live healthy and independent life for a long term. With the enhancement in managing infections and other complications and growing application of definitive therapies, the outcomes and long-term survival of PIDs have improved dramatically since the 1970s [13].

#### **9. Conclusion**

The investigation of PIDs has provided valuable insights to understand the specific gene defect that impairs the immune system. Flow cytometry and genetic testing enable to identify existing and novel phenotypes and genotypes as well as their impact on PIDs. The applications of flow cytometry and genetic technologies have expanded dramatically with more types of PID is defined, and the use of mass sequencing technologies has accelerated the identification of novel disorders. To efficiently use these complex assays, clinicians should have a good understanding of these methods and know how to interpret the results for diagnosis and disease management [33].

The management of patients with PID is based on three aspects of diagnosis: suspicious clinical manifestations, aberrant results of immune response, and the underlying genetic defect [4]. However, the diagnosis of PIDs may confront significant challenges: there are large numbers of variable types of PIDs to be recognized

#### *Primary Immunodeficiency DOI: http://dx.doi.org/10.5772/intechopen.89624*

and most of them have alike clinical presentations with common diseases; immunodeficiencies derived from multiple gene defects can share similar symptoms, and a defect in the same gene may have various clinical manifestations [1]. While severe forms of PIDs are relatively easier to be recognized, milder immunodeficiencies may not raise alertness until typical presentation occurs [68, 69]. Additionally, the criteria for constituting a PID diagnosis are subjective, for example, the degree of frequency and the severity of the infections for establishing the diagnosis are unclear, the association of PIDs with autoimmune disorder or malignancy is ambiguous, and some individuals may not have noticeable symptoms apart from laboratory findings. Furthermore, advanced laboratory examination such as specialized flow cytometric and genetic analysis is not always easy to access. All these factors may contribute to delayed or missed diagnosis of the diseases.

To combat the challenges, clinical warning signs of PIDs should be disseminated to all clinicians for raising earlier recognition of the diseases, and an immunologist must be consulted for proper diagnosis and management. Due to the complexity of clinical presentations and large number of disease types, the use of scoring system based on the codes of the international classification of PIDs [69] assisted by artificial intelligence may be beneficial for clinicians to differentiate these disorders from other diseases and raise initial recognition. The recent advances in understanding the human immune system, development of novel cellular and molecular assays, and collaborations from the international/national organizations have led to significant increase of clinical awareness and cases diagnosed and improvement of disease management and treatment outcomes for PIDs.
